Tzartos SJ

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Full name : Tzartos Socrates J

First name : Socrates J

Mail : Dept. of Biochemistry, Hellenic Pasteur Institute, 127, Vas. Sofias Avenue, Athens 11521

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Country : Greece

Email : stzartos@gmail.com

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References (124)

Title : Nicotinic cholinergic system and COVID-19: In silico evaluation of nicotinic acetylcholine receptor agonists as potential therapeutic interventions - Alexandris_2021_Toxicol.Rep_8_73
Author(s) : Alexandris N , Lagoumintzis G , Chasapis CT , Leonidas DD , Papadopoulos GE , Tzartos SJ , Tsatsakis A , Eliopoulos E , Poulas K , Farsalinos K
Ref : Toxicol Rep , 8 :73 , 2021
Abstract : SARS-CoV-2 infection was announced as a pandemic in March 2020. Since then, several scientists have focused on the low prevalence of smokers among hospitalized COVID-19 patients. These findings led to our hypothesis that the Nicotinic Cholinergic System (NCS) plays a crucial role in the manifestation of COVID-19 and its severe symptoms. Molecular modeling revealed that the SARS-CoV-2 Spike glycoprotein might bind to nicotinic acetylcholine receptors (nAChRs) through a cryptic epitope homologous to snake toxins, substrates well documented and known for their affinity to the nAChRs. This binding model could provide logical explanations for the acute inflammatory disorder in patients with COVID-19, which may be linked to severe dysregulation of NCS. In this study, we present a series of complexes with cholinergic agonists that can potentially prevent SARS-CoV-2 Spike glycoprotein from binding to nAChRs, avoiding dysregulation of the NCS and moderating the symptoms and clinical manifestations of COVID-19. If our hypothesis is verified by in vitro and in vivo studies, repurposing agents currently approved for smoking cessation and neurological conditions could provide the scientific community with a therapeutic option in severe COVID-19.
ESTHER : Alexandris_2021_Toxicol.Rep_8_73
PubMedSearch : Alexandris_2021_Toxicol.Rep_8_73
PubMedID: 33425684

Title : Crystal structure of a human neuronal nAChR extracellular domain in pentameric assembly: Ligand-bound alpha2 homopentamer - Kouvatsos_2016_Proc.Natl.Acad.Sci.U.S.A_113_9635
Author(s) : Kouvatsos N , Giastas P , Chroni-Tzartou D , Poulopoulou C , Tzartos SJ
Ref : Proc Natl Acad Sci U S A , 113 :9635 , 2016
Abstract : In this study we report the X-ray crystal structure of the extracellular domain (ECD) of the human neuronal alpha2 nicotinic acetylcholine receptor (nAChR) subunit in complex with the agonist epibatidine at 3.2 A. Interestingly, alpha2 was crystallized as a pentamer, revealing the intersubunit interactions in a wild type neuronal nAChR ECD and the full ligand binding pocket conferred by two adjacent alpha subunits. The pentameric assembly presents the conserved structural scaffold observed in homologous proteins, as well as distinctive features, providing unique structural information of the binding site between principal and complementary faces. Structure-guided mutagenesis and electrophysiological data confirmed the presence of the alpha2(+)/alpha2(-) binding site on the heteromeric low sensitivity alpha2beta2 nAChR and validated the functional importance of specific residues in alpha2 and beta2 nAChR subunits. Given the pathological importance of the alpha2 nAChR subunit and the high sequence identity with alpha4 (78%) and other neuronal nAChR subunits, our findings offer valuable information for modeling several nAChRs and ultimately for structure-based design of subtype specific drugs against the nAChR associated diseases.
ESTHER : Kouvatsos_2016_Proc.Natl.Acad.Sci.U.S.A_113_9635
PubMedSearch : Kouvatsos_2016_Proc.Natl.Acad.Sci.U.S.A_113_9635
PubMedID: 27493220

Title : Inflammation decreases the level of alpha7 nicotinic acetylcholine receptors in the brain mitochondria and makes them more susceptible to apoptosis induction - Lykhmus_2015_Int.Immunopharmacol_29(1)_148
Author(s) : Lykhmus O , Gergalova G , Zouridakis M , Tzartos SJ , Komisarenko S , Skok MV
Ref : Int Immunopharmacol , 29 :148 , 2015
Abstract : alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) are involved in regulating inflammatory reactions, as well as the cell viability. They are expressed in both the plasma membrane and mitochondria of eukaryotic cells. Previously we found that neuroinflammation resulted in the decrease of alpha7 nAChR density in the brain of mice and was accompanied by accumulation of amyloid-beta (Abeta) peptides and memory impairment. In the present paper, it is shown that inflammation induced by either regular bacterial lipopolysaccharide (LPS) injections or immunizations with alpha7 nAChR extracellular domain (1-208) affected also the brain cell mitochondria. Using various modifications of sandwich ELISA, we observed the decrease of alpha7 nAChRs and accumulation of Abeta(1-40) and Abeta(1-42) in mitochondria of immunized or LPS-treated mice compared to control ones. Mitochondria of treated mice responded with cytochrome c release to lower Ca(2+) concentrations than mitochondria of control mice and were less sensitive to its attenuation with alpha7 nAChR agonist PNU282987. It is concluded that inflammation decreases alpha7 nAChR expression in both mitochondria and cell plasma membrane and makes mitochondria more susceptible to apoptosis induction.
ESTHER : Lykhmus_2015_Int.Immunopharmacol_29(1)_148
PubMedSearch : Lykhmus_2015_Int.Immunopharmacol_29(1)_148
PubMedID: 25887272

Title : AChR-myasthenia gravis switching to double-seropositive several years after the onset - Zouvelou_2014_J.Neuroimmunol_267_111
Author(s) : Zouvelou V , Zisimopoulou P , Psimenou E , Matsigkou E , Stamboulis E , Tzartos SJ
Ref : Journal of Neuroimmunology , 267 :111 , 2014
Abstract : We report an early onset AChR-myasthenia gravis (MG) with biphasic clinical course. The clinical "switch" from AChR-MG to MuSK-MG emerged 16 years after the onset and 11 years after thymectomy. MuSK antibodies were detected only by cell-based assay and only upon clinical "switch", while AChR antibodies remained positive and at high titers during the whole disease course. Although the occurrence of AChR antibodies and MuSK antibodies in the same individual is rare, the re-assessment of the antibody status, using all available assays, is advisable when there is clinical indication.
ESTHER : Zouvelou_2014_J.Neuroimmunol_267_111
PubMedSearch : Zouvelou_2014_J.Neuroimmunol_267_111
PubMedID: 24388224

Title : Expression of human AChR extracellular domain mutants with improved characteristics - Lazaridis_2014_Int.J.Biol.Macromol_63_210
Author(s) : Lazaridis K , Zisimopoulou P , Giastas P , Bitzopoulou K , Evangelakou P , Sideri A , Tzartos SJ
Ref : Int J Biol Macromol , 63 :210 , 2014
Abstract : The muscle nicotinic acetylcholine receptor (AChR) has a central role in neuromuscular transmission, and is the major target in the autoimmune disease myasthenia gravis (MG). We created mutants of the extracellular domains (ECDs) of the human alpha1, beta1, delta and varepsilon AChR subunits, whereby their Cys-loop was exchanged for that of the acetylcholine binding protein. The mutants were expressed in Pichia pastoris and had improved solubility resulting in 2- to 43-fold higher expression yields compared to the wild type. An additional mutant was created for the alpha1 ECD restoring its glycosylation site within the Cys-loop and its alpha-bungarotoxin binding ability. Furthermore, we constructed dimeric and pentameric concatamers of the mutant ECDs. All concatamers were successfully expressed as soluble secreted proteins, although the pentamers had about 10-fold lower expression than the dimers and were more susceptible to fragmentation. Initial crystallizations with the mutant ECDs were promising, and we reproducibly obtained crystals of the beta1 ECD, diffracting at approximately 12A. Further optimization is underway to obtain crystals suitable for high resolution crystallography. The proteins described herein are useful tools in structural studies of the human muscle AChR and can be used in applications requiring high yields such as therapeutic adsorbents for MG autoantibodies.
ESTHER : Lazaridis_2014_Int.J.Biol.Macromol_63_210
PubMedSearch : Lazaridis_2014_Int.J.Biol.Macromol_63_210
PubMedID: 24246999

Title : Clonal heterogeneity of thymic B cells from early-onset myasthenia gravis patients with antibodies against the acetylcholine receptor - Vrolix_2014_J.Autoimmun_52_101
Author(s) : Vrolix K , Fraussen J , Losen M , Stevens J , Lazaridis K , Molenaar PC , Somers V , Bracho MA , Le Panse R , Stinissen P , Berrih-Aknin S , Maessen JG , Van Garsse L , Buurman WA , Tzartos SJ , De Baets MH , Martinez-Martinez P
Ref : J Autoimmun , 52 :101 , 2014
Abstract : Myasthenia gravis (MG) with antibodies against the acetylcholine receptor (AChR-MG) is considered as a prototypic autoimmune disease. The thymus is important in the pathophysiology of the disease since thymus hyperplasia is a characteristic of early-onset AChR-MG and patients often improve after thymectomy. We hypothesized that thymic B cell and antibody repertoires of AChR-MG patients differ intrinsically from those of control individuals. Using immortalization with Epstein-Barr Virus and Toll-like receptor 9 activation, we isolated and characterized monoclonal B cell lines from 5 MG patients and 8 controls. Only 2 of 570 immortalized B cell clones from MG patients produced antibodies against the AChR (both clones were from the same patient), suggesting that AChR-specific B cells are not enriched in the thymus. Surprisingly, many B cell lines from both AChR-MG and control thymus samples displayed reactivity against striated muscle proteins. Striational antibodies were produced by 15% of B cell clones from AChR-MG versus 6% in control thymus. The IgVH gene sequence analysis showed remarkable similarities, concerning VH family gene distribution, mutation frequency and CDR3 composition, between B cells of AChR-MG patients and controls. MG patients showed clear evidence of clonal B cell expansion in contrast to controls. In this latter aspect, MG resembles multiple sclerosis and clinically isolated syndrome, but differs from systemic lupus erythematosus. Our results support an antigen driven immune response in the MG thymus, but the paucity of AChR-specific B cells, in combination with the observed polyclonal expansions suggest a more diverse immune response than expected.
ESTHER : Vrolix_2014_J.Autoimmun_52_101
PubMedSearch : Vrolix_2014_J.Autoimmun_52_101
PubMedID: 24439114

Title : A comprehensive analysis of the epidemiology and clinical characteristics of anti-LRP4 in myasthenia gravis - Zisimopoulou_2014_J.Autoimmun_52_139
Author(s) : Zisimopoulou P , Evangelakou P , Tzartos J , Lazaridis K , Zouvelou V , Mantegazza R , Antozzi C , Andreetta F , Evoli A , Deymeer F , Saruhan-Direskeneli G , Durmus H , Brenner T , Vaknin A , Berrih-Aknin S , Frenkian Cuvelier M , Stojkovic T , DeBaets M , Losen M , Martinez-Martinez P , Kleopa KA , Zamba-Papanicolaou E , Kyriakides T , Kostera-Pruszczyk A , Szczudlik P , Szyluk B , Lavrnic D , Basta I , Peric S , Tallaksen C , Maniaol A , Tzartos SJ
Ref : J Autoimmun , 52 :139 , 2014
Abstract : Double-seronegative myasthenia gravis (dSN-MG, without detectable AChR and MuSK antibodies) presents a serious gap in MG diagnosis and understanding. Recently, autoantibodies against the low-density lipoprotein receptor-related protein 4 (LRP4) have been identified in several dSN-MG sera, but with dramatic frequency variation ( approximately 2-50%). We have developed a cell based assay (CBA) based on human LRP4 expressing HEK293 cells, for the reliable and efficient detection of LRP4 antibodies. We have screened about 800 MG patient sera from 10 countries for LRP4 antibodies. The overall frequency of LRP4-MG in the dSN-MG group (635 patients) was 18.7% but with variations among different populations (range 7-32.7%). Interestingly, we also identified double positive sera: 8/107 anti-AChR positive and 10/67 anti-MuSK positive sera also had detectable LRP4 antibodies, predominantly originating from only two of the participating groups. No LRP4 antibodies were identified in sera from 56 healthy controls tested, while 4/110 from patients with other neuroimmune diseases were positive. The clinical data, when available, for the LRP4-MG patients were then studied. At disease onset symptoms were mild (81% had MGFA grade I or II), with some identified thymic changes (32% hyperplasia, none with thymoma). On the other hand, double positive patients (AChR/LRP4-MG and MuSK/LRP4-MG) had more severe symptoms at onset compared with any single positive MG subgroup. Contrary to MuSK-MG, 27% of ocular dSN-MG patients were LRP4 antibody positive. Similarly, contrary to MuSK antibodies, which are predominantly of the IgG4 subtype, LRP4 antibodies were predominantly of the IgG1 and IgG2 subtypes. The prevalence was higher in women than in men (female/male ratio 2.5/1), with an average disease onset at ages 33.4 for females and 41.9 for males. Overall, the response of LRP4-MG patients to treatment was similar to published responses of AChR-MG rather than to MuSK-MG patients.
ESTHER : Zisimopoulou_2014_J.Autoimmun_52_139
PubMedSearch : Zisimopoulou_2014_J.Autoimmun_52_139
PubMedID: 24373505

Title : Double seronegative myasthenia gravis with low density lipoprotein-4 (LRP4) antibodies presenting with isolated ocular symptoms - Tsivgoulis_2014_J.Neurol.Sci_346_328
Author(s) : Tsivgoulis G , Dervenoulas G , Kokotis P , Zompola C , Tzartos JS , Tzartos SJ , Voumvourakis KI
Ref : Journal of Neurology Sci , 346 :328 , 2014
Abstract : The detection of low density lipoprotein-4 (LRP4) antibodies in double seronegative (dSN) myasthenia gravis (MG) patients has provided new insights in the diagnosis and treatment of MG. However, there are limited data regarding the clinical presentation and treatment response in dSN MG patients with LRP4-antibodies. We present a case series of three Caucasian dSN MG patients with positive LRP4-antibodies sharing a common ethnic background that presented with isolated ocular symptoms (MGFA I). The demographic and clinical characteristics, the diagnostic work-up as well as the treatment response during a follow-up period of 12-24 months are described in detail. All patients were treated successfully with acetylcholinesterase inhibitors (AcheI) and prednisone with two exhibiting full remission of their symptoms, while the remaining exhibited mild residual diplopia. Notably, we documented no signs of generalized disease progression, while no patient required immunosuppressive treatment. In conclusion, the distinct clinical phenotype of our patients highlights the clinical relevance of screening for LRP4-antibodies in patients presenting with isolated ocular MG independent of age and gender, since it may lead to the timely diagnosis of MG and prompt initiation of effective therapy with ACheI and corticosteroids.
ESTHER : Tsivgoulis_2014_J.Neurol.Sci_346_328
PubMedSearch : Tsivgoulis_2014_J.Neurol.Sci_346_328
PubMedID: 25248951

Title : Scale up and safety parameters of antigen specific immunoadsorption of human anti-acetylcholine receptor antibodies - Lagoumintzis_2014_J.Neuroimmunol_267_1
Author(s) : Lagoumintzis G , Zisimopoulou P , Trakas N , Grapsa E , Poulas K , Tzartos SJ
Ref : Journal of Neuroimmunology , 267 :1 , 2014
Abstract : Myasthenia gravis is an autoimmune disease usually caused by autoantibodies against the muscle nicotinic acetylcholine receptor (nAChR). Current treatments are not specific, and thus often cause side effects. Here, we elaborate on our previous findings on antigen specific immunoadsorption towards scaling up the method as well as testing whole blood apheresis. The average percent of plasma or whole blood immunoadsorption was up to 79.5%+/-2.9. Moreover, neither pyrogens were co-administered nor did complement activation occur after immunoadsorption. Thus, antigen-specific apheresis of anti-AChR autoantibodies seems a safe and effective treatment for myasthenia gravis that can be scaled up for clinical testing.
ESTHER : Lagoumintzis_2014_J.Neuroimmunol_267_1
PubMedSearch : Lagoumintzis_2014_J.Neuroimmunol_267_1
PubMedID: 24412396

Title : Crystal structures of free and antagonist-bound states of human alpha9 nicotinic receptor extracellular domain - Zouridakis_2014_Nat.Struct.Mol.Biol_21_976
Author(s) : Zouridakis M , Giastas P , Zarkadas E , Chroni-Tzartou D , Bregestovski PD , Tzartos SJ
Ref : Nat Struct Mol Biol , 21 :976 , 2014
Abstract : We determined the X-ray crystal structures of the extracellular domain (ECD) of the monomeric state of human neuronal alpha9 nicotinic acetylcholine receptor (nAChR) and of its complexes with the antagonists methyllycaconitine and alpha-bungarotoxin at resolutions of 1.8 A, 1.7 A and 2.7 A, respectively. The structure of the monomeric alpha9 ECD superimposed well with the structures of homologous proteins in pentameric assemblies, denoting native folding, despite the absence of a complementary subunit and transmembrane domain. The interaction motifs of both antagonists were similar to those in the complexes with homologous pentameric proteins, thus highlighting the major contribution of the principal side of alpha9 ECD to their binding. The structures revealed a functionally important beta7-beta10 strand interaction in alpha9-containing nAChRs, involving their unique Thr147, a hydration pocket similar to that of mouse alpha1 ECD and a membrane-facing network coordinated by the invariant Arg210.
ESTHER : Zouridakis_2014_Nat.Struct.Mol.Biol_21_976
PubMedSearch : Zouridakis_2014_Nat.Struct.Mol.Biol_21_976
PubMedID: 25282151

Title : Expression of a highly antigenic and native-like folded extracellular domain of the human alpha1 subunit of muscle nicotinic acetylcholine receptor, suitable for use in antigen specific therapies for Myasthenia Gravis - Niarchos_2013_PLoS.One_8_e84791
Author(s) : Niarchos A , Zouridakis M , Douris V , Georgostathi A , Kalamida D , Sotiriadis A , Poulas K , Iatrou K , Tzartos SJ
Ref : PLoS ONE , 8 :e84791 , 2013
Abstract : We describe the expression of the extracellular domain of the human alpha1 nicotinic acetylcholine receptor (nAChR) in lepidopteran insect cells (i-alpha1-ECD) and its suitability for use in antigen-specific therapies for Myasthenia Gravis (MG). Compared to the previously expressed protein in P. pastoris (y-alpha1-ECD), i-alpha1-ECD had a 2-fold increased expression yield, bound anti-nAChR monoclonal antibodies and autoantibodies from MG patients two to several-fold more efficiently and resulted in a secondary structure closer to that of the crystal structure of mouse alpha1-ECD. Our results indicate that i-alpha1-ECD is an improved protein for use in antigen-specific MG therapeutic strategies.
ESTHER : Niarchos_2013_PLoS.One_8_e84791
PubMedSearch : Niarchos_2013_PLoS.One_8_e84791
PubMedID: 24376846

Title : Antigen-specific apheresis of autoantibodies in myasthenia gravis - Lazaridis_2012_Ann.N.Y.Acad.Sci_1275_7
Author(s) : Lazaridis K , Zisimopoulou P , Lagoumintzis G , Skriapa L , Trakas N , Evangelakou P , Kanelopoulos I , Grapsa E , Poulas K , Tzartos SJ
Ref : Annals of the New York Academy of Sciences , 1275 :7 , 2012
Abstract : Myasthenia gravis (MG) is an autoimmune disorder affecting the neuromuscular junction, usually caused by autoantibodies against the acetylcholine receptor (AChR) or the muscle-specific kinase (MuSK). Our aim is the development of a therapy based on the selective extracorporeal elimination of anti-AChR or anti-MuSK antibodies. To this end, the extracellular domains of the AChR subunits and MuSK have been expressed in yeast to be used as adsorbents, after optimization, and to obtain large quantities of proteins with near-native structure. We have characterized these proteins with respect to their use as specific immunoadsorbents for MG autoantibodies, and have begun large-scale experiments in order to verify the feasibility of application of the method for therapy. Furthermore, we have initiated animal studies to test possible toxicity and safety issues of the adsorbents or the procedure itself. The successful completion of the scale-up and safety tests will allow the initiation of clinical trials.
ESTHER : Lazaridis_2012_Ann.N.Y.Acad.Sci_1275_7
PubMedSearch : Lazaridis_2012_Ann.N.Y.Acad.Sci_1275_7
PubMedID: 23278571

Title : Anti-MuSK- and anti-AChR-positive myasthenia gravis induced by d-penicillamine - Poulas_2012_J.Neuroimmunol_250_94
Author(s) : Poulas K , Koutsouraki E , Kordas G , Kokla A , Tzartos SJ
Ref : Journal of Neuroimmunology , 250 :94 , 2012
Abstract : BACKGROUND: Myasthenia gravis (MG) is an autoimmune disorder of the neuromuscular junction usually caused by antibodies to the nicotinic acetylcholine receptor (AChR) and occasionally to muscle-specific kinase (MuSK). D-penicillamine is a therapeutic agent for several diseases, but can also induce a number of immune-mediated disorders, including MG, as a side-effect. In most patients with D-penicillamine-induced MG, anti-AChR antibodies are detected, but the presence of anti-MuSK antibodies has not been reported previously. CASE: The case reported was a female patient who presented with myasthenic symptoms after D-penicillamine administration for scleroderma.
RESULTS: Both anti-AChR and anti-MuSK antibodies were identified in the patient's serum. The anti-MuSK antibodies were of the IgG4 subclass, as in idiopathic MG. Both types of antibody gradually disappeared after discontinuation of D-penicillamine. A significant improvement in symptoms was observed and the patient gradually became free of MG symptoms, without requiring any treatment for MG. Another four double-positive (anti-AChR and anti-MuSK antibodies) patients were identified during a retrospective study, but none had been treated with D-penicillamine. CONCLUSION: D-penicillamine can cause anti-AChR and anti-MuSK antibody-positive MG, a rare phenomenon which is reversed after discontinuation of D-penicillamine treatment.
ESTHER : Poulas_2012_J.Neuroimmunol_250_94
PubMedSearch : Poulas_2012_J.Neuroimmunol_250_94
PubMedID: 22683336

Title : Autoantibodies to lipoprotein-related protein 4 in patients with double-seronegative myasthenia gravis - Zhang_2012_Arch.Neurol_69_445
Author(s) : Zhang B , Tzartos JS , Belimezi M , Ragheb S , Bealmear B , Lewis RA , Xiong WC , Lisak RP , Tzartos SJ , Mei L
Ref : Archives of Neurology , 69 :445 , 2012
Abstract : OBJECTIVES: To determine whether patients with myasthenia gravis (MG) have serum antibodies to lipoprotein-related protein 4 (LRP4), a newly identified receptor for agrin that is essential for neuromuscular junction formation, and to establish whether such antibodies contribute to MG pathogenesis. DESIGN: Serum samples from patients with MG with known status of serum antibodies to the acetylcholine receptor (AChR) and muscle-specific kinase (MuSK) and serum samples from control subjects (healthy individuals and individuals with other diseases) were tested for antibodies to LRP4. Serum samples with such antibodies were tested to determine whether they had the ability to inhibit 2 different functions of LRP4 at the neuromuscular junction. SETTING: Serum samples were collected at the Hellenic Pasteur Institute and Wayne State University. Samples were tested for LRP4 autoantibodies at Georgia Health Sciences University. Other immunoreactivities of the samples were tested at the Hellenic Pasteur Institute, Athens, Greece, or processed through University Laboratories of the Detroit Medical Center, Michigan. Patients The study included 217 patients with MG, 76 patients with other neurologic or psychiatric diseases, and 45 healthy control subjects.
RESULTS: Anti-LRP4 antibodies were detected in 11 of 120 patients with MG without detectable anti-AChR or anti-MuSK antibodies (double seronegative) and in 1 of 36 patients without anti-AChR antibodies but with anti-MuSK antibodies, but they were not detected in any of the 61 patients with anti-AChR antibodies. No healthy control subjects and only 2 of the 76 control patients with neurologic disease had anti-LRP4 antibodies. Serum samples from patients with MG with anti-LRP4 antibodies were able to inhibit the LRP4-agrin interaction and/or alter AChR clustering in muscle cells.
CONCLUSIONS: Anti-LRP4 antibodies were detected in the serum of approximately 9.2% of patients with double-seronegative MG. This frequency is intermediate compared with 2 recent studies showing anti-LRP4 antibodies in 2% and 50% of patients with double-seronegative MG from different geographic locations. Together, these observations indicate that LRP4 is another autoantigen in patients with MG, and anti-LRP4 autoantibodies may be pathogenic through different immunopathogenic processes.
ESTHER : Zhang_2012_Arch.Neurol_69_445
PubMedSearch : Zhang_2012_Arch.Neurol_69_445
PubMedID: 22158716

Title : Genetics of myasthenia gravis: a case-control association study in the Hellenic population - Zagoriti_2012_Clin.Dev.Immunol_2012_484919
Author(s) : Zagoriti Z , Georgitsi M , Giannakopoulou O , Ntellos F , Tzartos SJ , Patrinos GP , Poulas K
Ref : Clin Dev Immunol , 2012 :484919 , 2012
Abstract : Myasthenia gravis (MG) is an heterogeneous autoimmune disease characterized by the production of autoantibodies against proteins of the postsynaptic membrane, in the neuromuscular junction. The contribution of genetic factors to MG susceptibility has been evaluated through family and twin studies however, the precise genetic background of the disease remains elusive. We conducted a case-control association study in 101 unrelated MG patients of Hellenic origin and 101 healthy volunteers in order to assess the involvement of common genetic variants in susceptibility to MG. We focused on three candidate genes which have been clearly associated with several autoimmune diseases, aiming to investigate their potential implication in MG pathogenesis. These are interferon regulatory factor 5 (IRF-5), TNFalpha-induced protein 3 (TNFAIP3), also known as A20, and interleukin-10 (IL-10), key molecules in the regulation of immune function. A statistical trend of association (P = 0.068) between IL-10 promoter single nucleotide polymorphisms (SNPs) and the subgroups of early and late-onset MG patients was revealed. No statistically significant differences were observed in the rest of the variants examined. As far as we are aware, this is the first worldwide attempt to address the possible association between IRF-5 and TNFAIP3 common genetic variants and the genetic basis of MG.
ESTHER : Zagoriti_2012_Clin.Dev.Immunol_2012_484919
PubMedSearch : Zagoriti_2012_Clin.Dev.Immunol_2012_484919
PubMedID: 23049601

