Gene_locus Report for: trybr-PEPTB

Whole-genome sequencing of the protozoan pathogen Trypanosoma cruzi revealed that the diploid genome contains a predicted 22,570 proteins encoded by genes, of which 12,570 represent allelic pairs. Over 50% of the genome consists of repeated sequences, such as retrotransposons and genes for large families of surface molecules, which include trans-sialidases, mucins, gp63s, and a large novel family (>1300 copies) of mucin-associated surface protein (MASP) genes. Analyses of the T. cruzi, T. brucei, and Leishmania major (Tritryp) genomes imply differences from other eukaryotes in DNA repair and initiation of replication and reflect their unusual mitochondrial DNA. Although the Tritryp lack several classes of signaling molecules, their kinomes contain a large and diverse set of protein kinases and phosphatases; their size and diversity imply previously unknown interactions and regulatory processes, which may be targets for intervention.

Serine oligopeptidases of trypanosomatids are emerging as important virulence factors and therapeutic targets in trypanosome infections. We report here the isolation and characterization of oligopeptidase B (OpdB) and its corresponding gene from Trypanosoma evansi, a pathogen of significant veterinary importance. The T. evansi opdB gene was present as a single copy per haploid genome containing an open reading frame of 2148 bp encoding a protein of 80.664 kDa. Purified OpdB hydrolyzed substrates with basic residues in P1 (k(cat)/K(m) for carbobenzyloxy-L-arginyl-L-arginyl-7-amido-4-methylcoumarin, 337 s(-1) x microm(-1)) and exhibited potent arginyl carboxypeptidase activity (k(cat)/K(m) for Val-Lys-Arg Arg-OH, 231 s(-1) x mM(-1)). While not secreted, T. evansi released OpdB into the plasma of infected hosts where it retained catalytic activity. Plasma OpdB levels correlated with blood parasitemia. In vitro, OpdB cleaved the peptide hormone atrial natriuretic factor (ANF) at four sites: Arg3 Arg4, Arg4 Ser5, Arg11 Ile12, and Arg27 Tyr28, thereby abrogating smooth muscle relaxant and prohypotensive properties of ANF. Circulating plasma ANF levels in T. evansi-infected rats were depressed from 130 to 8 pg x ml(-1), and plasma ANF levels inversely correlated with plasma OpdB activity. The in vitro half-life of ANF in rat plasma was reduced 300-fold in plasma from T. evansi-infected rodents, which contains high levels of OpdB activity. Addition of OpdB inhibitors to cell-free plasma from infected rodents significantly abrogated this ANF hydrolysis. Furthermore the in vivo ANF half-life was reduced 5-fold in T. evansi-infected rats. Thus, we propose a role for OpdB in peptide hormone dysregulation in trypanosomiasis, specifically in generating the depressed plasma levels of ANF in mammals infected with T. evansi.

Trypanosoma brucei contains a soluble serine oligopeptidase (OP-Tb) that is released into the host bloodstream during infection, where it has been postulated to participate in the pathogenesis of African trypanosomiasis. Here, we report the identification of a single copy gene encoding the T. brucei oligopeptidase and a homologue from the related trypanosomatid pathogen Leishmania major. The enzymes encoded by these genes belong to an emerging subgroup of the prolyl oligopeptidase family of serine hydrolases, referred to as oligopeptidase B. The trypanosomatid oligopeptidases share 70% amino acid sequence identity with oligopeptidase B from the intracellular pathogen Trypanosoma cruzi, which has a demonstrated role in mammalian host cell signaling and invasion. OP-Tb exhibited no activity toward the prolyl oligopeptidase substrate H-Gly-Pro-7-amido-4-methylcoumarin. Instead, it had activity toward substrates of trypsin-like enzymes, particularly those that have basic amino acids in both P(1) and P(2) (e.g. benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin k(cat)/K(m) = 529 s(-1) microM(-1)). The activity of OP-Tb was enhanced by reducing agents and by polyamines, suggesting that these agents may act as in vivo regulators of OP-Tb activity. This study provides the basis of the characterization of a novel subgroup of serine oligopeptidases from kinetoplastid protozoa with potential roles in pathogenesis.

