Title: Biochemical analysis of mutations in palmitoyl-protein thioesterase causing infantile and late-onset forms of neuronal ceroid lipofuscinosis Das AK, Lu JY, Hofmann SL Ref: Hum Mol Genet, 10:1431, 2001 : PubMed
Deficiency in a recently characterized lysosomal enzyme, palmitoyl-protein thioesterase (PPT), leads to a severe neurodegenerative disorder of children, infantile neuronal ceroid lipofuscinosis (NCL). Over 36 different mutations in the PPT gene have been described, and missense mutations have been interpreted in the light of the recently solved X-ray crystallographic structure of PPT. In the current study, we assessed the biochemical impact of mutations through the study of cells derived from patients and from the expression of recombinant PPT enzymes in COS and Sf9 cells. All missense mutations associated with infantile NCL showed no residual enzyme activity, whereas mutations associated with late-onset phenotypes showed up to 2.15% residual activity. Two mutations increased the K(m) of the enzyme for palmitoylated substrates and were located in positions that would distort the palmitate-binding pocket. An initiator methionine mutation (ATG-->ATA) in two late-onset patients was expressed at a significant level in COS cells, suggesting that the ATA codon may be utilized to a clinically important extent in vivo. The most common PPT nonsense mutation, R151X, was associated with an absence of PPT mRNA. Mannose 6-phosphate modification of wild-type and mutant PPT enzymes was grossly normal at the level of the phosphotransferase reaction. However, mutant PPT enzymes did not bind to mannose 6-phosphate receptors in a blotting assay. This observation was related to the failure of the mutant expressed enzymes to gain access to 'uncovering enzyme' (N-acetylglucosamine-1-phosphodiester alpha-N-acetyl glucosaminidase), presumably due to a block in transit out of the endoplasmic reticulum, where mutant enzymes are degraded.
Title: The crystal structure of palmitoyl protein thioesterase 1 and the molecular basis of infantile neuronal ceroid lipofuscinosis Bellizzi JJ, 3rd, Widom J, Kemp C, Lu JY, Das AK, Hofmann SL, Clardy J Ref: Proc Natl Acad Sci U S A, 97:4573, 2000 : PubMed
Mutations in palmitoyl-protein thioesterase 1 (PPT1), a lysosomal enzyme that removes fatty acyl groups from cysteine residues in modified proteins, cause the fatal inherited neurodegenerative disorder infantile neuronal ceroid lipofuscinosis. The accumulation of undigested substrates leads to the formation of neuronal storage bodies that are associated with the clinical symptoms. Less severe forms of PPT1 deficiency have been found recently that are caused by a distinct set of PPT1 mutations, some of which retain a small amount of thioesterase activity. We have determined the crystal structure of PPT1 with and without bound palmitate by using multiwavelength anomalous diffraction phasing. The structure reveals an alpha/beta-hydrolase fold with a catalytic triad composed of Ser115-His289-Asp233 and provides insights into the structural basis for the phenotypes associated with PPT1 mutations.
Title: Structural basis for the insensitivity of a serine enzyme (palmitoyl-protein thioesterase) to phenylmethylsulfonyl fluoride Das AK, Bellizzi JJ, 3rd, Tandel S, Biehl E, Clardy J, Hofmann SL Ref: Journal of Biological Chemistry, 275:23847, 2000 : PubMed
Palmitoyl-protein thioesterase-1 (PPT1) is a newly described lysosomal enzyme that hydrolyzes long chain fatty acids from lipid-modified cysteine residues in proteins. Deficiency in this enzyme results in a severe neurodegenerative storage disorder, infantile neuronal ceroid lipofuscinosis. Although the primary structure of PPT1 contains a serine lipase consensus sequence, the enzyme is insensitive to commonly used serine-modifying reagents phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate. In the current paper, we show that the active site serine in PPT1 is modified by a substrate analog of PMSF, hexadecylsulfonylfluoride (HDSF) in a specific and site-directed manner. The apparent K(i) of the inhibition was 125 micrometer (in the presence of 1.5 mm Triton X-100), and the catalytic rate constant for sulfonylation (k(2)) was 3.3/min, a value similar to previously described sulfonylation reactions. PPT1 was crystallized after inactivation with HDSF, and the structure of the inactive form was determined to 2.4 A resolution. The hexadecylsulfonyl was found to modify serine 115 and to snake through a narrow hydrophobic channel that would not accommodate an aromatic sulfonyl fluoride. Therefore, the geometry of the active site accounts for the reactivity of PPT1 with HDSF but not PMSF. These observations suggest a structural explanation as to why certain serine lipases are resistant to modification by commonly used serine-modifying reagents.
Title: Identification of three novel mutations of the palmitoyl-protein thioesterase-1 (PPT1) gene in children with neuronal ceroid-lipofuscinosis Waliany S, Das AK, Gaben A, Wisniewski KE, Hofmann SL Ref: Hum Mutat, 15:206, 2000 : PubMed
Eight unrelated children with progressive neurological deterioration and granular osmiophilic deposits (GROD) due to an underlying palmitoyl-protein thioesterase deficiency were analyzed for mutations in the PPT1 gene. Three novel mutations (G118D, Q291X and F84del) were identified. The novel Q291X mutation was observed in an African-American child. The G118D and Q291X mutations occurred in infantile-onset subjects. These two mutations would be predicted to have severe effects on enzyme activity. The novel F84del mutation involves an invariant phenylalanine residue. A missense mutation, Q177E, occurred in three subjects from two families with late-infantile NCL, confirming an association of the Q177E mutation with a late-infantile phenotype. Other previously described mutations were R151X (5/16 alleles), T75P (3/16 alleles), R164X (1/16 alleles), and V181M (1/16 alleles). The current study expands the spectrum of mutations in PPT1 deficiency and further confirms the broad range of age of onset of symptoms resulting from an enzyme deficiency previously associated only with infantile NCL.
Mutations in a newly described lysosomal enzyme, palmitoyl-protein thioesterase (PPT), were recently shown to be responsible for an autosomal recessive neurological disorder prevalent in Finland, infantile neuronal ceroid lipofuscinosis. The disease results in blindness, motor and cognitive deterioration, and seizures. Characteristic inclusion bodies (granular osmiophilic deposits [GROD]) are found in the brain and other tissues. The vast majority of Finnish cases are homozygous for a missense mutation (R122W) that severely affects PPT enzyme activity, and the clinical course in Finnish children is uniformly rapidly progressive and fatal. To define the clinical, biochemical, and molecular genetic characteristics of subjects with PPT deficiency in a broader population, we collected blood samples from U.S. and Canadian subjects representing 32 unrelated families with neuronal ceroid lipofuscinosis who had GROD documented morphologically. We measured PPT activity and screened the coding region of the PPT gene for mutations. In 29 of the families, PPT deficiency was found to be responsible for the neurodegenerative disorder, and mutations were identified in 57 out of 58 PPT alleles. One nonsense mutation (R151X) accounted for 40% of the alleles and was associated with severe disease in the homozygous state. A second mutation (T75P) accounted for 13% of the alleles and was associated with a late onset and protracted clinical course. A total of 19 different mutations were found, resulting in a broader spectrum of clinical presentations than previously seen in the Finnish population. Symptoms first appeared at ages ranging from 3 mo to 9 yr, and about half of the subjects have survived into the second or even third decades of life.