Title : Development of a highly sensitive diagnostic assay for muscle-specific tyrosine kinase (MuSK) autoantibodies in myasthenia gravis - Trakas_2011_J.Neuroimmunol_240-241_79
Author(s) : Trakas N , Zisimopoulou P , Tzartos SJ
Ref : Journal of Neuroimmunology , 240-241 :79 , 2011
Abstract : Autoimmune myasthenia gravis is usually characterized by the presence of autoantibodies against the acetylcholine receptor (~80-90% of patients) or muscle-specific tyrosine kinase (MuSK) (~5% of patients). In the remaining patients, no such antibodies (Abs) are detectable, but this could be due either to the presence of auto-Abs to yet unidentified antigens or to concentrations of circulating Abs below the threshold of the available assays. The most popular and sensitive assay for anti-MuSK Abs is a radioimmunoprecipitation assay (RIPA), which uses (125)I-labeled MuSK as test antigen. A serious limiting factor of the sensitivity of such RIPAs is that small volumes of test serum are required (maximum 5-20 mul) in order to avoid excessive background values. We have overcome this obstacle by the development of a two-step RIPA. This involves non-stringent affinity purification of anti-MuSK Abs from a large volume of patient's serum, followed by a regular RIPA step, with the isolated Abs, to determine the antibody titer. This two-step assay was shown to be 10-50 times more sensitive than the regular RIPA. All tested sera previously found to be positive in the regular RIPA and normal sera were also positive or negative in the two-step RIPA, respectively. Importantly, of seven tested sera previously characterized by regular RIPA as negative with titers that were above zero, but statistically not significantly higher from the background, two were found to be positive, while the others were clearly shown to be negative. We conclude that our diagnostic test can detect very low concentrations of circulating anti-MuSK Abs. The general principle of this two-step RIPA approach may also be applied to the detection of currently undetectable concentrations of circulating auto-Abs involved in other diseases.
ESTHER : Trakas_2011_J.Neuroimmunol_240-241_79
PubMedSearch : Trakas_2011_J.Neuroimmunol_240-241_79
PubMedID: 21993075

Title : Recent approaches to the development of antigen-specific immunotherapies for myasthenia gravis - Lagoumintzis_2010_Autoimmunity_43_436
Author(s) : Lagoumintzis G , Zisimopoulou P , Kordas G , Lazaridis K , Poulas K , Tzartos SJ
Ref : Autoimmunity , 43 :436 , 2010
Abstract : Acquired autoimmune myasthenia gravis (MG) is the most common disease that affects the neuromuscular junction (NMJ). MG is associated with autoantibodies (auto-Abs) to components of the NMJ. About 85-90% of MG patients have auto-Abs against the muscle nicotinic acetylcholine receptor (AChR), while about half of the remaining patients have auto-Abs against muscle-specific kinase. Auto-Abs, in combination with local deposition of complement, reduce the number of available post-synaptic nicotinic AChRs and thereby impair neuromuscular transmission. Current medications for MG are non-specific and include acetylcholinesterase inhibitors, immunosuppressants, plasma exchange, intravenous Ig administration and thymectomy. Treatments that selectively target the anti-AChR auto-Abs may prove to be more effective and free of side-effects. We here review two approaches aimed at the development of antigen-specific therapies for MG. The first is specific apheresis of Abs from patients' sera using immobilised recombinant AChR domains as immunoadsorbents. Indeed, we have recently shown that the combined recombinant extracellular domains of all human AChR subunits are capable of specifically immunoadsorbing the majority of pathogenic auto-Abs from several MG sera. The second therapeutic approach is the development of non-pathogenic anti-AChR monoclonal Abs that could potentially be used as protective agents by blocking the binding of patients' auto-Abs to the AChR.
ESTHER : Lagoumintzis_2010_Autoimmunity_43_436
PubMedSearch : Lagoumintzis_2010_Autoimmunity_43_436
PubMedID: 20187712

Title : A rapid, fluorescence-based assay for detecting antigenic modulation of the acetylcholine receptor on human cell lines - Keefe_2009_Cytometry.B.Clin.Cytom_76_206
Author(s) : Keefe D , Hess D , Bosco J , Tzartos SJ , Powell J , Lamsa J , Josiah S
Ref : Cytometry B Clin Cytom , 76 :206 , 2009
Abstract : BACKGROUND: Myasthenia gravis (MG) is an autoimmune disease affecting approximately 40,000 patients in the United States. One of the major mechanisms of disease pathology in MG is the binding, internalization, and eventual destruction of acetylcholine receptors (AChR) at the neuromuscular junction by cross-linking AChR-specific autoantibodies. This process, known as antigenic modulation, ultimately attenuates the ability of muscle cells to contract in response to signals from neurons, leading to muscle weakness and fatigue. For this reason, antigenic modulation of the AChR on cultured cells has become an important diagnostic tool for assessing the pathogenicity of AChR-specific autoantibodies. Traditionally, these assays have been done using radiolabeled AChR ligands such as (125)I alpha-bungarotoxin to determine relative AChR number. Here, we present a high-throughput immunofluorescent flow cytometry-based assay that can be used to quantify AChR levels on the cell surface and assess the efficacy of molecules designed to rescue antigenic modulation.
METHODS: AChR levels were quantified on human muscle cells before and after treatment with AChR antibodies via immunofluorescent labeling with the AChR monoclonal antibodies, mAb210 and mAb B3, followed by flow cytometry of EDTA-treated cells.
RESULTS: Using a novel, flow cytometry-based assay, antigenic modulation of the AChR was demonstrated on human cells using both AChR-specific monoclonal antibody and MG patient serum. The degree of antigenic modulation was dose responsive to antibody levels and could be reversed by preincubating antibodies with soluble AChR alpha subunit extracellular domain. SUMMARY: A rapid, nonradioactive assay was developed to determine the potential of AChR-specific antibodies in the serum of MG patients to bind and down-regulate the AChR. This assay can be used to assess the ability of putative therapeutics that rescue antigenic modulation and could be developed for the treatment of MG.
ESTHER : Keefe_2009_Cytometry.B.Clin.Cytom_76_206
PubMedSearch : Keefe_2009_Cytometry.B.Clin.Cytom_76_206
PubMedID: 18825779

Title : Epidemiological and immunological profile of muscle-specific kinase myasthenia gravis in Greece - Tsiamalos_2009_Eur.J.Neurol_16_925
Author(s) : Tsiamalos P , Kordas G , Kokla A , Poulas K , Tzartos SJ
Ref : Eur Journal of Neurology , 16 :925 , 2009
Abstract : OBJECTIVES: The purposes of this study were to determine the epidemiological characteristics of muscle-specific kinase-myasthenia gravis (MuSK-MG) in Greece and the IgG subclass of the anti-MuSK antibodies.
METHODS: This population-based study was performed on MuSK-MG patients in Greece between 1 January 1986 and 30 June 2006. Epidemiological and clinical data for 33 patients were collected. In addition, the distribution of anti-MuSK IgG autoantibody subclasses in the sera of 14 patients was determined by immunoprecipitation.
RESULTS: The average annual incidence was 0.32 patients/million population/year. On 1st July 2006, there were 33 prevalent cases, giving a point prevalence rate of 2.92/million (women 4.56 and men 1.25). In females, onset of MuSK-MG occurred after the age of 30, whilst, in males, the disease appears in any decade. The female:male incidence ratio was 3.33:1, whilst the prevalence ratio was 3.65:1. Most patients presented with involvement of the facial and bulbar muscles. Amongst about 800 MG patients seropositive for antibodies against either the AChR or MuSK, one patient was found to be seropositive for anti-MuSK antibodies and ambiguous for anti-acetylcholine receptor (anti-AChR) antibodies. The vast majority of anti-MuSK antibodies were IgG4, whilst total IgG4 levels in these patients were similar to those in two healthy controls.
CONCLUSIONS: The incidence and prevalence of MuSK-MG in Greece are amongst the highest reported previously for other countries. MuSK-MG in Greece affects both sexes, but mainly females. The main epidemiological indices were calculated. The vast majority of anti-MuSK antibodies were IgG4.
ESTHER : Tsiamalos_2009_Eur.J.Neurol_16_925
PubMedSearch : Tsiamalos_2009_Eur.J.Neurol_16_925
PubMedID: 19374661

Title : Design and expression of human alpha7 nicotinic acetylcholine receptor extracellular domain mutants with enhanced solubility and ligand-binding properties - Zouridakis_2009_Biochim.Biophys.Acta_1794_355
Author(s) : Zouridakis M , Zisimopoulou P , Eliopoulos E , Poulas K , Tzartos SJ
Ref : Biochimica & Biophysica Acta , 1794 :355 , 2009
Abstract : In order to facilitate structural studies of the extracellular domain (ECD) of human alpha7 nicotinic acetylcholine receptor (nAChR), we designed several mutants, since the wild-type-ECD forms large oligomers and microaggregates, and expressed them in the yeast Pichia pastoris. Mutant design was based on a 3D model of human alpha7-nAChR-ECD, constructed using as templates the X-ray crystal structure of the homologous acetylcholine-binding protein (AChBP) and the electron microscopy structure of the Torpedo alpha-nAChR-ECD. At least one mutant, mut10, carrying six single-point mutations (Phe3Tyr, Val69Thr, Cys116Ser, Ile165Thr, Val177Thr, Phe187Tyr) and the replacement of its Cys-loop with the corresponding and more hydrophilic AChBP Cys-loop, was expressed with a 4-fold higher expression yield (1.2 mg/L) than the wild-type alpha7-ECD, existing exclusively as a soluble oligomeric, probably pentameric, form, at concentrations up to at least 10 mg/mL, as judged by gel filtration and dynamic light scattering. This mutant displayed a significantly improved (125)I-alpha-bungarotoxin-binding affinity (K(d)=24 nM) compared to the wild-type-ECD (K(d)=70 nM), the binding being inhibited by unlabelled alpha-bungarotoxin, d-tubocurarine or nicotine (K(i) of 21.5 nM, 127 microM and 17.5 mM, respectively). Circular dichroism studies of mut10 revealed (a) a similar secondary structure composition ( approximately 5% alpha-helix, approximately 45% beta-sheet) to that of the AChBP, Torpedo alpha-nAChR-ECD, and mouse alpha1-nAChR-ECD, (b) a well-defined tertiary structure and (c) binding of small cholinergic ligands at micromolar concentrations. Furthermore, electron microscopy showed well-assembled, probably pentameric, particles of mut10. Finally, since deglycosylation did not alter its solubility or ligand-binding properties, mut10, in either its glycosylated or deglycosylated form, is a promising alpha7-ECD mutant for structural studies, useful for the rational drug design to treat alpha7-nAChR-related diseases.
ESTHER : Zouridakis_2009_Biochim.Biophys.Acta_1794_355
PubMedSearch : Zouridakis_2009_Biochim.Biophys.Acta_1794_355
PubMedID: 19059502

Title : Recent advances in understanding the structure of nicotinic acetylcholine receptors - Zouridakis_2009_IUBMB.Life_61_407
Author(s) : Zouridakis M , Zisimopoulou P , Poulas K , Tzartos SJ
Ref : IUBMB Life , 61 :407 , 2009
Abstract : Nicotinic acetylcholine receptors (nAChRs), members of the Cys-loop ligand-gated ion channels (LGICs) superfamily, are involved in signal transduction upon binding of the neurotransmitter acetylcholine or exogenous ligands, such as nicotine. nAChRs are pentameric assemblies of homologous subunits surrounding a central pore that gates cation flux, and are expressed at the neuromuscular junction and in the nervous system and several nonneuronal cell types. The 17 known nAChR subunits assemble into a variety of pharmacologically distinct receptor subtypes. nAChRs are implicated in a range of physiological functions and pathophysiological conditions related to muscle contraction, learning and memory, reward, motor control, arousal, and analgesia, and therefore present an important target for drug research. Such studies would be greatly facilitated by knowledge of the high-resolution structure of the nAChR. Although this information is far from complete, important progress has been made mainly based on electron microscopy studies of Torpedo nAChR and the high-resolution X-ray crystal structures of the homologous molluscan acetylcholine-binding proteins, the extracellular domain of the mouse nAChR alpha1 subunit, and two prokaryotic pentameric LGICs. Here, we review some of the latest advances in our understanding of nAChR structure and gating.
ESTHER : Zouridakis_2009_IUBMB.Life_61_407
PubMedSearch : Zouridakis_2009_IUBMB.Life_61_407
PubMedID: 19319967

Title : Mutant forms of the extracellular domain of the human acetylcholine receptor gamma-subunit with improved solubility and enhanced antigenicity. The importance of the Cys-loop - Bitzopoulou_2008_Biochim.Biophys.Acta_1784_1226
Author(s) : Bitzopoulou K , Kostelidou K , Poulas K , Tzartos SJ
Ref : Biochimica & Biophysica Acta , 1784 :1226 , 2008
Abstract : The muscle nicotinic acetylcholine receptor (AChR) is the prototype of the ligand-gated ion channels (or Cys-loop receptors), formed by 5 homologous subunits (alpha2betagammadelta or alpha2betagammaepsilon), and is the major autoantigen in the autoimmune disease, myasthenia gravis. Previously, we expressed the wild-type extracellular domain (ECD) of the gamma-subunit (gammaECD) of the AChR in yeast Pichia pastoris at 0.3-0.8 mg/L, in soluble but microaggregate form, to use as starting material for structural and antigenicity studies. To optimize these characteristics, we constructed and characterized four gammaECD variants: (a) mutants-1 (gammaC61S) and -2 (gammaC106S-C115S), where the non-conserved Cys of gammaECD were replaced by serines, (b) mutant-3 (gammaCysLoop), where the gamma Cys-loop region was substituted by the cognate region of the acetylcholine binding protein (AChBP) and (c) mutant-4 (gammaCysLoop-C106S-C115S), where both the C106S-C115S and Cys-loop mutations were combined. None of mutants-1 and -2 displayed any improvement, while mutant-3 and -4 were mostly in dimeric form and expressed at much higher levels (2.5 mg/L and 3.5 mg/L respectively). All four mutants and wild-type gammaECD were recognized by sera from myasthenic patients, but mutants-3 and -4 exhibited higher efficiency, compared to wild-type or mutants-1 and -2. These results suggest that the substitution of the Cys-loop region of any AChR ECD with the AChBP counterpart leads to AChR ECD of improved conformation, more suitable for structural and therapeutic studies.
ESTHER : Bitzopoulou_2008_Biochim.Biophys.Acta_1784_1226
PubMedSearch : Bitzopoulou_2008_Biochim.Biophys.Acta_1784_1226
PubMedID: 18502212

Title : Suppression of experimental myasthenia gravis by a B-cell epitope-free recombinant acetylcholine receptor - Yi_2008_Mol.Immunol_46_192
Author(s) : Yi HJ , Chae CS , So JS , Tzartos SJ , Souroujon MC , Fuchs S , Im SH
Ref : Mol Immunol , 46 :192 , 2008
Abstract : Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are antibody-mediated autoimmune diseases in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. Previously we have revealed that oral treatment with the less native recombinant fragment of the extracellular domain of the human AChR (Halpha1-205) suppressed ongoing EAMG, whereas the more native recombinant Trx-Halpha1-210 exacerbated EAMG. In this study, we speculated on the role of B-cell epitopes in oral tolerogens for the induction of oral tolerance in EAMG. We developed a B-cell epitope-free AChR fragment (BF-AChR) by removing two major B-cell epitopes (67-76 and 129-145) from Trx-Halpha1-210. BF-AChR exhibited a poor response to EAMG sera and to AChR-specific B- and T-cells while its parent fragment, Trx-Halpha1-210, showed much higher reactivity. Oral administration of BF-AChR ameliorated the symptoms in ongoing myasthenic rats accompanied by a significant decrease in AChR-specific humoral and Th1 cellular responses. The underlying mechanism for BF-AChR-induced oral tolerance was mediated by a shift from Th1 to regulatory T-cell (IL-10(+), CD4(+) TGF-beta(+) or Foxp3(+)) responses. This shift was assessed by changes in the cytokine profile and a deviation in the anti-AChR IgG isotypes from IgG2a/IgG2b to IgG1. Our results suggest that the removal of pathogenic B-cell epitopes from AChR fragments increases tolerogenicity by reducing the activation and proliferation of autoreactive B- and T-cells. Collectively, careful consideration of the immunogenicity of a tolerogen is necessary to induce successful oral tolerance in autoimmune disorders.
ESTHER : Yi_2008_Mol.Immunol_46_192
PubMedSearch : Yi_2008_Mol.Immunol_46_192
PubMedID: 18799218

Title : Antigen-specific apheresis of pathogenic autoantibodies from myasthenia gravis sera - Tzartos_2008_Ann.N.Y.Acad.Sci_1132_291
Author(s) : Tzartos SJ , Bitzopoulou K , Gavra I , Kordas G , Jacobson L , Kostelidou K , Lagoumintzis G , Lazos O , Poulas K , Sideris S , Sotiriadis A , Trakas N , Zisimopoulou P
Ref : Annals of the New York Academy of Sciences , 1132 :291 , 2008
Abstract : Myasthenia gravis (MG) is usually caused by autoantibodies against muscle nicotinic acetylcholine receptor (AChR), which is composed of five subunits (alpha(2)betagammadelta or alpha(2)betaepsilondelta). Current treatments, including plasmapheresis, are nonspecific, causing several side effects. We aim to develop an antigen-specific alternative to plasmapheresis, since the latter removes indispensable plasma components in addition to anti-AChR antibodies. We are developing a method for the selective depletion of the anti-AChR autoantibodies from patients' plasma through the construction of "immunoadsorbent" columns carrying AChR domains. We have expressed the extracellular domains (ECDs, amino acids approximately 1-210/220) of all human muscle AChR subunits in Pichia pastoris and, in preliminary experiments, in E. coli. The ECDs were immobilized (individually or mixed) on Sepharose beads, producing Sepharose-ECD columns, which were tested for their immunoadsorbing capacity on MG sera and shown to specifically eliminate major autoantibody fractions from several MG sera. The immobilized ECDs remained stable and did not dissociate from their matrix after incubation with serum, whereas the procedure was neither toxic nor immunogenic in two experimental rabbits. Testing the intact or antibody-depleted MG sera and the affinity purified autoantibodies showed that both the intact sera and the purified autoantibodies, but not the antibody-depleted sera, could induce AChR loss in cell cultures and experimental MG in rats. This preliminary study suggests that the myasthenic potency of MG sera is entirely due to their anti-AChR antibodies and therefore their depletion should be of therapeutic value. We conclude that ECD-mediated immunoadsorption can be used as an efficient, antigen-specific therapy for MG.
ESTHER : Tzartos_2008_Ann.N.Y.Acad.Sci_1132_291
PubMedSearch : Tzartos_2008_Ann.N.Y.Acad.Sci_1132_291
PubMedID: 18567880

Title : Model of the extracellular domain of the human alpha7 nAChR based on the crystal structure of the mouse alpha1 nAChR extracellular domain - Konstantakaki_2008_J.Mol.Graph.Model_26_1333
Author(s) : Konstantakaki M , Tzartos SJ , Poulas K , Eliopoulos E
Ref : J Mol Graph Model , 26 :1333 , 2008
Abstract : Neuronal nicotinic acetylcholine receptors (nAChRs) are important therapeutic targets for various diseases, including Alzheimer's disease, Parkinson's disease, and schizophrenia, as well as for cessation of smoking. Based on the recently determined crystal structure of the extracellular domain (ECD) of the mouse nAChR alpha1 subunit complexed with alpha-bungarotoxin at 1.94A resolution, we have constructed three-dimensional models of the ECD of the monomer, homodimer, and homopentamer of the human alpha7 nAChR and investigated in detail the interface between the two alpha7 subunits. The docking of the agonist in the ligand-binding pocket of the human alpha7 dimer was also performed and found consistent with results from labeling and mutagenesis experiments. Since the nAChR ligand-binding site is a useful target for mutagenesis studies and the rational design of drugs against diseases, these models provide useful information for future work.
ESTHER : Konstantakaki_2008_J.Mol.Graph.Model_26_1333
PubMedSearch : Konstantakaki_2008_J.Mol.Graph.Model_26_1333
PubMedID: 18329305

Title : Towards antigen-specific apheresis of pathogenic autoantibodies as a further step in the treatment of myasthenia gravis by plasmapheresis - Zisimopoulou_2008_J.Neuroimmunol_201-202_95
Author(s) : Zisimopoulou P , Lagoumintzis G , Kostelidou K , Bitzopoulou K , Kordas G , Trakas N , Poulas K , Tzartos SJ
Ref : Journal of Neuroimmunology , 201-202 :95 , 2008
Abstract : Myasthenia gravis (MG), a prototypic antibody-mediated autoimmune disease, presents an excellent target for scientific research aimed at a better understanding of the disease itself and the source that triggers an autoimmune reaction in an organism. MG is a neuromuscular disease caused mainly by an autoimmune response against the nicotinic acetylcholine receptor (AChR) which interferes with neuromuscular transmission. This review focuses on our studies on the extracellular domains of human muscle AChR subunits in an effort to develop an approach for the specific therapeutic apheresis of autoantibodies from patients' sera using the immobilized subunits as immunoadsorbents. The ability of the anti-AChR antibodies isolated by this technique, but not of the depleted sera, to induce disease is also described. This review is dedicated to the late Prof. John Newsom-Davis, who was the first to introduce the use of plasmapheresis for MG.
ESTHER : Zisimopoulou_2008_J.Neuroimmunol_201-202_95
PubMedSearch : Zisimopoulou_2008_J.Neuroimmunol_201-202_95
PubMedID: 18667243

Title : Isolation and functional characterization of anti-acetylcholine receptor subunit-specific autoantibodies from myasthenic patients: receptor loss in cell culture - Sideris_2007_J.Neuroimmunol_189_111
Author(s) : Sideris S , Lagoumintzis G , Kordas G , Kostelidou K , Sotiriadis A , Poulas K , Tzartos SJ
Ref : Journal of Neuroimmunology , 189 :111 , 2007
Abstract : The muscle nicotinic acetylcholine receptor (nAChR) is the major autoantigen in the autoimmune disease myasthenia gravis (MG), in which autoantibodies bind to, and cause loss of, nAChRs. Antibody-mediated nAChR loss is caused by the action of complement and by the acceleration of nAChR internalization caused by antibody-induced cross-linking of nAChR molecules (antigenic modulation). To obtain an insight into the role of the various anti-nAChR antibody specificities in MG, we have studied nAChR antigenic modulation caused by isolated anti-subunit autoantibodies. Autoantibodies against the nAChR alpha or beta subunits were isolated from four MG sera by affinity chromatography on columns carrying immobilized recombinant extracellular domains of human nAChR expressed in the yeast Pichia pastoris. The isolated anti-alpha and anti-beta autoantibodies, as well as untreated MG sera, induced nAChR antigenic modulation in TE671 cells. Partially antibody-depleted sera exhibited reduced modulating activity, whereas a serum completely depleted of anti-nAChR antibodies exhibited no nAChR modulation. Interestingly, the anti-alpha autoantibodies were, on average, approximately 4.3 times more effective than the anti-beta autoantibodies. The present work supports the notion that anti-nAChR autoantibodies may be the sole nAChR-reducing factor in anti-nAChR antibody-seropositive MG, and that anti-alpha-subunit autoantibodies are the dominant pathogenic autoantibody specificity.
ESTHER : Sideris_2007_J.Neuroimmunol_189_111
PubMedSearch : Sideris_2007_J.Neuroimmunol_189_111
PubMedID: 17617475

Title : Muscle and neuronal nicotinic acetylcholine receptors. Structure, function and pathogenicity - Kalamida_2007_FEBS.J_274_3799
Author(s) : Kalamida D , Poulas K , Avramopoulou V , Fostieri E , Lagoumintzis G , Lazaridis K , Sideri A , Zouridakis M , Tzartos SJ
Ref : Febs J , 274 :3799 , 2007
Abstract : Nicotinic acetylcholine receptors (nAChRs) are integral membrane proteins and prototypic members of the ligand-gated ion-channel superfamily, which has precursors in the prokaryotic world. They are formed by the assembly of five transmembrane subunits, selected from a pool of 17 homologous polypeptides (alpha1-10, beta1-4, gamma, delta, and epsilon). There are many nAChR subtypes, each consisting of a specific combination of subunits, which mediate diverse physiological functions. They are widely expressed in the central nervous system, while, in the periphery, they mediate synaptic transmission at the neuromuscular junction and ganglia. nAChRs are also found in non-neuronal/nonmuscle cells (keratinocytes, epithelia, macrophages, etc.). Extensive research has determined the specific function of several nAChR subtypes. nAChRs are now important therapeutic targets for various diseases, including myasthenia gravis, Alzheimer's and Parkinson's diseases, and schizophrenia, as well as for the cessation of smoking. However, knowledge is still incomplete, largely because of a lack of high-resolution X-ray structures for these molecules. Nevertheless, electron microscopy studies on 2D crystals of nAChR from fish electric organs and the determination of the high-resolution X-ray structure of the acetylcholine binding protein (AChBP) from snails, a homolog of the extracellular domain of the nAChR, have been major steps forward and the data obtained have important implications for the design of subtype-specific drugs. Here, we review some of the latest advances in our understanding of nAChRs and their involvement in physiology and pathology.
ESTHER : Kalamida_2007_FEBS.J_274_3799
PubMedSearch : Kalamida_2007_FEBS.J_274_3799
PubMedID: 17651090