Oligopeptidase B cleaves after basic amino acids in peptides up to 30 residues. As a virulence factor in bacteria and trypanosomatid pathogens that is absent in higher eukaryotes, this is a promising drug target. Here we present ligand-free open state and inhibitor-bound closed state crystal structures of oligopeptidase B from Trypanosoma brucei, the causative agent of African sleeping sickness. These (and related) structures show the importance of structural dynamics, governed by a fine enthalpic and entropic balance, in substrate size selectivity and catalysis. Peptides over 30 residues cannot fit the enzyme cavity, preventing the complete domain closure required for a key propeller Asp/Glu to fix the catalytic His and Arg in the catalytically competent conformation. This size exclusion mechanism protects larger peptides and proteins from degradation. Similar bacterial prolyl endopeptidase and archael acylaminoacyl peptidase structures demonstrate this mechanism is conserved among oligopeptidase family enzymes across all three domains of life.

BACKGROUND: Trypanosoma brucei gambiense is the causative agent of chronic Human African Trypanosomiasis or sleeping sickness, a disease endemic across often poor and rural areas of Western and Central Africa. We have previously published the genome sequence of a T. b. brucei isolate, and have now employed a comparative genomics approach to understand the scale of genomic variation between T. b. gambiense and the reference genome. We sought to identify features that were uniquely associated with T. b. gambiense and its ability to infect humans. METHODS AND FINDINGS: An improved high-quality draft genome sequence for the group 1 T. b. gambiense DAL 972 isolate was produced using a whole-genome shotgun strategy. Comparison with T. b. brucei showed that sequence identity averages 99.2% in coding regions, and gene order is largely collinear. However, variation associated with segmental duplications and tandem gene arrays suggests some reduction of functional repertoire in T. b. gambiense DAL 972. A comparison of the variant surface glycoproteins (VSG) in T. b. brucei with all T. b. gambiense sequence reads showed that the essential structural repertoire of VSG domains is conserved across T. brucei.
CONCLUSIONS: This study provides the first estimate of intraspecific genomic variation within T. brucei, and so has important consequences for future population genomics studies. We have shown that the T. b. gambiense genome corresponds closely with the reference, which should therefore be an effective scaffold for any T. brucei genome sequence data. As VSG repertoire is also well conserved, it may be feasible to describe the total diversity of variant antigens. While we describe several as yet uncharacterized gene families with predicted cell surface roles that were expanded in number in T. b. brucei, no T. b. gambiense-specific gene was identified outside of the subtelomeres that could explain the ability to infect humans.

African trypanosomes cause human sleeping sickness and livestock trypanosomiasis in sub-Saharan Africa. We present the sequence and analysis of the 11 megabase-sized chromosomes of Trypanosoma brucei. The 26-megabase genome contains 9068 predicted genes, including approximately 900 pseudogenes and approximately 1700 T. brucei-specific genes. Large subtelomeric arrays contain an archive of 806 variant surface glycoprotein (VSG) genes used by the parasite to evade the mammalian immune system. Most VSG genes are pseudogenes, which may be used to generate expressed mosaic genes by ectopic recombination. Comparisons of the cytoskeleton and endocytic trafficking systems with those of humans and other eukaryotic organisms reveal major differences. A comparison of metabolic pathways encoded by the genomes of T. brucei, T. cruzi, and Leishmania major reveals the least overall metabolic capability in T. brucei and the greatest in L. major. Horizontal transfer of genes of bacterial origin has contributed to some of the metabolic differences in these parasites, and a number of novel potential drug targets have been identified.