Title : Molecular modeling of the complex between Torpedo acetylcholine receptor and anti-MIR Fab198 - Konstantakaki_2007_Biochem.Biophys.Res.Commun_356_569
Author(s) : Konstantakaki M , Tzartos SJ , Poulas K , Eliopoulos E
Ref : Biochemical & Biophysical Research Communications , 356 :569 , 2007
Abstract : Myasthenia gravis is a neuromuscular disorder caused by an antibody-mediated autoimmune response to the muscle-type nicotinic acetylcholine receptor (AChR). The majority of monoclonal antibodies (mAbs) produced in rats immunized with intact AChR compete with each other for binding to an area of the alpha-subunit called the main immunogenic region (MIR). The availability of a complex between the AChR and Fab198 (Fab fragment of the anti-MIR mAb198) would help understand how the antigen and antibody interact and in designing improved antibody fragments that protect against the destructive activity of myasthenic antibodies. In the present study, we modeled the Torpedo AChR/Fab198 complex, based primarily on the recent 4A resolution structure of the Torpedo AChR. In order to computationally dock the two structures, we used the ZDOCK software. The total accessible surface area change of the complex compared to those of experimentally determined antigen-antibody complexes indicates an intermediate size contact surface. CDRs H3 and L3 seem to contribute most to the binding, while L2 seems to contribute least. These data suggest mutagenesis experiments aimed at validating the model and improving the binding affinity of Fab198 for the AChR.
ESTHER : Konstantakaki_2007_Biochem.Biophys.Res.Commun_356_569
PubMedSearch : Konstantakaki_2007_Biochem.Biophys.Res.Commun_356_569
PubMedID: 17376405

Title : Circular dichroism studies of extracellular domains of human nicotinic acetylcholine receptors provide an insight into their structure - Zouridakis_2007_Int.J.Biol.Macromol_41_423
Author(s) : Zouridakis M , Kostelidou K , Sotiriadis A , Stergiou C , Eliopoulos E , Poulas K , Tzartos SJ
Ref : Int J Biol Macromol , 41 :423 , 2007
Abstract : The extracellular domains (ECDs) of human nicotinic acetylcholine receptors (nAChRs) are of major pharmacological interest as drug targets in the autoimmune disease myasthenia gravis and in various neurological disorders. We have previously expressed and purified the human muscle alpha1-, beta1-, gamma- and epsilon-nAChR-ECDs, as well as the wild type and a mutant of neuronal alpha7-ECD, in yeast Pichia pastoris. The far-UV circular dichroism (CD) studies of these ECDs, presented here, revealed a major prevalence of beta-sheet ( approximately 40%) and a small proportion of alpha-helical ( approximately 5%) structure for all ECDs, in good agreement with the secondary structure composition of the Torpedo muscle-type nAChR-ECDs and in less, but considerable, agreement with that of the homologous invertebrate acetylcholine-binding proteins (AChBPs). The near-UV CD studies of these nAChR-ECDs indicated well-defined tertiary structures, as was previously suggested by biochemical and immunochemical studies. Furthermore, the binding of cholinergic ligands to the mutant of alpha7-ECD resulted in no changes in its secondary structure, but revealed significant local conformational changes. Our present studies probe the structure of human nAChR-ECDs for the first time and further suggest that our expressed proteins fold to a near-native conformation, thus being suitable for further structural studies.
ESTHER : Zouridakis_2007_Int.J.Biol.Macromol_41_423
PubMedSearch : Zouridakis_2007_Int.J.Biol.Macromol_41_423
PubMedID: 17659334

Title : Expression and characterization of soluble forms of the extracellular domains of the beta, gamma and epsilon subunits of the human muscle acetylcholine receptor - Kostelidou_2006_FEBS.J_273_3557
Author(s) : Kostelidou K , Trakas N , Zouridakis M , Bitzopoulou K , Sotiriadis A , Gavra I , Tzartos SJ
Ref : Febs J , 273 :3557 , 2006
Abstract : The nicotinic acetylcholine receptor (AChR) is a ligand-gated ion channel found in muscles and neurons. Muscle AChR, formed by five homologous subunits (alpha2 beta gamma delta or alpha2 beta gamma epsilon), is the major antigen in the autoimmune disease, myasthenia gravis (MG), in which pathogenic autoantibodies bind to, and inactivate, the AChR. The extracellular domain (ECD) of the human muscle alpha subunit has been heterologously expressed and extensively studied. Our aim was to obtain satisfactory amounts of the ECDs of the non-alpha subunits of human muscle AChR for use as starting material for the determination of the 3D structure of the receptor ECDs and for the characterization of the specificities of antibodies in sera from patients with MG. We expressed the N-terminal ECDs of the beta (amino acids 1-221; beta1-221), gamma (amino acids 1-218; gamma1-218), and epsilon (amino acids 1-219; epsilon1-219) subunits of human muscle AChR in the yeast, Pichia pastoris. beta1-221 was expressed at approximately 2 mg.L(-1) culture, whereas gamma1-218 and epsilon1-219 were expressed at 0.3-0.8 mg.L(-1) culture. All three recombinant polypeptides were glycosylated and soluble; beta1-221 was mainly in an apparently dimeric form, whereas gamma1-218 and epsilon1-219 formed soluble oligomers. CD studies of beta1-221 suggested that it has considerable beta-sheet secondary structure with a proportion of alpha-helix. Conformation-dependent mAbs against the ECDs of the beta or gamma subunits specifically recognized beta1-221 or gamma1-218, respectively, and polyclonal rabbit antiserum raised against purified beta1-221 bound to (125)I-labeled alpha-bungarotoxin-labeled human AChR. Moreover, immobilization of each ECD on Sepharose beads and incubation of the ECD-Sepharose matrices with MG sera caused a significant reduction in the concentrations of autoantibodies in the sera, showing specific binding to the recombinant ECDs. These results suggest that the expressed proteins present some near-native conformational features and are thus suitable for our purposes.
ESTHER : Kostelidou_2006_FEBS.J_273_3557
PubMedSearch : Kostelidou_2006_FEBS.J_273_3557
PubMedID: 16884496

Title : Effects of cytokines on acetylcholine receptor expression: implications for myasthenia gravis - Poea-Guyon_2005_J.Immunol_174_5941
Author(s) : Poea-Guyon S , Christadoss P , Le Panse R , Guyon T , de Baets M , Wakkach A , Bidault J , Tzartos SJ , Berrih-Aknin S
Ref : J Immunol , 174 :5941 , 2005
Abstract : Myasthenia gravis is an autoimmune disease associated with thymic pathologies, including hyperplasia. In this study, we investigated the processes that may lead to thymic overexpression of the triggering Ag, the acetylcholine receptor (AChR). Using microarray technology, we found that IFN-regulated genes are more highly expressed in these pathological thymic tissues compared with age- and sex-matched normal thymus controls. Therefore, we investigated whether proinflammatory cytokines could locally modify AChR expression in myoid and thymic epithelial cells. We found that AChR transcripts are up-regulated by IFN-gamma, and even more so by IFN-gamma and TNF-alpha, as assessed by real-time RT-PCR, with the alpha-AChR subunit being the most sensitive to this regulation. The expression of AChR protein was increased at the cytoplasmic level in thymic epithelial cells and at the membrane in myoid cells. To examine whether IFN-gamma could influence AChR expression in vivo, we analyzed AChR transcripts in IFN-gamma gene knock-out mice, and found a significant decrease in AChR transcript levels in the thymus but not in the muscle, compared with wild-type mice. However, up-regulation of AChR protein expression was found in the muscles of animals with myasthenic symptoms treated with TNF-alpha. Altogether, these results indicate that proinflammatory cytokines influence the expression of AChR in vitro and in vivo. Because proinflammatory cytokine activity is evidenced in the thymus of myasthenia gravis patients, it could influence AChR expression and thereby contribute to the initiation of the autoimmune anti-AChR response.
ESTHER : Poea-Guyon_2005_J.Immunol_174_5941
PubMedSearch : Poea-Guyon_2005_J.Immunol_174_5941
PubMedID: 15879086

Title : Isolation and characterization of human anti-acetylcholine receptor monoclonal antibodies from transgenic mice expressing human immunoglobulin loci - Protopapadakis_2005_Eur.J.Immunol_35_1960
Author(s) : Protopapadakis E , Kokla A , Tzartos SJ , Mamalaki A
Ref : European Journal of Immunology , 35 :1960 , 2005
Abstract : The isolation of human antibodies against muscle acetylcholine receptor (AChR), the autoantigen involved in myasthenia gravis (MG), is important for the development of therapeutically useful reagents. Monovalent antibody fragments from monoclonal antibodies against the main immunogenic region (MIR) of AChR protect the receptor from the destructive activity of MG autoantibodies. Human anti-AChR alpha-subunit antibody fragments with therapeutic potential have been isolated using phage display antibody libraries. An alternative approach for obtaining human mAb has been provided by the development of humanized mice. In this report, we show that immunization of transgenic mouse strains with the extracellular domain of the human AChR alpha-subunit results in antibody responses and isolation of hybridomas producing human mAb. Four specific IgM mAb were isolated and analyzed. mAb170 recognized the native receptor the best and was capable of inducing AChR antigenic modulation, suggesting its specificity for a pathogenic epitope. Moreover, the recombinant antigen-binding (Fab) fragment of this mAb competed with an anti-MIR mAb, revealing that its antigenic determinant lies in or near the MIR. Finally, Fab170 was able to compete with MG autoantibodies and protect the AChR against antigenic modulation induced by MG sera. This approach will be useful for isolating additional mAb with therapeutic potential against the other AChR subunits.
ESTHER : Protopapadakis_2005_Eur.J.Immunol_35_1960
PubMedSearch : Protopapadakis_2005_Eur.J.Immunol_35_1960
PubMedID: 15915538

Title : Future therapeutic strategies in autoimmune myasthenia gravis - Psaridi-Linardaki_2003_Ann.N.Y.Acad.Sci_998_539
Author(s) : Psaridi-Linardaki L , Mamalaki A , Tzartos SJ
Ref : Annals of the New York Academy of Sciences , 998 :539 , 2003
Abstract : Antibodies against muscle acetylcholine receptor (AChR) undoubtedly play a critical role in the pathology of most myasthenia gravis (MG) cases. Selective elimination of the majority of these antibodies should result in a considerable improvement of the MG symptoms. Such a specific elimination could be achieved by AChR-based immunoadsorbents. However, sufficient quantities of native human AChR are not available while bacterially expressed recombinant domains of the AChR are unable to bind satisfactorily MG antibodies. We have undertaken the production of the extracellular domains of human AChR subunits in eukaryotic systems, in native-like conformation, for their use as potent immunoadsorbents. The N-terminal extracellular domain (amino acids 1-210; alpha(1-210)) of the alpha(1) subunit of the human muscle AChR was expressed in the yeast Pichia pastoris. The polypeptide was water-soluble, glycosylated, and in monomer form. The alpha(1-210) bound 125I-alpha-bungarotoxin (125I-alpha-BTX) with a high affinity (Kd = 5.1 +/- 2.4 nM), and this binding was blocked by unlabeled d-tubocurarine and gallamine. Several conformation-dependent anti-AChR antibodies were able to bind alpha(1-210) as did antibodies from a large proportion of MG patients. The purified protein was subsequently immobilized on Sepharose-CNBr and was used to immunoadsorb anti-AChR antibodies from 64 MG sera. It eliminated more than 50% (50-94%) of the anti-AChR antibodies in 20% of the sera, whereas from another 30% of the sera it eliminated 20-60% of their anti-AChR antibodies. Work is in progress for the expression of the extracellular domain of all other muscle AChR subunits. It is expected that their combined use may eliminate the great majority of the anti-AChR antibodies from most MG patients.
ESTHER : Psaridi-Linardaki_2003_Ann.N.Y.Acad.Sci_998_539
PubMedSearch : Psaridi-Linardaki_2003_Ann.N.Y.Acad.Sci_998_539
PubMedID: 14592926

Title : Characterization of a fully human IgG1 reconstructed from an anti-AChR Fab -
Author(s) : Stassen MH , Machiels BM , Fostieri E , Tzartos SJ , Berrih-Aknin S , Bosmans E , Parren PW , De Baets MH
Ref : Annals of the New York Academy of Sciences , 998 :399 , 2003
PubMedID: 14592905

Title : Expression of soluble ligand- and antibody-binding extracellular domain of human muscle acetylcholine receptor alpha subunit in yeast Pichia pastoris. Role of glycosylation in alpha-bungarotoxin binding - Psaridi-Linardaki_2002_J.Biol.Chem_277_26980
Author(s) : Psaridi-Linardaki L , Mamalaki A , Remoundos M , Tzartos SJ
Ref : Journal of Biological Chemistry , 277 :26980 , 2002
Abstract : The N-terminal extracellular domain (amino acids 1-210; halpha-(1-210)) of the alpha subunit of the human muscle nicotinic acetylcholine receptor (AChR), bearing the binding sites for cholinergic ligands and the main immunogenic region, the major target for anti-AChR antibodies in patients with myasthenia gravis, was expressed in the yeast, Pichia pastoris. The recombinant protein was water-soluble and glycosylated, and fast protein liquid chromatography analysis showed it to be a monomer. halpha-(1-210) bound (125)I-alpha-bungarotoxin with a high affinity (K(d) = 5.1 +/- 2.4 nm), and this binding was blocked by unlabeled d-tubocurarine and gallamine (K(i) approximately 7.5 mm). Interestingly, (125)I-alpha-bungarotoxin binding was markedly impaired by in vitro deglycosylation of halpha-(1-210). Several monoclonal antibodies that show partial or strict conformation-dependent binding to the AChR were able to bind to halpha-(1-210), as did antibodies from a large proportion of myasthenic patients. These results suggest that the extracellular domain of the human AChR alpha subunit expressed in P. pastoris has an apparently near native conformation. The correct folding of the recombinant protein, together with its relatively high expression yield, makes it suitable for structural studies on the nicotinic acetylcholine receptor and for use as an autoantigen in myasthenia gravis studies.
ESTHER : Psaridi-Linardaki_2002_J.Biol.Chem_277_26980
PubMedSearch : Psaridi-Linardaki_2002_J.Biol.Chem_277_26980
PubMedID: 12015305

Title : Selective lesions of rabbit extraocular muscles injected with the anti-AChR immunotoxin saporin-mAb 73 - Campos_2002_Curr.Eye.Res_24_58
Author(s) : Campos EC , Schiavi C , Bolognesi A , Bellusci C , Lubelli C , Duca A , Polito L , Poulas K , Tzartos SJ , Stirpe F
Ref : Current Eye Research , 24 :58 , 2002
Abstract : PURPOSE: To evaluate the effects on extraocular muscles of a skeletal muscle-specific immunotoxin, saporin-mAb 73, as an alternative to botulinum toxin to induce a permanent correction of oculo-facial dystonias or some forms of ocular motility disorders.
METHODS: An immunotoxin was prepared with a monoclonal antibody (mAb 73) against acetylcholine receptors of skeletal muscle, linked to saporin, a type 1 ribosome-inactivating protein (RIP) from Saponaria officinalis. Sixteen New Zealand white rabbits were treated with a single injection of immunotoxin directly into the medial rectus muscle of one eye. Four different dosages of 2, 5, 20, or 50 ng saporin-mAb 73 were used. The rabbits were sacrificed at two, 7 and 14 days post-injection. The medial rectus muscle and the retractor bulbi muscle of both the injected and the fellow eyes were taken and serial sections were examined by light microscopy in a blinded manner.
RESULTS: Saporin-mAb 73, even at the dosage of 2 ng, brought about focal damage in the extraocular muscles of rabbits without histological changes in adjacent muscles. The histological examination revealed necrotic/apoptotic lesions restricted to the sites of inoculation and largely infiltrated by macrophages. No evident inflammatory reaction was detected at any time and neutrophils were substantially absent. At 14 days after injection, necrosis/apoptosis was still evident and the sclerotic reaction was minimal.
CONCLUSIONS: The immunotoxin saporin-mAb 73 injections into the extraocular muscles of rabbits caused focal damage to the muscles. There was no significant inflammatory reaction and muscle fiber loss was present even at the lower doses. Although the lesions were followed for only 14 days, our results suggest that saporin-mAb 73 has potential to cause safe focal muscle damage but longer-term follow-up are needed to investigate the persistence of muscle weakness.
ESTHER : Campos_2002_Curr.Eye.Res_24_58
PubMedSearch : Campos_2002_Curr.Eye.Res_24_58
PubMedID: 12187496

Title : Synthesis and binding affinities of 5-(3-pyridinyl)- and 5-(3-quinolinyl)-4-azahomoadamantanes to alpha7 nicotinic acetylcholine receptors - Tataridis_2002_Farmaco_57_979
Author(s) : Tataridis D , Kolocouris A , Fytas G , Kolocouris N , Foscolos GB , Poulas K , Tzartos SJ
Ref : Farmaco , 57 :979 , 2002
Abstract : A general synthetic route that can lead to nicotinic ligands bearing a variety of bulky aza-ring systems was developed. This methodology was applied to obtain 5-(3-pyridinyl)- and 5-(3-quinolinyl)-4-azahomoadamantanes 2a, 3a and 2b, 3b. The parent 5-(3-pyridinyl)-4-azahomoadamantane 2a (Ki = 5.0 microM) binds with about 100 times lower affinity than (+)-epibatidine 1 (Ki = 0.045 microM) to alpha7 nicotinic acetylcholine receptors (nAChRs). N-methyl substitution of 2a gives compound 3a which has about nine times lower binding affinity. The replacement of pyridinyl with a quinolinyl ring (compounds 2b, 3b) results in a dramatic reduction in potency (Ki > 1000 microM).
ESTHER : Tataridis_2002_Farmaco_57_979
PubMedSearch : Tataridis_2002_Farmaco_57_979
PubMedID: 12564471

Title : Conjugation of acetylcholine receptor-protecting Fab fragments with polyethylene glycol results in a prolonged half-life in the circulation and reduced immunogenicity - Trakas_2001_J.Neuroimmunol_120_42
Author(s) : Trakas N , Tzartos SJ
Ref : Journal of Neuroimmunology , 120 :42 , 2001
Abstract : Antibodies to the acetylcholine receptor (AChR) cause AChR loss, resulting in the disease, myasthenia gravis (MG). The majority of the pathogenic antibodies seem to be directed against the main immunogenic region (MIR) of the AChR. In contrast to the intact antibodies, Fab fragments of anti-AChR antibodies are not themselves pathogenic and such fragments of anti-MIR monoclonal antibodies (mAbs) protect the AChR in vitro and in vivo against the pathogenic antibodies. However, Fab fragments have a very short in vivo half-life and are immunogenic, obstacles which must be overcome before their clinical use can be envisaged. We investigated the effect of conjugating Fab fragments to polyethylene glycol (PEG), a method known to increase the in vivo half-life and reduce the immunogenicity of proteins. When the Fab' fragments of two rat anti-MIR mAbs (nos. 35 and 195) were conjugated to methoxy-PEG-maleimide, the conjugates retained about 10% of their AChR binding activity and efficiently protected the AChR against the binding and modulating activity of myasthenic antibodies. Their in vivo half-life in rats was approximately 15 times longer than that of the unconjugated Fab' fragment and they were much less immunogenic in mice. This work represents an important step towards the clinical use of AChR-protective anti-MIR Fabs, but further improvements are needed before their clinical use is attempted.
ESTHER : Trakas_2001_J.Neuroimmunol_120_42
PubMedSearch : Trakas_2001_J.Neuroimmunol_120_42
PubMedID: 11694318

Title : Nicotinic receptor subtypes in human brain ageing, Alzheimer and Lewy body diseases - Perry_2000_Eur.J.Pharmacol_393_215
Author(s) : Perry E , Martin-Ruiz C , Lee M , Griffiths M , Johnson M , Piggott M , Haroutunian V , Buxbaum JD , Nasland J , Davis K , Gotti C , Clementi F , Tzartos SJ , Cohen O , Soreq H , Jaros E , Perry R , Ballard C , McKeith I , Court J
Ref : European Journal of Pharmacology , 393 :215 , 2000
Abstract : Human brain ageing is associated with reductions in a variety of nicotinic receptors subtypes, whereas changes in age-related disorders including Alzheimer's disease or Parkinson's disease are more selective. In Alzheimer's disease, in the cortex there is a selective loss of the alpha4 (but not alpha3 or 7) subunit immunoreactivity and of nicotine or epibatidine binding but not alpha-bungarotoxin binding. Epibatidine binding is inversely correlated with clinical dementia ratings and with the level of Abeta1-42, but not related to plaque or tangle densities. In contrast, alpha-bungarotoxin binding is positively correlated with plaque densities in the entorhinal cortex. In human temporal cortex loss of acetylcholinesterase catalytic activity is positively correlated with decreased epibatidine binding and in a transgenic mouse model over expressing acetylcholinesterase, epibatidine binding is elevated. In Parkinson's disease, loss of striatal nicotine binding appears to occur early but is not associated with a loss of alpha4 subunit immunoreactivity. Tobacco use in normal elderly individuals is associated with increased alpha4 immunoreactivity in the cortex and lower densities of amyloid-beta plaques, and with greater numbers of dopaminergic neurons in the substantia nigra pars compacta. These findings indicate an early involvement of the alpha4 subunit in beta-amyloidosis but not in nigro-striatal dopaminergic degeneration.
ESTHER : Perry_2000_Eur.J.Pharmacol_393_215
PubMedSearch : Perry_2000_Eur.J.Pharmacol_393_215
PubMedID: 10771016

Title : Treatment of passively transferred experimental autoimmune myasthenia gravis using papain - Poulas_2000_Clin.Exp.Immunol_120_363
Author(s) : Poulas K , Tsouloufis T , Tzartos SJ
Ref : Clinical & Experimental Immunology , 120 :363 , 2000
Abstract : Antibody-mediated acetylcholine receptor (AChR) loss at the neuromuscular junction, the main cause of the symptoms of myasthenia gravis, is induced by bivalent or multivalent antibodies. Passive transfer of experimental autoimmune myasthenia gravis (EAMG) can be induced very efficiently in rats by administration of intact MoAbs directed against the main immunogenic region (MIR) of the AChR, but not by their monovalent Fab fragments. We tested whether papain, which has been used therapeutically in autoimmune and other diseases, is capable of preventing EAMG by in vivo cleavage of the circulating anti-AChR antibodies into Fab fragments. EAMG was induced in 4-week-old female Lewis rats by i.p. injection of anti-MIR mAb35. A total of 0.75 mg of papain was given as one or three injections 3-7 h after MoAb injection. The mAb35 + papain-treated animals developed mild weakness during the first 30 h and subsequently recovered, while all animals that received only mAb35 developed severe myasthenic symptoms and died within 24-30 h. Animals treated only with papain showed no apparent side effects for up to 2 months. Serum anti-AChR levels in mAb35 + papain-treated rats decreased within a few hours, whereas in non-papain-treated rats they remained high for at least 30 h. Muscle AChR in mAb35 + papain-treated animals was partially protected from antibody-mediated degradation. These results show that treatment of rats with papain can prevent passively transferred EAMG without any apparent harm to the animals, and suggest a potential therapeutic use for proteolytic enzymes in myasthenia gravis.
ESTHER : Poulas_2000_Clin.Exp.Immunol_120_363
PubMedSearch : Poulas_2000_Clin.Exp.Immunol_120_363
PubMedID: 10792389

Title : Relative spatial position of a snake neurotoxin and the reduced disulfide bond alpha (Cys192-Cys193) at the alpha gamma interface of the nicotinic acetylcholine receptor - Michalet_2000_J.Biol.Chem_275_25608
Author(s) : Michalet S , Teixeira F , Gilquin B , Mourier G , Servent D , Drevet P , Binder P , Tzartos SJ , Menez A , Kessler P
Ref : Journal of Biological Chemistry , 275 :25608 , 2000
Abstract : We determined the distances separating five functionally important residues (Gln(10), Lys(27), Trp(29), Arg(33), and Lys(47)) of a three-fingered snake neurotoxin from the reduced disulfide bond alpha(Cys(192)-Cys(193)) located at the alphagamma interface of the Torpedo nicotinic acetylcholine receptor. Each toxin position was substituted individually for a cysteine, which was then linked to a maleimido moiety through three different spacers, varying in length from 10 to 22 A. We estimated the coupling efficiency between the 15 toxin derivatives and the reduced cystine alpha(192-193) by gel densitometry of Coomassie Blue-stained gels. A nearly quantitative coupling was observed between alphaCys(192) and/or alphaCys(193) and all probes introduced at the tip of the first (position 10) and second (position 33) loops of Naja nigricollis alpha-neurotoxin. These data sufficed to locate the reactive thiolate in a "croissant-shaped" volume comprised between the first two loops of the toxin. The volume was further restrained by taking into account the absence or partial coupling of the other derivatives. Altogether, the data suggest that alphaCys(192) and/or alphaCys(193), at the alphagamma interface of a muscular-type acetylcholine receptor, is (are) located in a volume located between 11.5 and 15.5 A from the alpha-carbons at positions 10 and 33 of the toxin, under the tip of the toxin first loop and close to the second one.
ESTHER : Michalet_2000_J.Biol.Chem_275_25608
PubMedSearch : Michalet_2000_J.Biol.Chem_275_25608
PubMedID: 10807914

Title : The third-dimensional structure of the complex between an Fv antibody fragment and an analogue of the main immunogenic region of the acetylcholine receptor: a combined two-dimensional NMR, homology, and molecular modeling approach - Kleinjung_2000_Biopolymers_53_113
Author(s) : Kleinjung J , Petit MC , Orlewski P , Mamalaki A , Tzartos SJ , Tsikaris V , Sakarellos-Daitsiotis M , Sakarellos C , Marraud M , Cung MT
Ref : Biopolymers , 53 :113 , 2000
Abstract : Binding of autoantibodies to the acetylcholine receptor (AChR) plays a major role in the autoimmune disease Myasthenia gravis (MG). In this paper, we propose a structure model of a putative immunocomplex that gives rise to the reduction of functional AChR molecules during the course of MG. The model complex consists of the [G(70), Nle(76)] decapeptide analogue of the main immunogenic region (MIR), representing the major antigenic epitope of AChR, and the single chain Fv fragment of monoclonal antibody 198, a potent MG autoantibody. The structure of the complexed decapeptide antigen [G(70), Nle(76)]MIR was determined using two-dimensional nmr, whereas the antibody structure was derived by means of homology modeling. The final complex was constructed using calculational docking and molecular dynamics. We termed this approach "directed modeling," since the known peptide structure directs the prestructured antibody binding site to its final conformation. The independently derived structures of the peptide antigen and antibody binding site already showed a high degree of surface complementarity after the initial docking calculation, during which the peptide was conformationally restrained. The docking routine was a soft algorithm, applying a combination of Monte Carlo simulation and energy minimization. The observed shape complementarity in the docking process suggested that the structure assessments already led to anti-idiotypic conformations of peptide antigen and antibody fragment. Refinement of the complex by dynamic simulation yielded improved surface adaptation by small rearrangements within antibody and antigen. The complex presented herein was analyzed in terms of antibody-antigen interactions, properties of contacting surfaces, and segmental mobility. The structural requirements for AChR complexation by autoantibodies were explored and compared with experimental data from alanine scans of the MIR peptides. The analysis revealed that the N-terminal loop of the peptide structure, which is indispensable for antibody recognition, aligns three hydrophobic groups in a favorable arrangement leading to the burial of 40% of the peptide surface in the binding cleft upon complexation. These data should be valuable in the rational design of an Fv mutant with much improved affinity for the MIR and AChR to be used in therapeutic approaches in MG.
ESTHER : Kleinjung_2000_Biopolymers_53_113
PubMedSearch : Kleinjung_2000_Biopolymers_53_113
PubMedID: 10679615

Title : Reconstitution of conformationally dependent epitopes on the N-terminal extracellular domain of the human muscle acetylcholine receptor alpha subunit expressed in Escherichia coli: implications for myasthenia gravis therapeutic approaches - Tsouloufis_2000_Int.Immunol_12_1255
Author(s) : Tsouloufis T , Mamalaki A , Remoundos M , Tzartos SJ
Ref : Int Immunol , 12 :1255 , 2000
Abstract : Myasthenia gravis (MG) is an autoimmune disease, caused by autoantibodies against the muscle acetylcholine receptor (AChR), an oligomeric transmembrane glycoprotein composed of alpha(2)beta gamma delta subunits. The alpha subunit carries in its N-terminal extracellular domain the main immunogenic region (MIR), a group of conformationally dependent epitopes that seems to be a major target for the anti-AChR antibodies in MG patients. Detailed epitope studies on pathogenic anti-AChR antibodies have been hindered because the binding of most of these antibodies is conformationally dependent, which precludes the use of denatured AChR fragments. The N-terminal extracellular fragment, residues 1-207, of the human AChR alpha subunit was expressed in Escherichia coli in a denatured form, solubilized in a guanidinium hydrochloride-containing buffer, purified, and renatured using a refolding approach which employs a detergent and a cyclodextrin as 'artificial chaperones'. Compared with the non-refolded protein, the refolded molecule exhibited a dramatic improvement in terms of the binding of all anti-MIR mAb tested. Anti-MIR mAb that normally bind weakly to the denatured alpha subunit bound approximately 30-100 times better to the refolded polypeptide and other anti-MIR mAb that bind exclusively to completely conformationally dependent epitopes also bound quite efficiently. These results, in addition to providing a means for the thorough investigation of the antigenic structure of the AChR, show that the conformationally dependent MIR epitopes do not require the participation of the oligosaccharide moiety of the alpha subunit nor the contribution of neighboring subunits for antibody binding. Such AChR fragments may be used in structural studies of the AChR autoantigen, and should prove valuable in the understanding and development of therapeutic approaches for MG.
ESTHER : Tsouloufis_2000_Int.Immunol_12_1255
PubMedSearch : Tsouloufis_2000_Int.Immunol_12_1255
PubMedID: 10967020

Title : Severe congenital myasthenic syndrome due to homozygosity of the 1293insG epsilon-acetylcholine receptor subunit mutation - Sieb_2000_Ann.Neurol_48_379
Author(s) : Sieb JP , Kraner S , Schrank B , Reitter B , Goebel TH , Tzartos SJ , Steinlein OK
Ref : Annals of Neurology , 48 :379 , 2000
Abstract : Recently, a congenital myasthenic syndrome (CMS) with end-plate acetylcholine receptor (AChR) deficiency due to missense mutations in the genes for the AChR subunit was described. The first observed patient with this CMS was heteroallelic for the two epsilon-AChR subunit mutations epsilon1101insT and epsilon1293insG. This patient had only a moderate phenotype with mild muscle weakness and abnormal fatigue. We have now found homozygosity for the epsilon1293insG mutation in a severely affected CMS patient, who lost the ability to walk in midchildhood and shows profound weakness and muscle wasting. Our observation allows a genotype-phenotype correlation illustrating how differences in the AChR mutation haplotype can profoundly influence disease severity.
ESTHER : Sieb_2000_Ann.Neurol_48_379
PubMedSearch : Sieb_2000_Ann.Neurol_48_379
PubMedID: 10976646

Title : Prevention of passively transferred experimental autoimmune myasthenia gravis by Fab fragments of monoclonal antibodies directed against the main immunogenic region of the acetylcholine receptor - Papanastasiou_2000_J.Neuroimmunol_104_124
Author(s) : Papanastasiou D , Poulas K , Kokla A , Tzartos SJ
Ref : Journal of Neuroimmunology , 104 :124 , 2000
Abstract : The muscle acetylcholine receptor loss, responsible for the clinical symptoms of myasthenia gravis, is due mainly to mechanisms dependent on the bivalent character of the anti-receptor antibodies. In cell culture, univalent Fab fragments of monoclonal antibodies (mAbs) directed against the main immunogenic region (MIR) of the acetylcholine receptor are able to protect the receptor against the action of the intact antibodies. To investigate the potential therapeutic use of this approach, we examined the ability of the Fab fragment of anti-MIR mAb195 (Fab195) to protect the receptor in vivo against two anti-MIR mAbs. Because of the rapid clearance of Fab fragments from the circulation, Lewis rats were treated repeatedly with Fab195. The Fab fragment significantly protected muscle receptors against antibody-mediated loss and was very efficient in providing protection against clinical symptoms when its administration was commenced before, simultaneously with, or 2 h after, mAb injection. Twenty-four hours after mAb injection, the protected rats only showed mild myasthenic symptoms, whereas those which only received intact antibodies were moribund or dead. These results suggest that, once modified to ensure their low immunogenicity and a long half-life, anti-MIR Fab fragments might be useful in the specific immunotherapy of myasthenia gravis.
ESTHER : Papanastasiou_2000_J.Neuroimmunol_104_124
PubMedSearch : Papanastasiou_2000_J.Neuroimmunol_104_124
PubMedID: 10713351

Title : The crystal structure of the Fab fragment of a rat monoclonal antibody against the main immunogenic region of the human muscle acetylcholine receptor - Kontou_2000_Eur.J.Biochem_267_2389
Author(s) : Kontou M , Leonidas DD , Vatzaki EH , Tsantili P , Mamalaki A , Oikonomakos NG , Acharya KR , Tzartos SJ
Ref : European Journal of Biochemistry , 267 :2389 , 2000
Abstract : The crystal structure of the Fab fragment of a rat monoclonal antibody, number 192, with a very high affinity (Kd = 0.05 nM) for the main immunogenic region of the human muscle acetylcholine receptor (AChR), has been determined and refined to 2.4 A resolution by X-ray crystallographic methods. The overall structure is similar to a Fab (NC6.8) from a murine antibody, used as a search model in molecular replacement. Structural comparisons with known antibody structures showed that the conformations of the hypervariable regions H1, H2, L1, L2, L3 of Fab192 adopt the canonical structures 1, 1, 2, 1, and 1, respectively. The surface of the antigen-binding site is relatively planar, as expected for an antibody against a large protein antigen, with an accessible area of 2865 A2. Analysis of the electrostatic surface potential of the antigen-binding site shows that the bottom of the cleft formed in the center of the site appears to be negatively charged. The structure will be useful in the rational design of very high affinity humanized mutants of Fab192, appropriate for therapeutic approaches of the model autoimmune disease myasthenia gravis.
ESTHER : Kontou_2000_Eur.J.Biochem_267_2389
PubMedSearch : Kontou_2000_Eur.J.Biochem_267_2389
PubMedID: 10759865

Title : The conformation of the main immunogenic region on the alpha-subunit of muscle acetylcholine receptor is affected by neighboring receptor subunits - Fostieri_2000_FEBS.Lett_481_127
Author(s) : Fostieri E , Beeson D , Tzartos SJ
Ref : FEBS Letters , 481 :127 , 2000
Abstract : Myasthenia gravis (MG) is caused by autoantibodies to the acetylcholine receptor (AChR). Experiments with fetal (alpha(2)betagammadelta) and adult (alpha(2)betaepsilondelta) AChR and with recombinant subunit dimers showed that some monoclonal antibodies (mAbs) against the main immunogenic region (MIR), located on the alpha-subunit of the AChR, bind better to fetal AChR and to alphagamma subunit dimer than to adult AChR and alphaepsilon dimer and equally to both alphabeta and alphadelta. However, other anti-MIR mAbs prefer adult AChR and alphaepsilon dimer, bind well to alphabeta but weakly to alphadelta. These results suggest that the MIR conformation is affected by the neighboring gamma/epsilon- and delta-subunits and may contribute to understanding the antibody specificities in MG.
ESTHER : Fostieri_2000_FEBS.Lett_481_127
PubMedSearch : Fostieri_2000_FEBS.Lett_481_127
PubMedID: 10996310

Title : Monoclonal antibodies raised against human acetylcholine receptor bind to all five subunits of the fetal isoform - Jacobson_1999_J.Neuroimmunol_98_112
Author(s) : Jacobson L , Beeson D , Tzartos SJ , Vincent A
Ref : Journal of Neuroimmunology , 98 :112 , 1999
Abstract : The human muscle acetylcholine receptor (AChR) is an oligomeric membrane protein consisting of (alpha1)2,beta,delta,epsilon subunits in the adult form and (alpha 1)2,beta,gamma,delta in the fetal form. The adult AChR is the target for autoantibodies in myasthenia gravis (MG), and antibodies that block the function of fetal AChR can cross the placenta and paralyse the developing baby causing joint contractures. Monoclonal antibodies (mAbs) raised against purified AChR were characterised previously in terms of binding to five regions, three of which appeared to partially overlap, but the subunit localisation of the regions was not clearly established and they were assumed to be mainly on the immunodominant alpha subunits. We have studied binding of the mAbs to AChR subunit extracellular fragments expressed in E. coli, and to AChRs derived from TE671 cells and from fibroblast cell lines expressing human/Torpedo and Torpedo/mouse hybrid receptors. Using a combination of Western blotting and immunoprecipitation experiments, we demonstrate the subunit specificity of each mAb. The results confirm our previous observations but importantly show that only two of the regions are on the alpha subunit, the three others being on the beta, gamma and delta subunits of human AChR. Thus these mAbs should be useful in studies of AChR subunit expression in normal and diseased tissue, and to define further the binding sites of antibodies in MG patients.
ESTHER : Jacobson_1999_J.Neuroimmunol_98_112
PubMedSearch : Jacobson_1999_J.Neuroimmunol_98_112
PubMedID: 10430044

Title : Establishment of a human thymic myoid cell line. Phenotypic and functional characteristics - Wakkach_1999_Am.J.Pathol_155_1229
Author(s) : Wakkach A , Poea S , Chastre E , Gespach C , Lecerf F , De La Porte S , Tzartos SJ , Coulombe A , Berrih-Aknin S
Ref : American Journal of Pathology , 155 :1229 , 1999
Abstract : The subset of myoid cells is a normal component of the thymic stroma. To characterize these cells, we immortalized stromal cells from human thymus by using a plasmid vector encoding the SV40 T oncogene. Among the eight cell lines obtained, one had myoid characteristics including desmin and troponin antigens. This new line was designated MITC (myoid immortalized thymic cells). These cells expressed both the fetal and adult forms of muscle acetylcholine receptor (AChR) at the mRNA level, as well as the myogenic transcription factor MyoD1. alpha-Subunit AChR protein expression was detected by flow cytometry and the AChR was functional in patch-clamp studies. In addition, AChR expression was down-modulated by myasthenia gravis sera or by monoclonal antibody anti-AChR on MITC line similarly to TE671 rhabdomyosarcoma cells, making the MITC line an interesting tool for AChR antigenic modulation experiments. Finally, the MITC line expressed LFA-3, produced several cytokines able to act on T cells, and protected total thymocytes from spontaneous apoptosis in vitro. These results are compatible with a role of thymic myoid cells in some steps of thymocyte development. Therefore MITC line appears to be a useful tool to investigate the physiological role of thymic myoid cells.
ESTHER : Wakkach_1999_Am.J.Pathol_155_1229
PubMedSearch : Wakkach_1999_Am.J.Pathol_155_1229
PubMedID: 10514405

Title : Construction and characterization of a humanized single chain Fv antibody fragment against the main immunogenic region of the acetylcholine receptor - Papanastasiou_1999_J.Neuroimmunol_94_182
Author(s) : Papanastasiou D , Mamalaki A , Eliopoulos E , Poulas K , Liolitsas C , Tzartos SJ
Ref : Journal of Neuroimmunology , 94 :182 , 1999
Abstract : The single chain Fv fragment of mAb198 (scFv198) directed against the main immunogenic region (MIR) of the nicotinic acetylcholine receptor (AChR), can efficiently protect the AChR in muscle cell cultures against the destructive activity of human myasthenic autoantibodies. Humanization of the scFv198 antibody fragment should prove useful for therapeutic application by reducing its immunogenicity. Framework sequences from human immunoglobulins homologous to the rat scFv198 sequences were selected and a totally synthetic humanized scFv198 antibody fragment was constructed in vitro. Humanized VH and VL domains were synthesized using two overlapping sets of 225 bases long oligonucleotides overlap extension and polymerase chain reaction (PCR), then assembled into a full-length gene by overlap extension of single-stranded DNA (ssDNA) fragments and PCR. The initial humanized antibody fragment had a very low affinity for the AChR. Molecular modeling was then performed and four residues from the framework regions (FR) of the humanized VH domain were selected to be replaced by the corresponding amino acid from the rat sequence. Three mutants were constructed by overlap extension, using PCR. The humanized variant containing replacements at VH residues 27, 29, 30 and 71 showed very good recovery of AChR binding activity; its binding affinities for Torpedo or human AChR (K(D): 8.5 or 323 nM, respectively) being only four times lower than those of the parental scFv198 (K(D): 2 or 80 nM, respectively). This variant was able to protect the human AChR against the binding of anti-MIR mAb and anti-alpha autoantibodies from a myasthenic patient. It was also able to protect AChR against antigenic modulation induced by the anti-MIR mAb198.
ESTHER : Papanastasiou_1999_J.Neuroimmunol_94_182
PubMedSearch : Papanastasiou_1999_J.Neuroimmunol_94_182
PubMedID: 10376952

Title : Alpha4 but not alpha3 and alpha7 nicotinic acetylcholine receptor subunits are lost from the temporal cortex in Alzheimer's disease - Martin-Ruiz_1999_J.Neurochem_73_1635
Author(s) : Martin-Ruiz CM , Court JA , Molnar E , Lee M , Gotti C , Mamalaki A , Tsouloufis T , Tzartos SJ , Ballard C , Perry RH , Perry EK
Ref : Journal of Neurochemistry , 73 :1635 , 1999
Abstract : Neuronal nicotinic acetylcholine receptors labelled with tritiated agonists are reduced in the cerebral cortex in Alzheimer's disease (AD), but to date it has not been demonstrated which nicotinic receptor subunits contribute to this deficit. In the present study, autopsy tissue from the temporal cortex of 14 AD cases and 15 age-matched control subjects was compared using immunoblotting with antibodies against recombinant peptides specific for alpha3, alpha4, and alpha7 subunits, in conjunction with [3H]epibatidine binding. Antibodies to alpha3, alpha4, and alpha7 produced one major band on western blots at 59, 51, and 57 kDa, respectively. [3H]Epibatidine binding and alpha4-like immunoreactivity (using antibodies against the extracellular domain and cytoplasmic loop of the alpha4 subunit) were reduced in AD cases compared with control subjects (p < 0.02) and with a subgroup of control subjects (n = 9) who did not smoke prior to death (p < 0.05) for the former two parameters. [3H]Epibatidine binding and cytoplasmic alpha4-like immunoreactivity were significantly elevated in a subgroup of control subjects (n = 4) known to have smoked prior to death (p < 0.05). There were no significant changes in alpha3- or alpha7-like immunoreactivity associated with AD or tobacco use. The selective involvement of alpha4 has implications for understanding the role of nicotinic receptors in AD and potential therapeutic targets.
ESTHER : Martin-Ruiz_1999_J.Neurochem_73_1635
PubMedSearch : Martin-Ruiz_1999_J.Neurochem_73_1635
PubMedID: 10501210

Title : Detection of antibodies directed against the cytoplasmic region of the human acetylcholine receptor in sera from myasthenia gravis patients - Tzartos_1999_Clin.Exp.Immunol_116_146
Author(s) : Tzartos SJ , Remoundos M
Ref : Clinical & Experimental Immunology , 116 :146 , 1999
Abstract : The nicotinic acetylcholine receptor (AChR) is the autoantigen in the human autoimmune disease myasthenia gravis (MG). Anti-AChR antibodies in MG sera bind mainly to conformational epitopes, therefore the determination of their specificities requires the use of native AChR. Antibody competition studies suggest that most MG antibodies are directed against the extracellular part of the molecule, whereas antibodies directed against the cytoplasmic region of the AChR have not been detected. To determine whether even small quantities of such antibodies exist in MG sera, we performed competition experiments based on the inhibition by MG sera of the binding of MoAbs to the human AChR, rather than inhibition by MoAbs of the binding of MG sera performed earlier. When MoAbs directed against cytoplasmic epitopes on the alpha or beta subunits (alpha 373-380 and beta 354-360) were used as test MoAbs, 17% or 9% of MG sera inhibited the binding of the anti-alpha or anti-beta subunit MoAbs, respectively, by > or = 50%. Non-specific inhibition was excluded. These results suggest the presence, in several MG sera, of antibodies directed against cytoplasmic regions of the AChR; yet these antibodies seemed to represent a relatively small proportion of the total anti-AChR antibodies. The corresponding epitopes may be involved in the inducing mechanisms in certain MG cases, and knowledge of the presence of such antibodies may be useful in understanding the autoimmune mechanism involved in MG.
ESTHER : Tzartos_1999_Clin.Exp.Immunol_116_146
PubMedSearch : Tzartos_1999_Clin.Exp.Immunol_116_146
PubMedID: 10209519

Title : Association of acetylcholine receptor alpha-subunit gene expression in mixed thymoma with myasthenia gravis - Wilisch_1999_Neurology_52_1460
Author(s) : Wilisch A , Gutsche S , Hoffacker V , Schultz A , Tzartos SJ , Nix W , Schalke B , Schneider C , Muller-Hermelink HK , Marx A
Ref : Neurology , 52 :1460 , 1999
Abstract : OBJECTIVE: To investigate the association of MG with the transcription of muscular or neuronal acetylcholine receptor (AChR) subunit genes in thymomas. BACKGROUND: Many steps in the pathogenesis of MG have been elucidated but, with rare exceptions, its etiology is unknown. In patients with MG with thymoma, the tumor probably elicits autoimmunity to AChR, but it is enigmatic why MG develops in some patients but not in others.
METHODS: Reverse transcriptase (RT)-PCR, immunohistochemistry, and immunofluorescence studies were carried out to investigate AChR expression in 35 patients with thymoma. Statistical analysis was used to specify significant differences between thymoma subtypes.
RESULTS: Considering all thymomas (n = 35), no correlation was found between MG status and AChR gene expression as detected by RT-PCR. However, when histologically defined thymoma subtypes were studied separately, transcription of the muscular AChR P3A- alpha-subunit gene was significantly associated (alpha < 0.01) with the occurrence of MG in mixed thymomas (n = 17), but not in thymomas of the cortical type. For the other muscular AChR subunits (P3A+ alpha isoform, beta, gamma, delta, and epsilon) and the alpha2 and beta4 neuronal AChR subunits, no such correlation was detected.
CONCLUSIONS: Expression of the P3A AChR alpha-subunit gene might be important for the pathogenesis of MG in mixed thymomas, suggesting etiologic heterogeneity of paraneoplastic MG among patients with histologically different thymoma subtypes.
ESTHER : Wilisch_1999_Neurology_52_1460
PubMedSearch : Wilisch_1999_Neurology_52_1460
PubMedID: 10227635

Title : The fetal form of the acetylcholine receptor distinguishes rhabdomyosarcomas from other childhood tumors - Gattenloehner_1998_Am.J.Pathol_152_437
Author(s) : Gattenloehner S , Vincent A , Leuschner I , Tzartos SJ , Muller-Hermelink HK , Kirchner T , Marx A
Ref : American Journal of Pathology , 152 :437 , 1998
Abstract : The fetal nicotinic acetylcholine receptor (AChR) of muscle is an oligomeric membrane protein with subunit composition alpha2betadeltagamma. After birth, the adult form, in which an epsilon-subunit replaces the gamma-subunit, predominates, and expression of the fetal form is limited to thymic myoid cells, extraocular muscles, and denervated striated muscle. We looked for expression of AChR in rhabdomyosarcomas and other childhood tumors by reverse transcription polymerase chain reaction and immunohistochemistry. mRNA for the AChR gamma-subunit was detected in all embryonal and alveolar rhabdomyosarcomas tested (n = 16) and in some tumors with a rhabdomyomatous component (n = 2) but not in other nonrhabdomyomatous tumors of childhood and adults (n = 45). The fetal form of the AChR was detected immunohistochemically in five of eight embryonal and four of eight alveolar rhabdomyosarcomas and in two Wilms' tumors with a rhabdomyomatous component but not in other tumors or in normal muscle. We conclude that reverse transcription polymerase chain reaction for AChR gamma-subunit could be useful for the diagnosis of rhabdomyosarcoma of childhood and for the detection of micrometastases and minimal residual disease. In addition, the fetal AChR protein is the first extracellular tumor marker that can distinguish rhabdomyosarcomas from nonrhabdomyomatous tumors and from normal muscle. Our findings, therefore, imply that the fetal AChR may be a target for in vivo imaging and, as AChR internalization and degradation is increased by antibody-induced cross-linking, may also provide a sensitive and specific target for immunotherapeutic strategies.
ESTHER : Gattenloehner_1998_Am.J.Pathol_152_437
PubMedSearch : Gattenloehner_1998_Am.J.Pathol_152_437
PubMedID: 9466570

Title : Modulation of the anti-acetylcholine receptor response and experimental autoimmune myasthenia gravis by recombinant fragments of the acetylcholine receptor - Barchan_1998_Eur.J.Immunol_28_616
Author(s) : Barchan D , Asher O , Tzartos SJ , Fuchs S , Souroujon MC
Ref : European Journal of Immunology , 28 :616 , 1998
Abstract : Myasthenia gravis (MG) is a neuromuscular disorder of man caused by a humoral response to the acetylcholine receptor (AChR). Most of the antibodies in MG and in experimental autoimmune myasthenia gravis (EAMG) are directed to the extracellular portion of the AChR alpha subunit, and within it, primarily to the main immunogenic region (MIR). We have cloned and expressed recombinant fragments, corresponding to the entire extracellular domain of the AChR alpha subunit (H alpha1-210), and to portions of it that encompass either the MIR (H alpha1-121) or the ligand binding site of AChR (H alpha122-210), and studied their ability to interfere with the immunopathological anti-AChR response in vitro and in vivo. All fragments were expressed as fusion proteins with glutathione S-transferase. Fragments H alpha1-121 and H alpha1-210 protected AChR in TE671 cells against accelerated degradation induced by the anti-MIR monoclonal antibody (mAb)198 in a dose-dependent manner. Moreover, these fragments had a similar effect on the antigenic modulation of AChR by other anti-MIR mAb and by polyclonal rat anti-AChR antibodies. Fragments H alpha1-121 and H alpha1-210 were also able to modulate in vivo muscle AChR loss and development of clinical symptoms of EAMG, passively transferred to rats by mAb 198. Fragment H alpha122-210 did not have such a protective activity. Our results suggest that the appropriate recombinant fragments of the human AChR may be employed in the future for antigen-specific therapy of myasthenia.
ESTHER : Barchan_1998_Eur.J.Immunol_28_616
PubMedSearch : Barchan_1998_Eur.J.Immunol_28_616
PubMedID: 9521072