Whole-genome sequencing of the protozoan pathogen Trypanosoma cruzi revealed that the diploid genome contains a predicted 22,570 proteins encoded by genes, of which 12,570 represent allelic pairs. Over 50% of the genome consists of repeated sequences, such as retrotransposons and genes for large families of surface molecules, which include trans-sialidases, mucins, gp63s, and a large novel family (>1300 copies) of mucin-associated surface protein (MASP) genes. Analyses of the T. cruzi, T. brucei, and Leishmania major (Tritryp) genomes imply differences from other eukaryotes in DNA repair and initiation of replication and reflect their unusual mitochondrial DNA. Although the Tritryp lack several classes of signaling molecules, their kinomes contain a large and diverse set of protein kinases and phosphatases; their size and diversity imply previously unknown interactions and regulatory processes, which may be targets for intervention.

Serine oligopeptidases of trypanosomatids are emerging as important virulence factors and therapeutic targets in trypanosome infections. We report here the isolation and characterization of oligopeptidase B (OpdB) and its corresponding gene from Trypanosoma evansi, a pathogen of significant veterinary importance. The T. evansi opdB gene was present as a single copy per haploid genome containing an open reading frame of 2148 bp encoding a protein of 80.664 kDa. Purified OpdB hydrolyzed substrates with basic residues in P1 (k(cat)/K(m) for carbobenzyloxy-L-arginyl-L-arginyl-7-amido-4-methylcoumarin, 337 s(-1) x microm(-1)) and exhibited potent arginyl carboxypeptidase activity (k(cat)/K(m) for Val-Lys-Arg Arg-OH, 231 s(-1) x mM(-1)). While not secreted, T. evansi released OpdB into the plasma of infected hosts where it retained catalytic activity. Plasma OpdB levels correlated with blood parasitemia. In vitro, OpdB cleaved the peptide hormone atrial natriuretic factor (ANF) at four sites: Arg3 Arg4, Arg4 Ser5, Arg11 Ile12, and Arg27 Tyr28, thereby abrogating smooth muscle relaxant and prohypotensive properties of ANF. Circulating plasma ANF levels in T. evansi-infected rats were depressed from 130 to 8 pg x ml(-1), and plasma ANF levels inversely correlated with plasma OpdB activity. The in vitro half-life of ANF in rat plasma was reduced 300-fold in plasma from T. evansi-infected rodents, which contains high levels of OpdB activity. Addition of OpdB inhibitors to cell-free plasma from infected rodents significantly abrogated this ANF hydrolysis. Furthermore the in vivo ANF half-life was reduced 5-fold in T. evansi-infected rats. Thus, we propose a role for OpdB in peptide hormone dysregulation in trypanosomiasis, specifically in generating the depressed plasma levels of ANF in mammals infected with T. evansi.

Trypanosoma brucei contains a soluble serine oligopeptidase (OP-Tb) that is released into the host bloodstream during infection, where it has been postulated to participate in the pathogenesis of African trypanosomiasis. Here, we report the identification of a single copy gene encoding the T. brucei oligopeptidase and a homologue from the related trypanosomatid pathogen Leishmania major. The enzymes encoded by these genes belong to an emerging subgroup of the prolyl oligopeptidase family of serine hydrolases, referred to as oligopeptidase B. The trypanosomatid oligopeptidases share 70% amino acid sequence identity with oligopeptidase B from the intracellular pathogen Trypanosoma cruzi, which has a demonstrated role in mammalian host cell signaling and invasion. OP-Tb exhibited no activity toward the prolyl oligopeptidase substrate H-Gly-Pro-7-amido-4-methylcoumarin. Instead, it had activity toward substrates of trypsin-like enzymes, particularly those that have basic amino acids in both P(1) and P(2) (e.g. benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin k(cat)/K(m) = 529 s(-1) microM(-1)). The activity of OP-Tb was enhanced by reducing agents and by polyamines, suggesting that these agents may act as in vivo regulators of OP-Tb activity. This study provides the basis of the characterization of a novel subgroup of serine oligopeptidases from kinetoplastid protozoa with potential roles in pathogenesis.