Title : Congenital myasthenic syndromes in two kinships with end-plate acetylcholine receptor and utrophin deficiency - Sieb_1998_Neurology_50_54
Author(s) : Sieb JP , Dorfler P , Tzartos SJ , Wewer UM , Ruegg MA , Meyer D , Baumann I , Lindemuth R , Jakschik J , Ries F , Tzartos S
Ref : Neurology , 50 :54 , 1998
Abstract : We studied two families with five affected members suffering from ptosis and slowly progressive limb-girdle muscle weakness. All patients had abnormal decremental response on low-frequency nerve stimulation, but there were no repetitive responses to single stimuli. The patients improved on anti-acetylcholinesterase drugs. Intercostal muscle was obtained for special studies from one patient of each family. In vitro microelectrode studies were done in Patient 1. Miniature end-plate potentials were of low amplitude, and the quantal content of the evoked end-plate potentials was normal. Light microscopy revealed a marked type 1 fiber predominance. Acetylcholinesterase reactivity was dispersed over increased length of individual fibers in Patient 2. On morphometry of the end-plate ultrastructure, the number of secondary synaptic clefts per neuromuscular junction and the expansion of the postsynaptic area were markedly reduced. In Patient 1, but not in Patient 2, the envelopment of the nerve terminal by Schwann cell was increased. Acetylcholine-receptor (AChR) density was reduced as judged by the reduced immunoreactivity to antibodies against different receptor subunits. Immunohistochemical analysis of proteins known to be involved in orchestrating the end-plate structure showed deficiency of the AChR-associated protein utrophin. These patients appear to have a defect in the development or maintenance of the postsynaptic clefts; whether this defect results from or causes a reduced expression of utrophin or AChR is unclear.
ESTHER : Sieb_1998_Neurology_50_54
PubMedSearch : Sieb_1998_Neurology_50_54
PubMedID: 9443457

Title : Regulation of acetylcholine receptor gene expression in human myasthenia gravis muscles. Evidences for a compensatory mechanism triggered by receptor loss - Guyon_1998_J.Clin.Invest_102_249
Author(s) : Guyon T , Wakkach A , Poea S , Mouly V , Klingel-Schmitt I , Levasseur P , Beeson D , Asher O , Tzartos SJ , Berrih-Aknin S
Ref : J Clinical Investigation , 102 :249 , 1998
Abstract : Myasthenia gravis (MG) is a neuromuscular disorder mediated by antibodies directed against the acetylcholine receptor (nAChR) resulting in a functional nAChR loss. To analyze the molecular mechanisms involved at the muscular target site, we studied the expression of nAChR subunits in muscle biopsy specimens from MG patients. By using quantitative PCR with an internal standard for each subunit, we found that the levels of beta-, delta-, and epsilon-subunit mRNA coding for the adult nAChR were increased in severely affected MG patients, matching our previous data on the alpha-subunit. Messenger levels were highly variable in MG patients but not in controls, pointing to individual factors involved in the regulation of nAChR genes. The fetal subunit (gamma-chain) transcripts were almost undetectable in the extrajunctional region of MG muscle, suggesting that gene regulation in MG differs from that in the denervation model, in which nAChR gamma-subunit mRNA is reexpressed. Nicotinic AChR loss mediated by monoclonal anti-nAChR antibodies in both the TE671 muscle cell line and cultured normal human myotubes induces a similar increase in beta- alphand delta-subunit mRNA levels, suggesting the existence of a new muscular signaling pathway system coupled to nAChR internalization and independent of muscle electrical activity. These data demonstrate the existence of a compensatory mechanism regulating the expression of the genes coding for the adult nAChR in patients with MG.
ESTHER : Guyon_1998_J.Clin.Invest_102_249
PubMedSearch : Guyon_1998_J.Clin.Invest_102_249
PubMedID: 9649579

Title : Construction of single-chain Fv fragments of anti-MIR monoclonal antibodies -
Author(s) : Tzartos SJ , Tsantili P , Papanastasiou D , Mamalaki A
Ref : Annals of the New York Academy of Sciences , 841 :475 , 1998
PubMedID: 9668278

Title : Expression of human-Torpedo hybrid acetylcholine receptor (AChR) for analysing the subunit specificity of antibodies in sera from patients with myasthenia gravis (MG) - Loutrari_1997_Clin.Exp.Immunol_109_538
Author(s) : Loutrari H , Kokla A , Trakas N , Tzartos SJ
Ref : Clinical & Experimental Immunology , 109 :538 , 1997
Abstract : The nicotinic AChR, a pentamer composed of alpha2betagamma(or epsilon)delta subunits, is the autoantigen in the human autoimmune disease MG. Anti-AChR antibodies in MG sera bind mainly to conformational epitopes, therefore determination of their specificities requires the use of intact AChR. Indirect antibody competition studies have suggested that most MG antibodies are inhibited from binding to AChR by MoAb to the main immunogenic region (MIR) on the alpha-subunits. More recently, based on the knowledge that MG antibodies show little detectable cross-reaction with Torpedo AChR, we have shown, using mouse-Torpedo hybrid AChR, that most MG antibodies that detectably cross-react with the mouse AChR bind to the alpha-subunit. To analyse the whole anti-AChR antibody repertoire in MG sera, we expressed on stably transfected fibroblasts a novel human alpha+ Torpedo betagammadelta AChR and compared the antibody titres against human, Torpedo, and the hybrid AChR. Direct information was provided for the subunit specificity of several MoAbs and sera from 50 MG patients. On average, at least 48% of the anti-AChR antibodies in the sera were directed against the alpha-subunit. Interestingly, the anti-alpha-subunit antibodies predominated in low titre (0.6-7.4 nM) but not in high titre (10-386 nM) sera, where they comprised on average 68% versus 23% of the antibodies, respectively. Finally, the directly determined anti-alpha-subunit antibodies and the anti-MIR antibodies defined by antibody competition were significantly correlated, thus suggesting that at least a significant fraction of the anti-MIR antibodies in MG sera bind to the alpha-subunit.
ESTHER : Loutrari_1997_Clin.Exp.Immunol_109_538
PubMedSearch : Loutrari_1997_Clin.Exp.Immunol_109_538
PubMedID: 9328134

Title : Highly purified oligo-His tagged human recombinant alpha(1)-AChR is immunogenic in vivo and suitable for T cell stimulation in vitro in experimental and human myasthenia gravis - Voltz_1997_J.Neuroimmunol_80_131
Author(s) : Voltz R , Kamm C , Padberg F , Malotka J , Kerschensteiner M , Spuler S , Tzartos SJ , Dornmair K
Ref : Journal of Neuroimmunology , 80 :131 , 1997
Abstract : Using recombinantly expressed proteins for selection of antigen-specific T cell lines carries a high risk of selecting T cells specific for contaminating proteins. This risk is especially high for very hydrophobic proteins which are notoriously difficult to purify, such as the integral membrane protein acetylcholine receptor (AChR). We prepared a highly purified recombinant AChR by adding an oligo-histidine affinity-tag to the human alpha(1)-AChR and expressing it in E. coli. This allowed purification by Ni-NTA chromatography and subsequent electroelution from preparative SDS gel as purification steps, resulting in complete purity as assessed by silver stain on SDS-PAGE. This protein preparation induced fatal experimental allergic myasthenia gravis in Lewis rats. Furthermore, the protein could be used to select T cell lines from immunized Lewis rats and patients with myasthenia gravis. However, even with this highly purified protein, one of 8 Lewis rat T cell lines and 3 of 7 human T cell lines cross-reacted to E. coli control proteins. The results show that oligo-histidine tagged, highly purified human alpha(1)-AChR is highly immunogenic in vivo and in vitro.
ESTHER : Voltz_1997_J.Neuroimmunol_80_131
PubMedSearch : Voltz_1997_J.Neuroimmunol_80_131
PubMedID: 9413268

Title : Triphosgene: an efficient carbonylating agent for liquid and solid-phase aza-peptide synthesis. Application to the synthesis of two aza-analogues of the AChR MIR decapeptide - Andre_1997_J.Pept.Sci_3_429
Author(s) : Andre F , Marraud M , Tsouloufis T , Tzartos SJ , Boussard G
Ref : J Pept Sci , 3 :429 , 1997
Abstract : The N alpha/C alphaH exchange in aza-peptides has the advantage of preserving the side chain. Bis(trichloromethyl)carbonate or triphosgene is a solid, stable phosgene substitute which retains its high reactivity. Temperature and coupling times are greatly reduced with reference to other usually recommended carbonylating agents, while purity and yield are increased. It has been used, in both liquid- and solid-phase procedures, for the synthesis of various aza-analogues of dipeptides, tripeptides and decapeptides containing the alanine, aspartic acid and asparagine aza-residue.
ESTHER : Andre_1997_J.Pept.Sci_3_429
PubMedSearch : Andre_1997_J.Pept.Sci_3_429
PubMedID: 9467971

Title : Detection of antibody classes and subpopulations in myasthenia gravis patients using a new nonradioactive enzyme immunoassay - Gotti_1997_Muscle.Nerve_20_800
Author(s) : Gotti C , Balestra B , Mantegazza R , Tzartos SJ , Moretti M , Clementi F
Ref : Muscle & Nerve , 20 :800 , 1997
Abstract : To investigate the presence of antibodies in myasthenia gravis (MG) patients, we have developed a new reproducible and sensitive enzyme immunoassay (EIA-AChR), in which a beta subunit-specific monoclonal antibody (mAb 73) immobilizes fetal calf acetylcholine receptors (AChRs). We tested 92 MG patients (42 with positive and 50 with negative antibody titers), 60 healthy controls, and 40 controls with other autoimmune diseases. EIA-AChR detected immunoglobulin G (IgG) titers in all of the seropositive samples, with a significant correlation between these and those obtained using the traditional immunoprecipitation method. Moreover, 5 seronegative patients at immunoprecipitation assay were positive at EIA-AChR. EIA-AChR was also useful in revealing: (1) a seropositive patient subpopulation with generalized MG who had Abs directed against alpha-Bungarotoxin binding sites; and (2) patients with IgM directed against fetal calf AChR (detected in 13 seronegative and 16 seropositive MG patients, and in 6 of the patients with other autoimmune diseases).
ESTHER : Gotti_1997_Muscle.Nerve_20_800
PubMedSearch : Gotti_1997_Muscle.Nerve_20_800
PubMedID: 9179151

Title : Expression of acetylcholine receptor genes in human thymic epithelial cells: implications for myasthenia gravis - Wakkach_1996_J.Immunol_157_3752
Author(s) : Wakkach A , Guyon T , Bruand C , Tzartos SJ , Cohen-Kaminsky S , Berrih-Aknin S
Ref : J Immunol , 157 :3752 , 1996
Abstract : The intrathymic presence of the muscle acetylcholine receptor (AChR) is controversial, and the nature of the cell(s) expressing it is unclear. We thus analyzed the molecular expression of muscle AChR in human thymi. mRNA studies indicated that the two isoforms (P3A+ and P3A-) of the alpha-subunit were present in thymic extracts and in cultured thymic epithelial cells (TEC), while expression in thymocytes was low and not consistently detectable. The amount of mRNA coding for the alpha-subunit, evaluated by means of quantitative PCR, was about 20 times less in TEC than in muscle, and was similar in TEC from normal subjects and from patients with myasthenia gravis (MG). The beta- and epsilon-subunits present in adult AChR were also expressed in TEC (but not in thymocytes), while the embryonic subunit (gamma) was absent. In TEC cultures, the AChR alpha- and epsilon-subunit mRNA levels were down-regulated by forskolin, as also observed in the TE671 rhabdomyosarcoma cell line, suggesting similar regulation of AChR subunits in thymus and muscle. Protein expression was evidenced on TEC (but not on thymocytes), by Western blotting as well as by immunofluorescence, thus demonstrating AChR expression on human thymic epithelial cells. There was no difference in the expression of AChR between TEC from MG patients and controls, meaning that the expression of AChR subunits alone is not sufficient to explain the onset of MG.
ESTHER : Wakkach_1996_J.Immunol_157_3752
PubMedSearch : Wakkach_1996_J.Immunol_157_3752
PubMedID: 8871679

Title : Construction and application of a new class of sequential oligopeptide carriers (SOCn) for multiple anchoring of antigenic peptides--application to the acetylcholine receptor (AChR) main immunogenic region - Tsikaris_1996_Int.J.Biol.Macromol_19_195
Author(s) : Tsikaris V , Sakarellos C , Sakarellos-Daitsiotis M , Orlewski P , Marraud M , Cung MT , Vatzaki E , Tzartos SJ
Ref : Int J Biol Macromol , 19 :195 , 1996
Abstract : A new class of sequential oligopeptide carriers (SOCn), namely (Lys-Aib-Gly)n (n = 2-7), for anchoring antigenic peptides, is presented. These SOCn have been designed in order to assume a determined structural motif, exhibiting defined spatial orientations of the Lys-N epsilon H2 anchoring groups. The NMR study showed that SOCn adopt a rigid conformation with some regularity, initiated from the C-terminus of the carrier, while molecular dynamics simulation confirmed the occurrence of a distorted 3(10)-helix. It was also demonstrated, by 1HNMR, that all the antigenic peptides bound to the SOCn retain their original, folded active, structure and that probably they do not interact to each other. It is concluded that the beneficial structural elements of the SOCn impose a favorable disposition of the anchored peptides so that potent antigens with maximum molecular recognition are generated.
ESTHER : Tsikaris_1996_Int.J.Biol.Macromol_19_195
PubMedSearch : Tsikaris_1996_Int.J.Biol.Macromol_19_195
PubMedID: 8910060

Title : Epitope mapping by antibody competition. Methodology and Evaluation of the validity of the technique -
Author(s) : Tzartos SJ
Ref : Methods Mol Biol , 66 :55 , 1996
PubMedID: 8959704

Title : Characterisation, crystallisation and preliminary X-ray diffraction analysis of a Fab fragment of a rat monoclonal antibody with very high affinity for the human muscle acetylcholine receptor - Kontou_1996_FEBS.Lett_389_195
Author(s) : Kontou M , Vatzaki EH , Kokla A , Acharya KR , Oikonomakos NG , Tzartos SJ
Ref : FEBS Letters , 389 :195 , 1996
Abstract : The Fab fragment of a rat monoclonal antibody (no. 192) with very high affinity for the main immunogenic region of the human muscle nicotinic acetylcholine receptor (AChR) has been purified, characterised and crystallised using vapour diffusion techniques. Its Kd for human AChR was determined to be 5 X 10(-11) M. Its cross-reactivity pattern suggests that residue alpha23 of the AChR strongly affects its epitope. Crystals suitable for X-ray analysis, obtained by micro- and macroseeding techniques, belong to the orthorhombic space group C222(1) and they diffract to 2.8 A resolution using synchrotron radiation. The unit cell dimensions are alpha=83.4 A, b=110.0 A and c=212.2 A and there are two Fab molecules per asymmetric unit.
ESTHER : Kontou_1996_FEBS.Lett_389_195
PubMedSearch : Kontou_1996_FEBS.Lett_389_195
PubMedID: 8766828

Title : Antibodies to acetylcholinesterase cross-reacting with thyroglobulin in myasthenia gravis and Graves's disease - Mappouras_1995_Clin.Exp.Immunol_100_336
Author(s) : Mappouras DG , Philippou G , Haralambous S , Tzartos SJ , Balafas A , Souvatzoglou A , Lymberi P
Ref : Clinical & Experimental Immunology , 100 :336 , 1995
Abstract : In the present study we analysed by ELISA the ability of sera from 50 patients with myasthenia gravis (MG), 20 with Hashimoto's thyroiditis (HT), 53 with Graves' disease (GD) and 36 healthy controls (CR) to react with acetylcholinesterase (AChE) from Electrophorus electricus and human thyroglobulin (Tg). Significantly increased anti-AChE activity was exhibited by a high proportion of MG (IgG 36%) and GD (IgG 21%) sera, while increased anti-Tg activity was detected in all three patient groups (MG, IgG 26% and IgA 26%; HT, IgG 85% and IgA 40%; and GD, IgG 51%). Interestingly, a significant proportion of MG and GD sera exhibited both IgG anti-AChE and anti-Tg activities (MG, 18%; P < 0.001; and GD, 15%; P < 0.001, versus CR, 0%). This bi-reactivity was exhibited by anti-AChE antibodies cross-reacting with Tg (anti-AChE/Tg activity); (i) serum anti-AChE activity was effectively inhibited by soluble Tg, and (ii) affinity-purified anti-Tg antibodies cross-reacted with AChE. Cross-reactivity seems to be a property of pathological (auto)antibodies; induced (rabbit) antibodies to AChE or Tg were highly monospecific. Analysis of clinical data showed that increased IgG anti-AChE/Tg activity was well associated with: (i) overlapping GD in MG (P < 0.02), and (ii) ophthalmopathy in GD (P < 0.01). In contrast, no correlation was noted in MG between anti-AChE activity units and anti-Tg activity units or acetylcholine receptor antibody titres. The clinical significance of anti-AChE/Tg antibodies remains to be elucidated.
ESTHER : Mappouras_1995_Clin.Exp.Immunol_100_336
PubMedSearch : Mappouras_1995_Clin.Exp.Immunol_100_336
PubMedID: 7743674

Title : Monoclonal antibodies as site-specific probes for the acetylcholine-receptor delta-subunit tyrosine and serine phosphorylation sites - Tzartos_1995_Eur.J.Biochem_228_463
Author(s) : Tzartos SJ , Kouvatsou R , Tzartos E
Ref : European Journal of Biochemistry , 228 :463 , 1995
Abstract : Phosphorylation of the nicotinic acetylcholine receptor (AChR) has been implicated in the assembly, clustering, regulation of function and degradation rate of the molecule. Torpedo AChR is phosphorylated at eight cytoplasmic residues, four of which are on the delta subunit. We have precisely mapped the epitopes of eleven monoclonal antibodies (mAbs) directed against the Torpedo AChR delta-subunit regions delta 350-396 and delta 484-493, which are therefore exposed on the surface of the intact AChR, and now have four highly specific tools for analysing the role of delta-subunit phosphorylation. More than 160 synthetic peptides attached to polyethylene rods were used for epitope mapping. Four mAbs bound within the region delta 350-380, which contains all of the delta-subunit phosphorylation sites (Ser361, Ser362, Tyr372 and Ser377). Specifically, the epitope for mAb 134 (delta 365-375) contains Tyr372, the epitope(s) for mAbs 139 and 166 (delta 376-381) contains Ser377, while the epitope for mAb 146 (delta 350-359) is close to Ser361 and Ser362 and includes parts of the corresponding phosphorylation consensus sequences. Using peptide analogues with single residue substitutions, Tyr372 was found to be essential for the binding of mAb 134 and Ser377 was found contributing to the binding of mAbs 139 and 166. Finally, tyrosine phosphorylation of Torpedo AChR selectively inhibited binding of mAb 134. These data, and the availability of the defined mAb probes, should facilitate the study of the functional role of single AChR phosphorylation sites.
ESTHER : Tzartos_1995_Eur.J.Biochem_228_463
PubMedSearch : Tzartos_1995_Eur.J.Biochem_228_463
PubMedID: 7705363

Title : Monoclonal antibodies against the acetylcholine receptor gamma-subunit as site specific probes for receptor tyrosine phosphorylation - Tzartos_1995_FEBS.Lett_363_195
Author(s) : Tzartos SJ , Tzartos E , Tzartos JS
Ref : FEBS Letters , 363 :195 , 1995
Abstract : Tyrosine phosphorylation of the nicotinic acetylcholine receptor (AChR) may be involved in AChR desensitization and clustering. Torpedo AChR gamma-subunit is phosphorylated at Tyr365. Using overlapping synthetic peptides, we have precisely mapped the epitopes of five anti-gamma-subunit monoclonal antibodies (mAbs) and found that the epitope(s) for the mAbs 154, 165 and 168 (gamma 365-370) all contain Tyr365. mAb 168 is a known blocker of AChR channel function. Using peptide analogues, Tyr365 was found to be indispensable for mAb165 binding; furthermore its binding was selectively inhibited by in vitro AChR tyrosine phosphorylation. The possible connection between gamma-subunit phosphorylation and regulation of AChR function and the proven usefulness of these mAbs as tools should facilitate functional studies of AChR gamma-subunit phosphorylation.
ESTHER : Tzartos_1995_FEBS.Lett_363_195
PubMedSearch : Tzartos_1995_FEBS.Lett_363_195
PubMedID: 7537227

Title : Specific immune complexes augment in vitro acetylcholine receptor-specific T-cell proliferation - Melms_1993_Neurology_43_583
Author(s) : Melms A , Weissert R , Klinkert WE , Schalke BC , Tzartos SJ , Wekerle H
Ref : Neurology , 43 :583 , 1993
Abstract : We investigated the interaction between acetylcholine receptor (AChR)-specific T-helper cells from patients with myasthenia gravis and murine monoclonal anti-AChR antibodies. At optimal antigen concentration, anti-AChR antibodies neither enhanced nor impaired T-cell responses. However, at substimulatory antigen concentration, addition of anti-AChR antibodies substantially enhanced the proliferation of AChR-specific T cells. In spite of low amounts of antigen, immune complex formation allowed highly efficient capture and uptake of antigen via Fc receptors on antigen-presenting cells, which could be inhibited by an antibody to Fc receptors. Immune complex-mediated stimulation of sensitized AChR-specific T lymphocytes in vivo may contribute to the exacerbation of the disease, and demonstrates the interaction between T and B lymphocytes in myasthenia gravis.
ESTHER : Melms_1993_Neurology_43_583
PubMedSearch : Melms_1993_Neurology_43_583
PubMedID: 7680803

Title : Increased gene expression of acetylcholine receptor and myogenic factors in passively transferred experimental autoimmune myasthenia gravis - Asher_1993_J.Immunol_151_6442
Author(s) : Asher O , Kues WA , Witzemann V , Tzartos SJ , Fuchs S , Souroujon MC
Ref : J Immunol , 151 :6442 , 1993
Abstract : The passive transfer of myasthenia gravis by injection of mAb against muscle acetylcholine receptor (AChR) alpha-subunit, results in increased expression of AChR subunit genes, mainly at synaptic regions. The gene expression of AChR and of other muscle-specific proteins is regulated in a similar manner in passively transferred experimental autoimmune myasthenia gravis (EAMG) and in AChR-induced EAMG. Administration of AChR-specific mAb leads to a significant reduction in muscle AChR content and to an elevation in the mRNA levels corresponding to the adult, synaptic type of the receptor, as shown by Northern blot and in situ hybridization analyses. The mRNA levels of the myogenic factors myogenin and MRF4 are also increased moderately, whereas MyoD transcript levels remain unchanged. Thus, passive transfer of EAMG by mAb directed to defined epitopes of AChR alpha-subunit provides a suitable model for analyzing and following the cascade of molecular events triggered by anti-AChR immunopathologic antibodies and may shed light on the regulatory mechanisms underlying the human disease as well.
ESTHER : Asher_1993_J.Immunol_151_6442
PubMedSearch : Asher_1993_J.Immunol_151_6442
PubMedID: 8245477

Title : Cyclic lactam analogues containing the main immunogenic region of Torpedo acetylcholine receptor - Detsikas_1993_Pept.Res_6_17
Author(s) : Detsikas E , Tsikaris V , Sakarellos-Daitsiotis M , Sakarellos C , Cung MT , Marraud M , Vatzaki E , Tzartos SJ
Ref : Pept Res , 6 :17 , 1993
Abstract : The majority of autoantibodies against the nicotinic acetylcholine receptor (AChR) bind to an extracellular region of the AChR's alpha-subunit, named main immunogenic region (MIR), with the sequence W67-N-P-A-DY-G-G-I-K76 for the Torpedo californica electric organ. We report on the synthesis and the biological and 1H-NMR studies of two cyclic MIR compounds--namely, [D71,K76]-MIR-NH2 and Ac-[Orn68,D71,A76]-MIR-NH2. The relatively small chemical shift differences between [D71,K76]-MIR-NH2 and the biologically active [A76]-analogue suggest that both MIR derivatives possess similar conformations. Thus, the observed limited anti-MIR MAb binding capacity of [D71,K76]-MIR-NH2 is attributed to the D71,K76 side-chain blockage, through lactam. Formation of the Orn68,D71 cycle in the Ac-[Orn68,D71,A76]-MIR-NH2 preserves, unchanged, the low antigenicity of the linear Ac-[Orn68,A76]-MIR-NH2, thus confirming the key role of position 68. The low temperature coefficient value of A70-NH and the observed NOE effect between P69-C delta H2 and A70-NH in Ac-[Orn68,D71,A76]-MIR-NH2 argue in favor of a type I beta-turn in the Trp67-Orn-P-A70 sequence. However, the N-terminus beta-folding and the Orn68,D71 cycle appear ineffective for optimal antibody molecular recognition.
ESTHER : Detsikas_1993_Pept.Res_6_17
PubMedSearch : Detsikas_1993_Pept.Res_6_17
PubMedID: 7679937

Title : Bacterial expression of a single-chain Fv fragment which efficiently protects the acetylcholine receptor against antigenic modulation caused by myasthenic antibodies - Mamalaki_1993_Eur.J.Immunol_23_1839
Author(s) : Mamalaki A , Trakas N , Tzartos SJ
Ref : European Journal of Immunology , 23 :1839 , 1993
Abstract : Monoclonal antibodies (mAb) against the main immunogenic region (MIR) of the acetylcholine receptor (AChR) are very potent in inducing antigenic modulation of the AChR in animals and in muscle cell cultures. A recombinant antibody fragment of the rat anti-MIR mAb198 was cloned by polymerase chain reaction and expressed as soluble single-chain Fv fragment (scFv198) in E. coli and affinity purified. DNA sequencing was used to define the VH (IB) and VL (K2) chain gene usage. scFv198 was found immunologically and biologically active. Its binding affinity for the Torpedo AChR (KD = 2 +/- 0.6 nM) was very similar with that of the intact mAb198 (KD = 1.8 +/- 0.6 nM) while for the human AChR (KD = 80.7 +/- 16.6 nM) it was about four times lower than that of the intact mAb198 (KD = 21.6 +/- 6.6 nM). This fragment was capable of efficiently protecting the AChR in human cell cultures, against antigenic modulation caused by the intact mAb198 or by the antibodies from a myasthenic patient. The produced scFv198 fragment is, therefore, potentially useful in therapeutic applications for myasthenia gravis after appropriate genetic manipulations.
ESTHER : Mamalaki_1993_Eur.J.Immunol_23_1839
PubMedSearch : Mamalaki_1993_Eur.J.Immunol_23_1839
PubMedID: 8344344

Title : Passive transfer of experimental myasthenia gravis via antigenic modulation of acetylcholine receptor - Loutrari_1992_Eur.J.Immunol_22_2449
Author(s) : Loutrari H , Kokla A , Tzartos SJ
Ref : European Journal of Immunology , 22 :2449 , 1992
Abstract : Antigenic modulation of acetylcholine receptor (AChR) is considered to contribute to the reduction of endplate AChR in myasthenia gravis (MG). Yet, the pathogenic significance of this mechanism is unclear. To investigate the in vivo role of AChR antigenic modulation we examined the ability of bivalent F(ab')2 and monovalent Fab fragments of monoclonal antibody (mAb) 35 to passively transfer experimental autoimmune MG (EAMG) in rats. mAb 35 which binds at the main immunogenic region (MIR) of the AChR causes severe EAMG without being involved in channel function. Compared to the intact mAb, F(ab')2 35 proved to be less potent but still capable of inducing moderate EAMG, whereas Fab 35 were totally ineffective. Furthermore, both intact and F(ab')2 35 induced mild EAMG in complement-depleted rats. These results (a) provide evidence that antigenic modulation of endplate AChR is sufficient to generate passive transfer of EAMG and (b) further support the pathogenic potential of the anti-MIR antibodies in MG.
ESTHER : Loutrari_1992_Eur.J.Immunol_22_2449
PubMedSearch : Loutrari_1992_Eur.J.Immunol_22_2449
PubMedID: 1516631

Title : Use of Torpedo-mouse hybrid acetylcholine receptors reveals immunodominance of the alpha subunit in myasthenia gravis antisera - Loutrari_1992_Eur.J.Immunol_22_2949
Author(s) : Loutrari H , Tzartos SJ , Claudio T
Ref : European Journal of Immunology , 22 :2949 , 1992
Abstract : The nicotinic acetylcholine receptor (AChR), a pentameric complex of alpha 2 beta gamma delta subunits, is the autoantigen in the human autoimmune disease myasthenia gravis (MG). Anti-AChR antibodies are found in approximately 90% of MG patients and using indirect methods (competitive binding to solubilized AChR), peptides, or synthetic peptides, the majority of these antibodies have been shown to bind to the AChR alpha subunit. In order to determine directly the AChR subunit specificities of MG antibodies, we employed as antigens a novel set of hybrid AChR composed of species cross-reacting and non-cross-reacting subunits stably expressed in fibroblasts. Sequence similarities of homologous subunits among species can vary widely, with mammalian subunits having 87%-96% identity and Torpedo-mammalian subunits having 54%-80% identity. These findings are reflected in antigenic specificities, with human anti-AChR antisera frequently recognizing mouse AChR but rarely recognizing Torpedo. By establishing separate cell lines stably expressing all-Torpedo, all-mouse, and different combinations of Torpedo and mouse subunits, we were able to provide the first direct evidence of a predominant anti-alpha subunit specificity in MG antisera. Functional hybrid AChR stably expressed in an intact cell membrane provide us with a system that best mimics the in vivo environment of the MG antibody in a binding assay. Such a system allows us to investigate a perplexing observation in the field: a poor correlation between the patient's clinical status and antibody titer. Those antibodies which can interfere with AChR function, such as ones with the ability to cross-link AChR and induce their accelerated internalization and degradation (antigenic modulation) might represent a subpopulation of MG antibodies important in disease induction or maintenance. In this report, we demonstrate that wild-type and hybrid AChR expressed in fibroblasts can be antigenically modulated by intermolecular cross-linking antibodies as AChR are in native muscle cells. Because we can monitor dynamic interactions between AChR and MG antibodies, this system may allow us to define crucial pathogenic epitopes in MG by expressing hybrid, chimeric, and mutant AChR.
ESTHER : Loutrari_1992_Eur.J.Immunol_22_2949
PubMedSearch : Loutrari_1992_Eur.J.Immunol_22_2949
PubMedID: 1385157

Title : Interactions of an immunogenic decapeptide fragment of the neuromuscular acetylcholine receptor (AChR) with a monoclonal anti-AChR antibody -
Author(s) : Marraud M , Demange P , Cung MT , Tsikaris V , Sakarellos C , Vatzaki E , Tzartos SJ
Ref : Biochemical Society Transactions , 20 :837 , 1992
PubMedID: 1487075

Title : A shared epitope in the acetylcholine receptor-alpha subunit and fast troponin I of skeletal muscle. Is it important for myasthenia gravis? - Osborn_1992_Am.J.Pathol_140_1215
Author(s) : Osborn M , Marx A , Kirchner T , Tzartos SJ , Plessman U , Weber K
Ref : American Journal of Pathology , 140 :1215 , 1992
Abstract : The monoclonal antibody MAb 155, isolated by Tzartos et al, recognizes the alpha subunit of acetylcholine receptor (AChR) and stains type II skeletal muscle fibers but does not decorate heart muscle. In addition it reacts with most myasthenia gravis-associated thymomas. The authors show by immunoblotting techniques that the myofibrillar antigen is a 23 kd protein and by partial protein sequence data identify it as fast troponin I. Fast troponin I from various species contains the sequence EEKSGMEGRK close to the C-terminal end at positions 165 to 174. The first lysine (K) is crucial for MAb 155 reactivity since its substitution by methionine and leucine in slow troponin I and cardiac troponin I, respectively, abolishes MAb 155 reactivity. The epitope identified on troponin I is homologous in sequence with the MAb 155 epitope on the AChR alpha subunit established by direct peptide binding as KSAIEGIK (positions 373-380). The authors consider whether fortuitously shared epitopes can be responsible for the high level of autoantibodies to AChR and to muscle proteins seen in many MG patients.
ESTHER : Osborn_1992_Am.J.Pathol_140_1215
PubMedSearch : Osborn_1992_Am.J.Pathol_140_1215
PubMedID: 1374594

Title : B-T lymphocyte interactions in experimental autoimmune myasthenia gravis: autoantibody mediated up-regulation of the response of acetylcholine receptor-specific T lymphocytes - Zhang_1992_Immunology_77_571
Author(s) : Zhang Y , Tzartos SJ
Ref : Immunology , 77 :571 , 1992
Abstract : Twenty-six monoclonal antibodies of five different isotypes and reactive against distinct parts (alpha, beta, gamma, delta-subunits) of the nicotinic acetylcholine receptor (AChR) from Torpedo californica were screened for their capacity to enhance activation of AChR-specific CD4+ autoreactive T cells. The T-cell line (LR) used in this study recognized an epitope (98-116) on the alpha-subunit of Torpedo AChR. Four monoclonal antibodies bearing a gamma 2b isotype and recognizing an epitope on the Torpedo AChR alpha-subunit, especially the main immunogenic region (MIR), were able to enhance T-cell activation in a dose-response manner. Four further gamma 2b isotype monoclonal antibodies, recognizing epitopes other than the AChR alpha-subunit, had no effect. Monoclonal antibodies of other isotypes (IgM, IgG1, IgG2a, IgG2c), irrespective of their subunit specificity, were unable to influence the T-cell response. Thus, the enhancement requires a IgG2b isotype, and both the antibody and the T-cell recognize an epitope on the same subunit. We have previously shown that AChR-specific B cells are directly able to present antigen to AChR-specific T-cell lines in a privileged way. The present data demonstrate that B cells are also capable of enhancing indirectly the immunogenicity of autoantigens via their humoral antibodies.
ESTHER : Zhang_1992_Immunology_77_571
PubMedSearch : Zhang_1992_Immunology_77_571
PubMedID: 1283599

Title : A striational muscle antigen and myasthenia gravis-associated thymomas share an acetylcholine-receptor epitope - Marx_1992_Dev.Immunol_2_77
Author(s) : Marx A , Osborn M , Tzartos SJ , Geuder KI , Schalke B , Nix W , Kirchner T , Muller-Hermelink HK
Ref : Dev Immunol , 2 :77 , 1992
Abstract : The coincidence of autoantibodies against the acetylcholine receptor (AChR) and muscle striational antigens (SA) is a characteristic finding in thymoma-associated myasthenia gravis (MG), but their origins are still unresolved. Some common muscle antigens that were shown to be targets of anti-SA autoantibodies in thymoma-associated MG have also been detected in normal or neoplastic thymic epithelial cells, suggesting that the release of (eventually altered) antigens from the thymic tumors could elicit SA autoimmunity. In contrast to this model, we report here that titin, which is a recently reported target of SA autoimmunity, is not expressed in thymomas. In addition, we show that skeletal muscle type-II fibers exhibit a striational immunoreactivity with monoclonal antibody mAb155, which was previously identified to label a very immunogenic cytoplasmic epitope of the AChR and neoplastic epithelial cells of MG-associated thymomas. We conclude from these findings that titin autoimmunity in thymoma-associated MG is either due to a molecular mimicry mechanism involving tumor antigens (other than titin) or is a secondary phenomenon following release of titin from muscle. Based on the common immunoreactivity of the AChR, a striational antigen and thymoma, we suggest as the pathogenetic mechanism of thymoma-associated MGa "circulus vitiosus" in which SA autoimmunity could help maintain the AChR autoimmunity that is primarily elicited by the thymomas.
ESTHER : Marx_1992_Dev.Immunol_2_77
PubMedSearch : Marx_1992_Dev.Immunol_2_77
PubMedID: 1379503

Title : Precise epitope mapping of monoclonal antibodies to the cytoplasmic side of the acetylcholine receptor alpha subunit. Dissecting a potentially myasthenogenic epitope - Tzartos_1992_Eur.J.Biochem_207_915
Author(s) : Tzartos SJ , Remoundos MS
Ref : European Journal of Biochemistry , 207 :915 , 1992
Abstract : The epitopes for twelve monoclonal antibodies against the cytoplasmic side of the acetylcholine receptor (AChR) alpha subunit were precisely mapped using over 300 continuously overlapping synthetic peptides attached on poly(ethylene) rods. mAb cross-reactive between Torpedo and human AChR generally bound to the homologous peptides from both species. Epitopes 4-10-residues long were identified. One mAb could bind to either arm on both sides of a beta-turn structure. Five mAb bound to a very-immunogenic cytoplasmic epitope on alpha 373-380 (VICE-alpha). Three of the mAb against VICE-alpha were earlier found to cross-react with non-AChR protein(s), present in thymomas from myasthenia gravis patients but absent in thymomas from non-myasthenics. Since VICE-alpha has a potentially crucial pathogenic role, the antigenic role of each residue within it was subsequently studied by 55 analogues, most having single amino acid substitutions. All the mAb against VICE-alpha bound similarly but not identically to the analogues, thus explaining their known binding heterogeneity. Lys373 proved indispensable for mAb binding. Ile376, Glu377, Gly378 and Lys380 were quite critical, while Ser374, Ala375 and Val379 seemed rather inactive. These data should prove instructive in searches for VICE-alpha-like epitopes carrying autoantigens with potential involvement in myasthenia gravis and should further expand the applications of the anti-(AChR) mAb in AChR studies.
ESTHER : Tzartos_1992_Eur.J.Biochem_207_915
PubMedSearch : Tzartos_1992_Eur.J.Biochem_207_915
PubMedID: 1379917

Title : 2D-NMR and molecular dynamics analysis of the Torpedo californica acetylcholine receptor alpha 67-76 fragment and of its [Ala76]-analogue - Cung_1992_Pept.Res_5_14
Author(s) : Cung MT , Tsikaris V , Demange P , Papadouli I , Tzartos SJ , Sakarellos C , Marraud M
Ref : Pept Res , 5 :14 , 1992
Abstract : The alpha 67-76 fragment (Trp67-Asn68-Pro69-Ala70-Asp71-Tyr72 -Gly73-Gly74- Ile75-Lys76) of the Torpedo californica acetylcholine receptor (AChR) is selectively recognized by antibodies against the main immunogenic region of the AChR. The antibody binding capacity of its [Ala76]-analogue is usually higher than that of the natural fragment. A conformational analysis of these two decapeptides has been carried out in Me2SO by 2D-NMR and molecular dynamics using the SYBYL and BIOGROMOS programs. The natural sequence presents the most numerous and strongest NOE connectivities and is accordingly less flexible than the [Ala76]-analogue. Due to the flexible orientation of the side chains in both peptides, the NOE backbone side chain and side chain-side chain connectivities have not been introduced as distance constraints in the molecular dynamics calculations. It appeared that the N-terminal heptapeptide in both sequences assumes two very similar folded conformations, whereas the Ala substitution induces conformational flexibility in the C-terminal tripeptide sequence. The most flexible [Ala76]-analogue is the most tightly bound to the monoclonal mAb6 anti-AChR antibody, and the transferred NOEs from the bound to the free peptide in D2O reveal some similarity with the intrinsic NOEs for the free natural sequence in Me2SO, suggesting that the bound conformation of the [Ala76]-analogue could not be very different from that of the free natural fragment.
ESTHER : Cung_1992_Pept.Res_5_14
PubMedSearch : Cung_1992_Pept.Res_5_14
PubMedID: 1623299

Title : Paratope- and framework-related cross-reactive idiotopes on anti-acetylcholine receptor antibodies - Verschuuren_1991_J.Immunol_146_941
Author(s) : Verschuuren JJ , Graus YM , Tzartos SJ , van Breda Vriesman PJ , De Baets MH
Ref : J Immunol , 146 :941 , 1991
Abstract : Cross-reactive idiotopes are a possible target for therapeutical interventions in autoimmune diseases. To investigate their role in the pathogenesis of experimental autoimmune myasthenia gravis (EAMG) we analyzed the Id of rat anti-AChR mAb 6, 35, 61, 65 and a control myeloma protein IR27. Anti-Id 6, 35, 61, 65 bound in a direct binding assay with various affinity to all rat anti-AChR mAb that were tested. Anti-Id IR27 recognized none of the anti-AChR mAb. The specificity of these crossreactions was confirmed by inhibition studies with anti-AChR mAb and two control rat myeloma proteins (IR27 and IR241). In addition, the Id expression on mAb D6, a mouse anti-human AChR mAb was recognized by anti-Id 6, 35, and 65. Anti-Id, except anti-Id IR27, bound to affinity purified IgG from the sera of rats with EAMG, but not to preimmune Lewis IgG. These results suggest extensive sharing of idiotopes among anti-AChR mAb, which are also present in EAMG serum. Anti-AChR mAb against the main immunogenic region (6, 35, 65) from different rat strains, shared at least one paratope-related cross-reactive idiotopes. In the view of the fact that anti-main immunogenic region antibodies might form a predominant fraction of the polyclonal response against AChR, it is conceivable that an anti-Id recognizing these antibodies could have therapeutical applications as for example a selective immune absorbent or in immunotoxin therapy.
ESTHER : Verschuuren_1991_J.Immunol_146_941
PubMedSearch : Verschuuren_1991_J.Immunol_146_941
PubMedID: 1988504

Title : In vivo effects of neonatal administration of antiidiotype antibodies on experimental autoimmune myasthenia gravis - Verschuuren_1991_Autoimmunity_10_173
Author(s) : Verschuuren JJ , Graus YM , van Breda Vriesman PJ , Tzartos SJ , De Baets MH
Ref : Autoimmunity , 10 :173 , 1991
Abstract : The in vivo effects of neonatal administration of varying doses of anti-idiotype antibodies on serum anti-acetylcholine receptor (AChR) antibody titers, idiotype expression, and disease severity was studied in experimental autoimmune myasthenia gravis. Polyclonal affinity purified anti-idiotype antibodies and monoclonal anti-idiotype antibodies directed at anti-AChR monoclonal antibody 65 were administered in dosages varying from the nanogram to the microgram range. Mab 65 is directed against the main immunogenic region of mammalian AChR. In 1 out of 4 experiments administration of a nanogram dosage of anti-idiotype antibodies led to an enhanced anti-AChR antibody response after immunization with AChR. But no enhancing effect on idiotype expression could be demonstrated during this experiment. Adoptive transfer of spleen cells from rats pretreated with a nanogram dosage of anti-idiotype antibodies resulted in an significantly increased antibody response against rat AChR after immunization. From these experiments we conclude that in vivo administration of polyclonal or monoclonal anti-idiotypes does not reproduceably modify the serum antibody level against the acetylcholine receptor, nor influences the idiotype profile of the immune response. Secondly, the idiotype mediated manipulation of the immune response against large antigens, like the acetylcholine receptor, is clearly more complicated than that against small haptens. Adoptive transfer models, might be helpful in analysing the possibilities of anti-idiotype treatment in myasthenia gravis in more detail.
ESTHER : Verschuuren_1991_Autoimmunity_10_173
PubMedSearch : Verschuuren_1991_Autoimmunity_10_173
PubMedID: 1756222

Title : Two-dimensional 1H-NMR study of antigen-antibody interactions: binding of synthetic decapeptides to an anti-acetylcholine receptor monoclonal antibody - Cung_1991_Biopolymers_31_769
Author(s) : Cung MT , Demange P , Marraud M , Tsikaris V , Sakarellos C , Papadouli I , Kokla A , Tzartos SJ
Ref : Biopolymers , 31 :769 , 1991
Abstract : Two-dimensional NMR experiments [correlated spectroscopy (COSY) and two-dimensional transferred nuclear Overhauser enhancement spectroscopy (TR-NOESY)] have been applied to study the interactions of a monoclonal antibody (mAb) directed to the main immunogenic region (MIR) of the acetylcholine receptor (AChR), and four synthetic decapeptides from the MIR. The decapeptides were the Torpedo AChR alpha 67-76 fragment (W67-N68-P69-A70-D71-Y72-G73-+ ++G74-I75-K76) and its three [A69], [A73], and [A76] analogues. The results led to the following conclusions: (1) the magnitude of the TR-NOE cross peaks does not depend only on the structuration of the peptide in the bound state, but also on restrictions of the mobility, i.e., on the correlation time tau c, which can be different for every residue; (2) the binding capacity of the synthetic peptides to mAbs measured by radioimmunoassay is directly correlated to the NOE magnitude; and (3) the combined interpretation of the COSY and TR-NOESY experiments gives a qualitative information about the nature and the overall conformation of the sequence which is in contact with the mAb binding site.
ESTHER : Cung_1991_Biopolymers_31_769
PubMedSearch : Cung_1991_Biopolymers_31_769
PubMedID: 1932573

Title : Visualization and functional testing of acetylcholine receptor-like molecules in cochlear outer hair cells - Plinkert_1990_Hear.Res_44_25
Author(s) : Plinkert PK , Gitter AH , Zimmermann U , Kirchner T , Tzartos SJ , Zenner HP
Ref : Hearing Research , 44 :25 , 1990
Abstract : The efferent nerve endings at outer hair cells (OHCs) have been suggested to regulate active mechanical processes in the cochlea. The discovery of acetylcholine (ACh)-producing and -degrading enzymes in these synapses gave rise to the speculation that ACh might be one of the efferent transmitters. However, there has as yet been no identification and characterization of any corresponding receptor in OHCs which is required for further clarification of this question. In the present paper existence, location and first characterization of acetylcholine receptors (AChRs) in OHCs are reported. Using two anti-AChR monoclonal antibodies, AChR epitopes were found forming a cup at the basal end of the OHCs opposite to the efferent nerve endings. Furthermore, the studied molecules could be shown to extend through the cell membrane. In addition, the denervated OHC AChR-epitopes seem to move by lateral diffusion. Application of Carbachol and ACh to the basal pole of OHCs induced a weak, reversible cell contraction. Pharmacological controls revealed, that hte motile responses were mediated by the AChRs.
ESTHER : Plinkert_1990_Hear.Res_44_25
PubMedSearch : Plinkert_1990_Hear.Res_44_25
PubMedID: 2324016

Title : Characterization of a protein with an acetylcholine receptor epitope from myasthenia gravis-associated thymomas - Marx_1990_Lab.Invest_62_279
Author(s) : Marx A , O'Connor R , Geuder KI , Hoppe F , Schalke B , Tzartos SJ , Kalies I , Kirchner T , Muller-Hermelink HK
Ref : Lab Invest , 62 :279 , 1990
Abstract : Immunohistochemical studies have shown that almost all thymomas of myasthenia gravis patients contain at least one protein sharing an antigenic determinant with the nicotinic acetylcholine receptor (AchR) of human muscle. We describe the characterization of this protein (p153) which has a molecular weight of 153 and an isoelectric point of 5.0. By treatment of p153 with endoglycosidases, no significant glycosylation has been detected. Immunologically, p153 crossreacts with monoclonal antibodies against the amino acid sequence 371-378 of the alpha-chain of the AchR. No cross-reactivity to the main immunogenic region of the AchR nor an alpha-bungarotoxin binding site are found. By Western blotting, p153 was generally neither detectable in normal tissues nor extrathymic tumors with the exception of paraganglioma and neuroblastoma. In conclusion, the structure of p153 is apparently unrelated to the AchR from muscle or the alpha-bungarotoxin binding proteins from thymoma. Since there is no evidence for an AchR expression in thymoma, the antigenic homology of p153 with the nicotinic AchR might be relevant for triggering an intrathymomatous autosensitization of maturing T cells and could be responsible for the high association of thymomas with myasthenia gravis.
ESTHER : Marx_1990_Lab.Invest_62_279
PubMedSearch : Marx_1990_Lab.Invest_62_279
PubMedID: 1690313

Title : Neonatal myasthenia gravis: antigenic specificities of antibodies in sera from mothers and their infants - Tzartos_1990_Clin.Exp.Immunol_80_376
Author(s) : Tzartos SJ , Efthimiadis A , Morel E , Eymard B , Bach JF
Ref : Clinical & Experimental Immunology , 80 :376 , 1990
Abstract : Transient neonatal myasthenia gravis (MG) is a human model of passively transferred MG. In an effort to understand the characteristics of the most pathogenic antibodies in MG, we studied the fine antigenic specificities of anti-AChR antibodies in sera from 21 MG mothers (nine of which had transiently transferred the disease) and 17 of their infants. Although in a few cases significant differences in antibody specificities were observed between mothers and infants, whether myasthenic or not, generally the antigenic specificities of the antibodies in sera from infants were very similar to those of their mothers. Furthermore, no characteristic differences were detected between the antibody repertoires of mothers who transferred the disease and those who did not.
ESTHER : Tzartos_1990_Clin.Exp.Immunol_80_376
PubMedSearch : Tzartos_1990_Clin.Exp.Immunol_80_376
PubMedID: 1695559

Title : Main immunogenic region of Torpedo electroplax and human muscle acetylcholine receptor: localization and microheterogeneity revealed by the use of synthetic peptides - Tzartos_1990_J.Neurochem_54_51
Author(s) : Tzartos SJ , Loutrari H , Tang F , Kokla A , Walgrave SL , Milius RP , Conti-Tronconi BM
Ref : Journal of Neurochemistry , 54 :51 , 1990
Abstract : Most anti-nicotinic acetylcholine receptor (AChR) antibodies in myasthenia gravis are directed against an immunodominant epitope or epitopes [main immunogenic region (MIR)] on the AChR alpha-subunit. Thirty-two synthetic peptides, corresponding to the complete Torpedo alpha-subunit sequence and to a segment of human muscle alpha-subunit, were used to map the epitopes for 11 monoclonal antibodies (mAbs) directed against the Torpedo and/or the human MIR and for a panel of anti-AChR mAbs directed against epitopes on the alpha-subunit other than the MIR. A main constituent loop of the MIR was localized within residues alpha 67-76. Residues 70 and 75, which are different in the Torpedo and human alpha-subunits, seem to be crucial in determining the binding profile for several mAbs whose binding to the peptides correlated very well with their binding pattern to native Torpedo and human AChRs. This strongly supports the identification of the peptide loop alpha 67-76 as the actual location of the MIR on the intact AChR molecule. Residues 75 and 76 were necessary for binding of some mAbs and irrelevant for others, in agreement with earlier suggestions that the MIR comprises overlapping epitopes. Structural predictions for the sequence segment alpha 67-76 indicate that this segment has a relatively high segmental mobility and a very strong turning potential centered around residues 68-71. The most stable structure predicted for this segment, in both the Torpedo and human alpha-subunits, is a hairpin loop, whose apex is a type I beta-turn and whose arms are beta-strands. This loop is highly hydrophilic, and its apex is negatively charged. All these structural properties have been proposed as characteristic of antibody binding sites. We also localized the epitopes for mAbs against non-MIR regions. Among these, the epitope for a monoclonal antibody (mAb 13) that noncompetitively inhibits channel function was localized within residues alpha 331-351.
ESTHER : Tzartos_1990_J.Neurochem_54_51
PubMedSearch : Tzartos_1990_J.Neurochem_54_51
PubMedID: 1688377

Title : Proteins with epitopes of the acetylcholine receptor in epithelial cell cultures of thymomas in myasthenia gravis - Marx_1989_Am.J.Pathol_134_865
Author(s) : Marx A , Kirchner T , Hoppe F , O'Connor R , Schalke B , Tzartos SJ , Muller-Hermelink HK
Ref : American Journal of Pathology , 134 :865 , 1989
Abstract : Thymomas from 12 patients with myasthenia gravis (MG) were investigated for the presence of epitopes of the alpha-subunit of the nicotinic acetylcholine receptor (AchR) using monoclonal antibodies (MAb) reacting against the AchR. In all but two of the tumors epitopes corresponding to antigenic determinants located on the cytoplasmic side of the AchR were identified. From eight thymomas cell lines were established that have been kept in culture for up to 6 months. The cultured cells expressed the same AchR-epitopes as did the primary tumors. During early passages the percentage of epithelial cells positive for the AchR epitopes approximately mirrored the percentage of positive cells in the original tumors. With passaging the relative number of positive cells usually declined but in some cultures an increase was observed. Three cell lines that showed extensive staining with an MAb against the AchR were radiolabeled to characterize the antigen. From protein extracts of these three cell lines proteins of 45 kd and 156 kd molecular weight (MW) were precipitated. These proteins are different from other proteins described in the context of both thymomas and MG. The negative reactivity with MAb against other epitopes of the alpha-subunit, especially against the main immunogenic region (MIR), speaks in favor of membrane-associated proteins of only limited crossreactivity to the AchR. A previous study found an almost exclusive occurrence of these AchR-epitopes in thymomas associated with MG, but not in other thymomas of similar histologic type. The expression of the proteins described here could therefore play a role in the triggering of the autoimmune process against the AchR of the motor, endplate in MG patients.
ESTHER : Marx_1989_Am.J.Pathol_134_865
PubMedSearch : Marx_1989_Am.J.Pathol_134_865
PubMedID: 2468286

Title : Acetylcholine receptor epitope in proteins of myasthenia gravis-associated thymomas and non-thymic tissues - Marx_1989_Thymus_14_171
Author(s) : Marx A , O'Connor R , Tzartos SJ , Kalies I , Kirchner T , Muller-Hermelink HK
Ref : Thymus , 14 :171 , 1989
Abstract : Immunohistochemical studies have shown that almost all thymomas of myasthenia gravis (MG) patients contain proteins which share antigenic determinants with the nicotinic acetylcholine receptor (AChR) of human muscle. Here we describe one of the proteins (p153) which (1) is not part of a compound structure, (2) has a MW of 153 kd, (3) has an isoelectric point of 5.0, and (4) is probably free of sugar residues. The protein does not bind mAb to the main immunogenic region of the AChR and has no alpha-bungarotoxin (alpha-btx) binding site. p153 was not found in both normal tissues and a variety of tumours. However, the epitope defined by mAb155 also occurs in at least two other proteins (from muscle and TE671 cells) which have MW different from 153 kd and which are unrelated to AChR. Experiments presented elsewhere [Geuder et al., this volume] show that there are no proteins in thymomas which share an extensive molecular homology with the AChR. All these findings suggest that p153 is unrelated to the AChR. As p153 is the only protein demonstrated in thymomas which is significantly correlated with MG and which shares an antigenic determinant with AChR, p153 is a candidate protein determining the AChR-specificity of the autoimmune process in MG.
ESTHER : Marx_1989_Thymus_14_171
PubMedSearch : Marx_1989_Thymus_14_171
PubMedID: 2482999

Title : Fab fragments of monoclonal antibodies protect the human acetylcholine receptor against antigenic modulation caused by myasthenic sera - Sophianos_1989_J.Autoimmun_2_777
Author(s) : Sophianos D , Tzartos SJ
Ref : J Autoimmun , 2 :777 , 1989
Abstract : The human cell line TE671 produces large amounts of muscle nicotinic acetylcholine receptor (AChR). TE671 cells were used to determine the specificity of antibodies which can increase the internalization rate of AChR (antigenic modulation) and to test procedures for protecting AChR against this mechanism. The half-life of AChR both in the absence and the presence of anti-AChR antibodies was very similar to that of AChR on human muscle cell cultures. The relative contribution of different anti-AChR antibody fractions to the total antigenic modulation capacity of human myasthenic sera was investigated by competition experiments between Fab fragments of anti-AChR monoclonal antibodies (MoAbs) and intact antibodies (MoAb or myasthenic sera). Fab fragments, which do not induce antigenic modulation, were allowed to shield the corresponding regions of the AChR. Intact antibodies were subsequently added. It was found that protection of the main immunogenic region (MIR), but not of a region on the beta-subunit, essentially blocked the modulatory effect of the intact anti-MIR MoAbs, and approximately 80% of that of myasthenic sera. These data suggest that anti-MIR antibodies are mainly responsible for the loss of human AChR via antigenic modulation. Furthermore the observation that Fab fragments of anti-MIR MoAbs can efficiently protect AChR against antigenic modulation may have therapeutic implications.
ESTHER : Sophianos_1989_J.Autoimmun_2_777
PubMedSearch : Sophianos_1989_J.Autoimmun_2_777
PubMedID: 2619869

Title : Monoclonal antibodies against the main immunogenic region of the acetylcholine receptor. Mapping on the intact molecule - Kordossi_1989_J.Neuroimmunol_23_35
Author(s) : Kordossi AA , Tzartos SJ
Ref : Journal of Neuroimmunology , 23 :35 , 1989
Abstract : About two-thirds of the antibodies to the nicotinic acetylcholine receptor (AChR) in myasthenic patients, and in rats immunized with intact AChR, bind to the main immunogenic region (MIR) on the alpha-subunit. We tested all available anti-MIR monoclonal antibodies (mAbs) by competition experiments for binding on the intact AChR from Torpedo electric organ and human muscle. Practically complete competition between all possible paired combinations of anti-MIR mAbs was found. As a consequence, the MIR must be a very concrete and small region. Furthermore, the location of the MIR relative to some other less immunogenic regions was also determined.
ESTHER : Kordossi_1989_J.Neuroimmunol_23_35
PubMedSearch : Kordossi_1989_J.Neuroimmunol_23_35
PubMedID: 2723040

Title : Two-dimensional 1H-NMR study of synthetic peptides containing the main immunogenic region of the Torpedo acetylcholine receptor - Cung_1989_Biopolymers_28_465
Author(s) : Cung MT , Marraud M , Hadjidakis I , Bairaktari E , Sakarellos C , Kokla A , Tzartos SJ
Ref : Biopolymers , 28 :465 , 1989
Abstract : A comparative 1H-NMR spectral study of a synthetic decapeptide containing the main immunogenic region of the Torpedo acetylcholine receptor (AChR; WNPADYGGIK, representing the alpha 67-76 fragment of Torpedo AChR) with four analogous peptides (WNP3-D5YGGIK, WNPAA5YGGIK, WNPADYGGA9K, and WNPD4DYGGV9K) has been carried out in dimethyl sulfoxide. One- and two-dimensional nmr experiments [correlated spectroscopy (COSY), relayed COSY, and phase-sensitive nuclear Overhauser enhancement spectroscopy (NOESY)] were performed to obtain complete assignments of the proton resonances. The presence of strong and multiple short- and long-range NOEs, and especially a strong long-range NOE between the two Asn2-C alpha H and Gly7-C alpha H protons, argues in favor of a rigid folded structure in all five cases. Temperature dependence measurements indicate the existence of three intramolecular interactions involving the Asp3, Gly8, and Lys10 amide protons.
ESTHER : Cung_1989_Biopolymers_28_465
PubMedSearch : Cung_1989_Biopolymers_28_465
PubMedID: 2470436

Title : Pathogenesis of myasthenia gravis. Acetylcholine receptor-related antigenic determinants in tumor-free thymuses and thymic epithelial tumors - Kirchner_1988_Am.J.Pathol_130_268
Author(s) : Kirchner T , Tzartos SJ , Hoppe F , Schalke B , Wekerle H , Muller-Hermelink HK
Ref : American Journal of Pathology , 130 :268 , 1988
Abstract : The authors describe an immunohistologic study of acetylcholine receptor (AChR)-related antigenic determinants in tumor-free thymuses of myasthenia gravis (MG) patients (13 cases) and nonmyasthenic controls (10 cases) and in thymic epithelial tumors of patients with MG (8 cases) and without MG (6 cases). Monoclonal antibodies (MAbs) to the cytoplasmic part and to the extracellular main immunogenic region (MIR) of the alpha subunit of AChRs were used. Their intrathymic binding sites were defined by double-immunostaining, and compared with alpha-bungarotoxin (alpha-Bgt) labeling demonstrated by fluorescence microscopy. Tumor-free thymuses of MG patients and control patients contained cytoplasmic AChR epitopes and alpha-Bgt binding sites on myoid cells and some epithelial cells. Only myoid cells also expressed extracellular MIR epitopes, suggesting that they bear complete AChRs, and are important targets for the autoimmune attack in tumor-free MG thymus. Evidence that AChR-related antigenic determinants of epithelial cells are also significant for MG is provided by our findings in thymic epithelial tumors. All eight tumors with MG but only two out of six tumors without MG showed cytoplasmic AChR epitopes and alpha-Bgt binding sites on neoplastic epithelial cells. Myoid cells and MIR epitopes did not occur in the neoplasms, but in some tumor-free thymic remnants beside thymomas. It is assumed that nonneoplastic and neoplastic thymic epithelial cells contain only incomplete AChRs or AChR-like molecules. The different expression of AChR epitopes in thymic epithelial tumors and tumor-free thymuses might explain some of the heterogeneous region specificities of anti-AChR antibodies in sera of MG patients with and without thymoma.
ESTHER : Kirchner_1988_Am.J.Pathol_130_268
PubMedSearch : Kirchner_1988_Am.J.Pathol_130_268
PubMedID: 2449082

Title : Localization of the main immunogenic region of human muscle acetylcholine receptor to residues 67-76 of the alpha subunit - Tzartos_1988_Proc.Natl.Acad.Sci.U.S.A_85_2899
Author(s) : Tzartos SJ , Kokla A , Walgrave SL , Conti-Tronconi BM
Ref : Proc Natl Acad Sci U S A , 85 :2899 , 1988
Abstract : The majority of antibodies to the acetylcholine receptor (AcChoR), both in the human disease myasthenia gravis and in its experimental models, are directed against an extracellular area of the AcChoR alpha subunit called the main immunogenic region (MIR). We have studied the binding of anti-AcChoR monoclonal antibodies (mAbs) to 26 synthetic peptides corresponding to the hydrophilic parts of the human AcChoR alpha subunit. The binding sites for eight anti-MIR mAbs and for eight anti-alpha-subunit, non-anti-MIR mAbs were localized. Anti-MIR mAbs bound to one peptide corresponding to residues 63-80 of the human alpha subunit. A second panel of peptides corresponding to the various parts of the alpha-subunit segment 63-80 was synthesized. Anti-MIR antibodies bound to a peptide that contained the alpha-subunit sequence 67-76. Thus, a main constituent loop of the MIR is localized between residues 67 and 76 of the alpha subunit.
ESTHER : Tzartos_1988_Proc.Natl.Acad.Sci.U.S.A_85_2899
PubMedSearch : Tzartos_1988_Proc.Natl.Acad.Sci.U.S.A_85_2899
PubMedID: 3362855

Title : Determination of antibody binding sites on the three-dimensional and primary structure of acetylcholine receptor -
Author(s) : Tzartos SJ , Kordossi A , Walgrave SL , Kokla A , Conti-Tronconi BM
Ref : Monogr Allergy , 25 :20 , 1988
PubMedID: 2459612

Title : Fine antigenic specificities of antibodies in sera from patients with D-penicillamine-induced myasthenia gravis - Tzartos_1988_Clin.Exp.Immunol_74_80
Author(s) : Tzartos SJ , Morel E , Efthimiadis A , Bustarret AF , D'Anglejan J , Drosos AA , Moutsopoulos HA
Ref : Clinical & Experimental Immunology , 74 :80 , 1988
Abstract : A small fraction of patients with rheumatoid arthritis and other diseases on D-penicillamine treatment may develop antibodies against the acetylcholine receptor (AChR) and symptoms of myasthenia gravis (MG). The mechanism leading to this phenomenon is not known. We have studied the fine antigenic specificities of the anti-AChR antibodies in 19 D-penicillamine-induced MG (pen-MG) patients and compared them with those of antibodies from 204 idiopathic MG patients (the data for 122 obtained from earlier experiments). Antigenic specificities of the circulating antibodies were determined by the capacity of monoclonal antibodies (MoAbs), against certain determinants on the AChR, to inhibit binding of the serum antibodies to the AChR. Monoclonal antibodies against alpha, beta and gamma subunits were used. The anti-AChR antibody patterns of pen-MG patients were very similar to those of idiopathic MG patients. Antibodies to the main immunogenic region, which is located on the extracellular surface of the alpha-subunit, were the predominant group. The variations of antibody specificities in serial sera collected from individual patients at different times were usually small, as were those of idiopathic MG. These results strongly suggest that the antibody repertoire in the sera of idiopathic and pen-MG patients is very similar.
ESTHER : Tzartos_1988_Clin.Exp.Immunol_74_80
PubMedSearch : Tzartos_1988_Clin.Exp.Immunol_74_80
PubMedID: 2464451

Title : Mapping the main immunogenic region and toxin-binding site of the nicotinic acetylcholine receptor - Barkas_1987_Science_235_77
Author(s) : Barkas T , Mauron A , Roth B , Alliod C , Tzartos SJ , Ballivet M
Ref : Science , 235 :77 , 1987
Abstract : The alpha-chain of the nicotinic acetylcholine receptor carries the binding sites both for cholinergic ligands and for most experimentally induced or naturally occurring antibodies to the native receptor. By means of expression cloning in Escherichia coli, fusion proteins were derived from specific fragments of a complementary DNA encoding the mouse alpha-chain, allowing the mapping of the toxin-binding site to residues 160-216 and the main immunogenic region to residues 6-85. This approach permits the independent study of different functional domains of a complex receptor molecule and should be generally applicable to other proteins for which complementary DNA clones are available.
ESTHER : Barkas_1987_Science_235_77
PubMedSearch : Barkas_1987_Science_235_77
PubMedID: 2432658

Title : Acetylcholine receptor epitopes on epithelial cells of thymoma in myasthenia gravis -
Author(s) : Kirchner T , Hoppe F , Muller-Hermelink HK , Schalke B , Tzartos SJ
Ref : Lancet , 1 :218 , 1987
PubMedID: 2433555

Title : Passive transfer of experimental autoimmune myasthenia gravis by monoclonal antibodies to the main immunogenic region of the acetylcholine receptor - Tzartos_1987_J.Neuroimmunol_15_185
Author(s) : Tzartos SJ , Hochschwender S , Vasquez P , Lindstrom JM
Ref : Journal of Neuroimmunology , 15 :185 , 1987
Abstract : Experimental autoimmune myasthenia gravis (EAMG) was passively transferred to rats by injecting monoclonal antibodies (mAbs) directed at the main immunogenic region (MIR) of the nicotinic acetylcholine receptor (AChR). The MIR is located on the extracellular part of the AChR alpha-subunit. All four mAbs directed at the MIR which were tested were very efficient in inducing EAMG: within 2 days the rats became moribund or very weak and their muscle AChR content decreased to about 50% of normal. These mAbs are of two different IgG subclasses (IgG1 and IgG2a) and derived from rats immunized with AChR from either fish electric organs or mammalian muscles. One mAb directed at the extracellular side of the beta-subunit did not cause AChR loss or induce symptoms of EAMG. mAbs to the cytoplasmic side were, as expected, ineffective.
ESTHER : Tzartos_1987_J.Neuroimmunol_15_185
PubMedSearch : Tzartos_1987_J.Neuroimmunol_15_185
PubMedID: 3495549

Title : Human T-helper lymphocytes in myasthenia gravis recognize the nicotinic receptor alpha subunit - Hohlfeld_1987_Proc.Natl.Acad.Sci.U.S.A_84_5379
Author(s) : Hohlfeld R , Toyka KV , Tzartos SJ , Carson W , Conti-Tronconi BM
Ref : Proc Natl Acad Sci U S A , 84 :5379 , 1987
Abstract : Myasthenia gravis is a human disease caused by an autoimmune response against the nicotinic acetylcholine receptor (AcChoR). Since the molecular structure of AcChoR is well known, myasthenia gravis is an excellent system for studying the recognition of a complex membrane antigen in the human immune system. Human T-helper (TH) cell lines reactive to the AcChoR were isolated from four myasthenic patients by selection with native AcChoR from Torpedo californica. The selected TH cells could efficiently recognize native and fully denatured AcChoR. The vast majority of the TH-stimulating AcChoR epitopes were located on the denatured alpha subunit of AcChoR. Antibody competition experiments using a panel of rat anti-AcChoR monoclonal antibodies showed that 39-45% of the autoantibodies present in the sera of these same patients bound to the conformation-sensitive "main immunogenic region" (MIR), also located on the alpha subunit. However, AcChoR-induced stimulation of the T cells could not be inhibited with up to 20-fold molar excess of different rat anti-MIR monoclonal antibodies. These results suggest that the Torpedo AcChoR alpha subunit contains conformation-insensitive epitopes that play a role in the autosensitization of TH cells and that seem to be physically separated from the MIR. The specificity of the TH cell response may contribute to directing the B-cell response to other alpha-subunit determinants, such as the MIR itself.
ESTHER : Hohlfeld_1987_Proc.Natl.Acad.Sci.U.S.A_84_5379
PubMedSearch : Hohlfeld_1987_Proc.Natl.Acad.Sci.U.S.A_84_5379
PubMedID: 2955417

Title : Conformation of cytoplasmic segments of acetylcholine receptor alpha- and beta-subunits probed by monoclonal antibodies: sensitivity of the antibody competition approach - Kordossi_1987_EMBO.J_6_1605
Author(s) : Kordossi AA , Tzartos SJ
Ref : EMBO Journal , 6 :1605 , 1987
Abstract : The conformation of the cytoplasmic side of Torpedo marmorata acetylcholine receptor (AChR) was investigated by 22 monoclonal antibodies (mAbs) binding to known sites on the amino acid sequences 339-378 and 336-469 of the AChR alpha- and beta-subunits respectively. Competitions among these mAbs for binding on the intact AChR were compared with their competition for binding on the SDS-denatured subunits and with their corresponding epitopes previously determined on the primary structure of the subunits. We found the following: The three approaches correlated very well suggesting that these mAbs bind on the intact AChR at the same sequences determined by synthetic peptides and not on irrelevant discontinuous epitopes; this finding supports conclusions of Ratnam et al. (1986a) that the amphipathic helix M5 is exposed on the cytoplasmic side of the AChR. The subunit segments alpha 339-378 and beta 336-469 seem to be extended over large distances on the cytoplasmic surface of the AChR. The cytoplasmic surface of beta-subunit has a very immunogenic region. The mAb-competition technique is very sensitive since mAbs to epitopes separated by only about seven residues did not exclude each other, and mAbs to overlapping epitopes exhibited differential competitions with other mAbs.
ESTHER : Kordossi_1987_EMBO.J_6_1605
PubMedSearch : Kordossi_1987_EMBO.J_6_1605
PubMedID: 2440678

Title : Characteristics of monoclonal antibodies to denatured Torpedo and to native calf acetylcholine receptors: species, subunit and region specificity - Tzartos_1986_J.Neuroimmunol_10_235
Author(s) : Tzartos SJ , Langeberg L , Hochschwender S , Swanson LW , Lindstrom JM
Ref : Journal of Neuroimmunology , 10 :235 , 1986
Abstract : Seventy-five monoclonal antibodies (mAbs) to sodium dodecyl sulfate-denatured Torpedo californica (66 mAbs) and intact fetal calf (9 mAbs) acetylcholine receptor (AChR) were produced. These mAbs were characterized for subunit, region and species specificity, for Ig class and subclass, for protein A binding and for antigen-crosslinking capacity. Fourteen were identified as anti-alpha, 35 were anti-beta, 8 were anti-gamma and 15 were anti-delta. None of the 11 anti-alpha derived from denatured AChR bound to the main immunogenic region (MIR) as judged by antibody competition assays. This contrasts with previous results using mAbs against native AChr, the majority of which bind to the MIR. Thirty-eight mAbs crossreacted with some or all of the tested AChRs from fish electric organs and mammalian muscles in addition to the immunogen. Eight anti-alpha, anti-beta and 1 anti-delta mAbs showed good to excellent autoantibody activity. Analysis by sucrose gradient centrifugation of some AChR-mAb complexes revealed that some mAbs form intermolecular and others form intramolecular crosslinkings of the AChR. The described mAbs have proven valuable tools in AChR and myasthenia gravis research.
ESTHER : Tzartos_1986_J.Neuroimmunol_10_235
PubMedSearch : Tzartos_1986_J.Neuroimmunol_10_235
PubMedID: 3484485

Title : Antigenic modulation of human myotube acetylcholine receptor by myasthenic sera. Serum titer determines receptor internalization rate - Tzartos_1986_J.Immunol_136_3231
Author(s) : Tzartos SJ , Sophianos D , Zimmerman K , Starzinski-Powitz A
Ref : J Immunol , 136 :3231 , 1986
Abstract : Antibodies to the acetylcholine receptor (AChR) added to AChR-bearing muscle cells cross-link the receptors, thus increasing their internalization and degradation rate (antigenic modulation). This mechanism contributes to AChR loss in myasthenia gravis. Until recently, antigenic modulation has been studied in animal tissues, where only a small fraction of human anti-AChR antibodies bind. In the present study, we examined the antigenic modulation of AChR by using patients' sera and cultures of human muscle cells. We aimed to see whether antigenic modulation correlates better with disease severity or with antibody titer. Antibody-containing sera from 29 myasthenic patients in various states of the disease and with different antibody titers against AChR were tested. Control sera from six healthy individuals were also tested. Our experiments showed that all myasthenic sera affected the overall AChR content on the human myotube surface, causing a 49 to 82% loss, whereas control sera had no effect. Although at fixed serum volumes there was some correlation between disease severity and AChR loss, this effect was clearly due to differences in antibody titers. In fact, the antigenic modulation depended mainly on the final concentration of the antibody present. Thus, intrinsic factors other than antibodies to AChR may determine or influence the patients' susceptibility to the disease.
ESTHER : Tzartos_1986_J.Immunol_136_3231
PubMedSearch : Tzartos_1986_J.Immunol_136_3231
PubMedID: 3958494

Title : Decrease in acetylcholine-receptor content of human myotube cultures mediated by monoclonal antibodies to alpha, beta and gamma subunits - Tzartos_1986_FEBS.Lett_196_91
Author(s) : Tzartos SJ , Starzinski-Powitz A
Ref : FEBS Letters , 196 :91 , 1986
Abstract : One of the two main causes of acetylcholine-receptor loss in myasthenia gravis is antigenic modulation, i.e. accelerated internalization and degradation rate by antibody-crosslinking. This phenomenon has been studied only in animal tissues. Therefore, we tested antigenic modulation of the acetylcholine receptor on human embryonic myotubes in cultures. Several monoclonal antibodies to the alpha, beta and gamma subunits of the receptor reduced its concentration, in some cases down to one-third of the control. Some of these antibodies only form complexes of one antibody with two receptor molecules; consequently such small complexes are sufficient to accelerate internalization of the human acetylcholine receptor. This technique might be proved valuable for clinical screening of sera from myasthenic patients.
ESTHER : Tzartos_1986_FEBS.Lett_196_91
PubMedSearch : Tzartos_1986_FEBS.Lett_196_91
PubMedID: 2417888

Title : Role of the main immunogenic region of acetylcholine receptor in myasthenia gravis. An Fab monoclonal antibody protects against antigenic modulation by human sera - Tzartos_1985_J.Immunol_134_2343
Author(s) : Tzartos SJ , Sophianos D , Efthimiadis A
Ref : J Immunol , 134 :2343 , 1985
Abstract : Antigenic modulation of acetylcholine receptor (AChR), i.e., acceleration of its internalization and degradation rate by antibody-cross-linking, is considered to be one of the two main causes of AChR loss in myasthenia gravis (MG). The majority of the antibodies to AChR are directed to the main immunogenic region (MIR) on the alpha-subunit of the receptor. We here examine the relative contribution of the anti-MIR antibody fraction (as well as of another fraction) to the antigenic modulation caused by MG patients' sera. Fab fragments of an anti-MIR monoclonal antibody (mAb) or a mAb to the beta-subunit (neither of which causes antigenic modulation) were allowed to shield their corresponding regions on the AChR on the mouse muscle cell line BC3H1. The 27 MG sera subsequently added thus bound to all other regions except to the protected one, and the resulting antigenic modulation was measured. The anti-MIR mAb protected the AChR by 68 +/- 16%. This is interpreted as the contribution to antigenic modulation of the anti-MIR antibody fraction in the human sera. This percentage correlated very well with the occurrence of the anti-MIR antibodies in the same sera. The anti-beta mAb gave only small protection of the AChR. No significant pattern differences were observed between sexes, early and recent onset of the disease, or high and low antibody titers. It is concluded that as far as it concerns the one of the pathogenic mechanisms in MG, i.e., the antigenic modulation, the MIR seems to be the main pathogenic region. The observation that a single mAb can efficiently protect the AChR in this system may prove to be of therapeutic interest.
ESTHER : Tzartos_1985_J.Immunol_134_2343
PubMedSearch : Tzartos_1985_J.Immunol_134_2343
PubMedID: 3973387

Title : Spectrotypic analysis of antibodies to acetylcholine receptors in experimental autoimmune myasthenia gravis - Bionda_1984_Clin.Exp.Immunol_57_41
Author(s) : Bionda A , De Baets MH , Tzartos SJ , Lindstrom JM , Weigle WO , Theophilopoulos AN
Ref : Clinical & Experimental Immunology , 57 :41 , 1984
Abstract : We have studied the isoelectric focusing pattern of antibodies expressed in rats with experimental autoimmune myasthenia gravis (EAMG) induced by immunization with acetylcholine receptors (AChR) purified from Torpedo californica. Sera or tissue eluates were obtained at intervals in the course of disease and subjected to isoelectric focusing. Subsequently, the focused antibodies were detected by autoradiography of gels labelled with 125I-alpha-bungarotoxin conjugated AChR. Reverse electrofocusing was used to separate complexes of antibody and AChR formed in vivo, thereby allowing detection of the full spectrotype (banding pattern). As little as 1.1 X 10(-12) moles of monoclonal antibodies (MoAbs) to AChR yielded distinct bands of radiolabelled antigen binding by this technique. The anti-AChR MoAbs studied showed a multitude of bands localized in neutral to alkaline position. The clonotypes expressed in late post-immunization sera were compared to early sera. The spectrotypes of immunized Lewis and Brown Norway rats were not identical. In early sera most of the isoelectric focusing bands were specific for T. californica AChR, whereas in late sera further expansion of the repertoire produced bands that reacted with rat muscle AChR as well. The focused bands that bound rat AChR also bound T. californica AChR. The anti-AChR antibodies eluted from muscles of rats with EAMG showed similar binding patterns to anti-receptor antibodies in rats' sera. These results indicate that the antibody specificities detected in serum are the same specificities which are effective in binding to muscle AChR in vivo. Minor specificities of serum anti-receptor antibodies are not disproportionally represented in the antibodies actually bound at the neuromuscular junction in EAMG.
ESTHER : Bionda_1984_Clin.Exp.Immunol_57_41
PubMedSearch : Bionda_1984_Clin.Exp.Immunol_57_41
PubMedID: 6611231

Title : Structure and transmembrane nature of the acetylcholine receptor in amphibian skeletal muscle as revealed by cross-reacting monoclonal antibodies - Sargent_1984_J.Cell.Biol_98_609
Author(s) : Sargent PB , Hedges BE , Tsavaler L , Clemmons L , Tzartos SJ , Lindstrom JM
Ref : Journal of Cell Biology , 98 :609 , 1984
Abstract : A collection of 126 monoclonal antibodies (mAbs) made against acetylcholine receptors (AChRs) from the electric organs of Torpedo californica or Electrophorus electricus was tested for cross-reactivity with AChRs in cryostat sections of skeletal muscle from Rana pipiens and Xenopus laevis by indirect immunofluorescence. 49 mAbs (39%) cross-reacted with AChRs from Rana, and 25 mAbs (20%) cross-reacted with AChRs from Xenopus. mAbs specific for each of the four subunits of electric organ AChR (alpha, beta, gamma, delta) cross-reacted with AChRs from each amphibian species. mAbs cross-reacting with Xenopus AChRs were, with one exception, a subset of the mAbs cross-reacting with Rana AChRs. The major difference detected between the two species was in binding by mAbs specific for the main immunogenic region (MIR) of the alpha-subunit. Whereas 22 of 33 anti-MIR mAbs tested cross-reacted with Rana AChRs, only one of these mAbs cross-reacted with Xenopus AChRs. Some (32) of the cross-reacting mAbs were tested for binding to AChRs in intact muscle. 21 of these mAbs bound to AChRs only when membranes were made permeable with saponin. Electron microscopy using immunoperoxidase or colloidal gold techniques revealed that these mAbs recognize cytoplasmic determinants and that mAbs that do not require saponin in order to bind AChRs in intact muscle recognize extracellular determinants. These results suggest that AChRs in skeletal muscle of Rana and Xenopus are composed of subunits corresponding to the alpha-, beta-, gamma-, and delta-subunits of AChRs from fish electric organs. The subunit specificity of mAbs whose binding was examined by electron microscopy suggests that parts of each subunit (alpha, beta, gamma, delta) are exposed on the cytoplasmic surface and that, as in AChRs from fish electric organs and mammalian muscle, the MIR on alpha-subunits of Rana AChRs is exposed on the extracellular surface.
ESTHER : Sargent_1984_J.Cell.Biol_98_609
PubMedSearch : Sargent_1984_J.Cell.Biol_98_609
PubMedID: 6363425

Title : Lipid-dependent recovery of alpha-bungarotoxin and monoclonal antibody binding to the purified alpha-subunit from Torpedo marmorata acetylcholine receptor. Enhancement by noncompetitive channel blockers - Tzartos_1984_J.Biol.Chem_259_11512
Author(s) : Tzartos SJ , Changeux JP
Ref : Journal of Biological Chemistry , 259 :11512 , 1984
Abstract : Recently the purified alpha-subunit from Torpedo marmorata acetylcholine receptor was shown to bind alpha-bungarotoxin with a KD approximately 3 nM in the presence of sodium dodecyl sulfate (Tzartos, S.J., and Changeux, J.P. (1983) EMBO J. 2, 381-387). Here we describe a further significant step toward renaturation of the alpha-subunit as judged by toxin and monoclonal antibody binding. Purified T. marmorata receptor subunits were diluted with 1% lipids (asolectin) plus 0.5% Na+ cholate. An anion-exchange resin eliminated most of the detergents, leaving approximately 0.1% Na+ cholate and the lipids. After this treatment, about 20% of the alpha-subunit recovered (but not the beta-, gamma-, or delta-subunit) exhibited a high affinity for radioiodinated alpha-bungarotoxin with a KD approximately 0.5 nM. The 34,000- and 27,000-dalton proteolytic peptides of the alpha-subunit conserved this lipid-dependent toxin binding. Unlabeled alpha-toxins, hexamethonium, and carbamylcholine competed with alpha-bungarotoxin for the renatured alpha-subunit. Noncompetitive channel blockers doubled the lipid-dependent toxin-binding capacity of the alpha-subunit but had no effect on the 27,000-dalton peptide. The binding of several monoclonal antibodies to the main immunogenic region (which is particularly sensitive to denaturation) significantly increased. In particular, binding of antibody 16 changed from 1% to denatured to 100% to the lipid-renaturated alpha-subunit. The binding of these antibodies was lost with the lipid-renatured 34,000- and 27,000-dalton peptides.
ESTHER : Tzartos_1984_J.Biol.Chem_259_11512
PubMedSearch : Tzartos_1984_J.Biol.Chem_259_11512
PubMedID: 6088547

Title : Immunohistochemical localization of monoclonal antibodies to the nicotinic acetylcholine receptor in chick midbrain - Swanson_1983_Proc.Natl.Acad.Sci.U.S.A_80_4532
Author(s) : Swanson LW , Lindstrom JM , Tzartos SJ , Schmued LC , O'Leary DD , Cowan WM
Ref : Proc Natl Acad Sci U S A , 80 :4532 , 1983
Abstract : We used the indirect immunofluorescence method to determine the crossreactivity of a library of 57 monoclonal antibodies (mAbs) against each of the subunits of the nicotinic acetylcholine receptor (nAcChoR) isolated from Torpedo and Electrophorus electric organs or from fetal calf and human muscle, with specific neural elements in the midbrain of the chick. Out of 17 mAbs that recognized motor end plates on chick muscle, 14 produced a similar pattern of labeling in the midbrain: the neuronal perikarya and dendrites in the lateral spiriform nucleus (SpL) were intensely labeled, and there was moderate labeling of fibers in certain of the deeper layers of the optic tectum, which disappeared after the SpL was destroyed electrolytically. Two lines of evidence suggest that the mAbs may be crossreacting with nAcChoRs in the midbrain. First, all of the mAbs that stained the SpL also stained neuromuscular junctions in skeletal muscle, whereas none of the 40 mAbs that failed to stain end plates crossreacted with the SpL; second, in vitro immunological studies and blocking experiments on tissue sections (in which unlabeled mAbs were used to block the staining of a directly fluorescein-treated mAb) indicated the presence of mAbs specific for unique antigenic determinants on all four of the subunits (alpha, beta, gamma, and delta) from Torpedo nAcChoR in chick midbrain and muscle. On the other hand, the distribution of mAb staining in the optic tectum does not closely parallel that of either acetylcholinesterase staining or of 125I-labeled alpha-bungarotoxin binding; no toxin binding has been observed autoradiographically in the SpL, but the nucleus does contain moderately dense acetylcholinesterase staining. Take together, our observations suggest that there may be a cholinergic input to the SpL and that the projection fibers from the SpL to the optic tectum (which are also stained with an antiserum to [Leu]enkephalin) may contain presynaptic nAcChoRs. It is clear, however, that the distribution of the putative nAcChoRs, alpha-bungarotoxin binding sites, and acetylcholinesterase staining in the avian midbrain are quite different, although they do overlap to some degree in the deeper layers of the optic tectum.
ESTHER : Swanson_1983_Proc.Natl.Acad.Sci.U.S.A_80_4532
PubMedSearch : Swanson_1983_Proc.Natl.Acad.Sci.U.S.A_80_4532
PubMedID: 6192437

Title : Characterization of the mRNA for mouse muscle acetylcholine receptor alpha subunit by quantitative translation in vitro - Sebbane_1983_J.Biol.Chem_258_3294
Author(s) : Sebbane R , Clokey G , Merlie JP , Tzartos SJ , Lindstrom JM
Ref : Journal of Biological Chemistry , 258 :3294 , 1983
Abstract : The nicotinic acetylcholine receptor of mammalian skeletal muscle is a multisubunit membrane glycoprotein whose synthesis is regulated by developmental and physiological cues. We report here the identification and characterization of the primary translation product of alpha subunit mRNA. The alpha subunit synthesized in rabbit reticulocyte lysate is approximately 2000 larger in apparent molecular weight than the native alpha subunit polypeptide found in acetylcholine receptor. Evidence from peptide maps and the effect of co-translational incubation with dog pancreas microsomes suggests that the in vitro product differs in two ways from native alpha subunit: 1) it is synthesized with an NH2-terminal signal peptide which is removed in vivo, and 2) the in vitro product is not glycosylated. We have characterized the alpha subunit mRNA activity by using a quantitative the membrane-bound polysome fraction. It is poly(A+) and approximately 2000 nucleotides long. Finally, we have shown that in BC3H-1 cells, alpha subunit mRNA is regulated developmentally. We detected a 10-fold increase in the relative abundance of alpha subunit mRNA in cells which had undergone the transition from log phase growth to differentiated myoblast.
ESTHER : Sebbane_1983_J.Biol.Chem_258_3294
PubMedSearch : Sebbane_1983_J.Biol.Chem_258_3294
PubMedID: 6826561

Title : Use of monoclonal antibodies to study acetylcholine receptors from electric organs, muscle, and brain and the autoimmune response to receptor in myasthenia gravis -
Author(s) : Lindstrom JM , Tzartos SJ , Gullick W , Hochschwender S , Swanson L , Sargent P , Jacob M , Montal M
Ref : Cold Spring Harbor Symposium on Quantitative Biology , 48 Pt 1 :89 , 1983
PubMedID: 6586364

Title : Demonstration of a main immunogenic region on acetylcholine receptors from human muscle using monoclonal antibodies to human receptor - Tzartos_1983_FEBS.Lett_158_116
Author(s) : Tzartos SJ , Langeberg L , Hochschwender S , Lindstrom JM
Ref : FEBS Letters , 158 :116 , 1983
Abstract : Eleven cloned hybridomas which secrete antibodies to acetylcholine receptors from human muscle have been prepared. All of these monoclonal antibodies to have the same basic specificity as shown by competition for binding to the main immunogenic region on the receptor, but these antibodies differ in fine specificity as shown by reaction with denatured receptor subunits and interspecies cross-reaction.
ESTHER : Tzartos_1983_FEBS.Lett_158_116
PubMedSearch : Tzartos_1983_FEBS.Lett_158_116
PubMedID: 6862030

Title : Transmembrane orientation of an early biosynthetic form of acetylcholine receptor delta subunit determined by proteolytic dissection in conjunction with monoclonal antibodies - Anderson_1983_J.Neurosci_3_1773
Author(s) : Anderson DJ , Blobel G , Tzartos SJ , Gullick W , Lindstrom JM
Ref : Journal of Neuroscience , 3 :1773 , 1983
Abstract : The transmembrane topology of acetylcholine receptor (AChR) delta subunit, synthesized in vitro and co-translationally integrated into dog pancreas rough microsomal membranes, was studied using limited proteolysis and domain-specific immunoprecipitation. Forty-four kilodaltons (kd) of the 65-kd delta subunit comprise a single fragment that is inaccessible to exhaustive proteolytic digestion from the cytoplasmic surface of the membrane by trypsin, chymotrypsin, thermolysin, and pronase. Previously, we have shown that this 44-kd "protected" fragment contains the amino terminus of the intact molecule and all of the core oligosaccharides (Anderson, D.J., P. Walter, and G. Blobel (1982) J. Cell Biol. 93: 501-506). Here we demonstrate that this domain can be further dissected into a 26-kd fragment, together with low molecular weight material, when the membranes are rendered permeable to trypsin by low concentrations of deoxycholate (Kreibich, G., P. Debey, and D. D. Sabatini (1973) J. Cell Biol. 58: 436-462). This 26-kd fragment contains all of the core oligosaccharides present on the intact subunit and therefore constitutes at least part, if not all, of the extracellular domain. The remaining low molecular weight material may derive from the membrane-embedded domain; our data imply that as much as 18 kd may be internal to the lipid bilayer. On the other hand, part of the cytoplasmic pole of AChR-delta can be recovered as a discrete, 12-kd fragment upon mild trypsinization of intact vesicles. We have used this 12-kd fragment to identify anti-AChR-delta monoclonal antibodies (mAbs) that react with the cytoplasmic domain of this subunit. Partial proteolytic fragmentation of the AChR in vitro translation products, in topologically well defined rough microsomes, may be used as a general assay to characterize the domain specificity of anti-AChR mAbs. For example, in the case of AChR-beta, we were able to identify two mAbs that recognize extracellular and cytoplasmic fragments, respectively.
ESTHER : Anderson_1983_J.Neurosci_3_1773
PubMedSearch : Anderson_1983_J.Neurosci_3_1773
PubMedID: 6193254

Title : Inhibition of glycosylation with tunicamycin blocks assembly of newly synthesized acetylcholine receptor subunits in muscle cells - Merlie_1982_J.Biol.Chem_257_2694
Author(s) : Merlie JP , Sebbane R , Tzartos SJ , Lindstrom JM
Ref : Journal of Biological Chemistry , 257 :2694 , 1982
Abstract : We have characterized the oligosaccharide chains of the alpha subunit of acetylcholine receptor of the clonal mouse muscle cell line BC3H-1 by their sensitivity to end-beta-N-acetylglucosaminidase H and by comparison of the native glycosylated polypeptide with the nonglycosylated form made in tunicamycin-treated cells. These studies indicate that the native alpha subunit has a single N-asparagine-linked oligosaccharide chain of the "high mannose" or "simple" type. Furthermore, these results considered in light of our previous characterization of the alpha subunit synthesized in vitro suggest that the alpha subunit contains no "complex"-type N-linked oligosaccharide chains. We have investigated the role of glycosylation in the biogenesis of the acetylcholine receptor. Receptor biogenesis in normal cells involves the assembly of newly synthesized alpha subunits into a form active for binding alpha-bungarotoxin. This process is only 30% efficient and is complete by 30 min postsynthesis. When glycosylation is inhibited by tunicamycin, alpha subunit synthesis is inhibited only slightly but assembly into an alpha-bungarotoxin binding species is reduced dramatically.
ESTHER : Merlie_1982_J.Biol.Chem_257_2694
PubMedSearch : Merlie_1982_J.Biol.Chem_257_2694
PubMedID: 7061443

Title : Specificities of antibodies to acetylcholine receptors in sera from myasthenia gravis patients measured by monoclonal antibodies - Tzartos_1982_Proc.Natl.Acad.Sci.U.S.A_79_188
Author(s) : Tzartos SJ , Seybold ME , Lindstrom JM
Ref : Proc Natl Acad Sci U S A , 79 :188 , 1982
Abstract : The pattern of antibody specificities in sera from patients with myasthenia gravis (MG) was determined by the ability of monoclonal antibodies against defined determinants on the acetylcholine receptor molecule to inhibit binding of the serum antibodies to receptor from human muscle. We found that MG patients produce fundamentally the same pattern of specificities as that produced by animals immunized with receptor purified from fish electric organs or mammalian muscle. Most of the antibodies are directed at the "main immunogenic region' which is located on the extracellular surface of the alpha subunit and is distinct from the acetylcholine binding site. Regions on the beta and gamma subunits near the main immunogenic region are also significantly immunogenic. In one patient the proportions of antibodies to various regions are constant over time despite changes in total antibody amount and clinical state. Between patients there is no obvious correlation between antibody specificities and clinical state. These data suggest that the autoimmune response in MG is stimulated by human receptor rather than a crossreacting (e.g., viral) antigen and that in both MG and experimental autoimmune MG the pattern of specificities produced is determined by the inherently immunogenic structural features of the receptor molecule. They also suggest that the wide differences in clinical state sometimes observed between patients with the same total concentration of antireceptor antibody are due primarily to differences in endogenous factors which affect the safety factor for neuromuscular transmission rather than to the presence of especially pathogenic antireceptor specificities.
ESTHER : Tzartos_1982_Proc.Natl.Acad.Sci.U.S.A_79_188
PubMedSearch : Tzartos_1982_Proc.Natl.Acad.Sci.U.S.A_79_188
PubMedID: 6948300

Title : Production and assay of antibodies to acetylcholine receptors -
Author(s) : Lindstrom JM , Einarson B , Tzartos SJ
Ref : Methods Enzymol , 74 Pt C :432 , 1981
PubMedID: 7321891

Title : Structure and function of the acetylcholine receptor molecule studied using monoclonal antibodies -
Author(s) : Lindstrom JM , Tzartos SJ , Gullick W
Ref : Annals of the New York Academy of Sciences , 377 :1 , 1981
PubMedID: 6176164

Title : Monoclonal antibodies as probes of acetylcholine receptor structure. 2. Binding to native receptor - Conti-Tronconi_1981_Biochemistry_20_2181
Author(s) : Conti-Tronconi B , Tzartos SJ , Lindstrom JM
Ref : Biochemistry , 20 :2181 , 1981
Abstract : Binding of monoclonal antibodies top Torpedo californica acetylcholine receptor monomers solubilized in Triton X-100 was studied by centrifugation on sucrose gradients. Antibodies to alpha subunits were of two types. One type formed complexes of one antibody and one receptor monomer, independent of antibody/receptor ratio. We conclude that the binding sites for these antibodies are oriented on the two alpha subunits per monomer in such a way that each could be bound by one of the two binding sites of a single immunoglobulin molecule. Most antibodies were of this type. The other type of monoclonal antibody formed complexes of several sizes, including antibody cross-linked receptors, depending on the ratio of antibody to receptor. We conclude that the binding sites for these antibodies are oriented in such a way that the two alpha subunits per monomer could not be cross-linked by a single antibody molecule. A monoclonal antibody of this type raised against Electrophorus electricus receptors was used to show that this receptor also has two alpha subunits per monomer. This antibody cross-reacted with receptor from fetal calf muscle and was able to induce modulation of receptor in muscle cells in culture. This suggests that muscle receptor also has two alpha subunits and that the antibody can cross-link receptor in the plane of the membrane, as it does in solution, and thereby form complexes which enhance endocytosis and increase the rate of receptor destruction. The rate of antigenic modulation decreases at high antibody/receptor ratios, as expected if un-cross-linked complexes of two antibodies and one receptor were not destroyed at a faster rate. Antibodies which cross-link alpha subunits within a receptor monomer are frequent but would not be expected to be able to induce antigenic modulation. This provides one mechanism by which antisera of equivalent antireceptor titer might differ in their ability to induce antigenic modulation. An antibody which binds to denatured delta and gamma subunits forms complexes of only one antibody and one receptor monomer, independent of antibody ratio, as do antibodies thought to cross-link the two alpha subunits in a monomer. It apparently cross-links delta and gamma subunits within the monomer. Some of the monoclonal antibodies to alpha subunits can bind simultaneously to receptor, while the binding of others is mutually exclusive.
ESTHER : Conti-Tronconi_1981_Biochemistry_20_2181
PubMedSearch : Conti-Tronconi_1981_Biochemistry_20_2181
PubMedID: 6786327

Title : Monoclonal antibodies as probes of acetylcholine receptor structure. 1. Peptide mapping - Gullick_1981_Biochemistry_20_2173
Author(s) : Gullick WJ , Tzartos SJ , Lindstrom JM
Ref : Biochemistry , 20 :2173 , 1981
Abstract : The isolated subunits of the acetylocholine receptor from Torpedo californica were digested with proteolytic enzymes, and the resulting polypeptide fragments were analyzed by gel electrophoresis. We have identified those fragments which contain carbohydrate and those from the alpha subunit which are labelled with the acetylcholine binding site specific reagent [4-(N-maleimido)benzyl]tri[3H]methylammonium iodide. We have tested several monoclonal antibodies raised to the acetylcholine receptor from torpedo, some of which react with the denatured subunits [Tzartos, S.J., & Lindstrom, J.M. (1980) Proc. Natl. Acad. Sci. U.S.A.77, 755; Tzartos, S.J., & Lindstrom, J.M. (1981) in Monoclonal antibodies in Endocrine Research (Fellows, R., & Eisenbarth, G., Eds.) Raven Press (in press)]. The binding specificities of these antibodies to radioiodinated proteolytically generated fragments of the alpha subunit were determined by immunoprecipitation followed by gel electrophoresis. The antibodies tested fell into at least three main groups on the basis of their binding specificities. These antibodies were also tested for their capacity to bind to acetylcholine receptor solubilized in Triton X-100, sodium cholate, or sodium cholate supplemented with exogenous lipids. A monoclonal antibody raised to the denatured delta subunit, was tested for its ability to select radioiodinated proteolytic fragments of these subunits. These molecules provide probes for many sites on the acetylcholine receptor with affinities and specificities comparable to alpha-neurotoxins.
ESTHER : Gullick_1981_Biochemistry_20_2173
PubMedSearch : Gullick_1981_Biochemistry_20_2173
PubMedID: 6786326

Title : Mapping of surface structures of electrophorus acetylcholine receptor using monoclonal antibodies - Tzartos_1981_J.Biol.Chem_256_8635
Author(s) : Tzartos SJ , Rand DE , Einarson BL , Lindstrom JM
Ref : Journal of Biological Chemistry , 256 :8635 , 1981
Abstract : Forty monoclonal antibodies to acetylcholine receptor from the electric organs of Electrophorus electricus have been characterized by immunoglobulin isotype, affinity for receptor, and specificity for species, subunit, and determinants within subunits. Using these antibodies, nine immunogenic regions on the receptor molecule were distinguished. Most of these are species specific, and are located on various subunits of the acetylcholine receptor. The least species-specific region forms the "main immunogenic region" (MIR). Most monoclonal antibodies and most antibodies in conventional antisera are directed at this region. The MIR is located on the extracellular surface of the alpha subunits and is homologous to the MIR which we previously described on Torpedo californica receptor. An homologous MIR is also a characteristic feature of receptor from mammalian muscle. The possible immunological and structural significance of the MIR is discussed.
ESTHER : Tzartos_1981_J.Biol.Chem_256_8635
PubMedSearch : Tzartos_1981_J.Biol.Chem_256_8635
PubMedID: 6167581

Title : Acetylcholine receptors from Torpedo and Electrophorus have similar subunit structures - Lindstrom_1980_Biochemistry_19_1454
Author(s) : Lindstrom JM , Cooper J , Tzartos SJ
Ref : Biochemistry , 19 :1454 , 1980
Abstract : Previously, acetylcholine receptor purified from the electric organs of electric eels (Electrophorus electricus) and electric rays (Torpedo californica) (torpedo) had appeared to differ in subunit structure. Receptor from torpedo has the subunit structure alpha 2 beta gamma delta, but subunits corresponding only to alpha, beta, and gamma had been observed in receptor from eel. Here we report that if membrane fragments of eel electric organ are prepared and detergent extracted in the presence of iodoacetamide, then receptor purified from the extract contains a fourth subunit. Using monoclonal antibodies as well as conventional antisera, we show that the newly recognized subunit of receptor from eel corresponds to the delta subunit of torpedo. A monoclonal antibody to the delta subunit of torpedo cross-reacts with the gamma subunit and shows a similar cross-reaction between the delta' and gamma' subunits of receptor from eel, indicating the presence of an unexpected structural similarity. Although the function of the beta, gamma, and delta subunits remains unknown, these results support the concept that receptors from the electric organs of several species and probably also from muscle share a similarly complex subunit structure.
ESTHER : Lindstrom_1980_Biochemistry_19_1454
PubMedSearch : Lindstrom_1980_Biochemistry_19_1454
PubMedID: 7388004

Title : Monoclonal antibodies used to probe acetylcholine receptor structure: localization of the main immunogenic region and detection of similarities between subunits - Tzartos_1980_Proc.Natl.Acad.Sci.U.S.A_77_755
Author(s) : Tzartos SJ , Lindstrom JM
Ref : Proc Natl Acad Sci U S A , 77 :755 , 1980
Abstract : Seventeen cell lines producing monoclonal antibodies against Torpedo californica (torpedo) acetylcholine receptor (AcChoR) and its subunits were established. By using these antibodies as probes, we identified: (i) a similar antigenic determinant on alpha and beta torpedo subunits, (ii) a similar antigenic determinant on gamma and delta subunits, (iii) antigenic determinants unique for alpha or beta torpedo AcChoR subunits, (iv) a small region on the alpha subunit that dominates the immunogenicity of native torpedo AcChoR in rats (a monoclonal antibody directed at this region could bind to rat AcChoR in vivo and cause passive experimental autoimmune myasthenia gravis), and (v) antigenic determinants on torpedo subunits recognized in AcChoR from other species. The unexpected similarities between alpha and beta and between gamma and delta subunits raise the possibility that the complex four-subunit structure of AcChoR was derived from a simpler precursor and suggests that these antigenic similarities might reflect some structural and functional homologies.
ESTHER : Tzartos_1980_Proc.Natl.Acad.Sci.U.S.A_77_755
PubMedSearch : Tzartos_1980_Proc.Natl.Acad.Sci.U.S.A_77_755
PubMedID: 6153804