Biochemical analysis of mutations in palmitoyl-protein thioesterase causing infantile and late-onset forms of neuronal ceroid lipofuscinosis

Deficiency in a recently characterized lysosomal enzyme, palmitoyl-protein thioesterase (PPT), leads to a severe neurodegenerative disorder of childre n, infantile neuronal ceroid lipofuscinosis (NCL). Over 36 different mutati ons in the PPT gene have been described, and missense mutations have been i nterpreted in the light of the recently solved X-ray crystallographic struc ture of PPT. In the current study, we assessed the biochemical impact of mu tations through the study of cells derived from patients and from the expre ssion of recombinant PPT enzymes in COS and Sf9 cells. All missense mutatio ns associated with infantile NCL showed no residual enzyme activity, wherea s mutations associated with late-onset phenotypes showed up to 2.15% residu al activity. Two mutations increased the K-m of the enzyme for palmitoylate d substrates and were located in positions that would distort the palmitate -binding pocket. An initiator methionine mutation (ATG-->ATA) in two late-o nset patients was expressed at a significant level in COS cells, suggesting that the ATA codon may be utilized to a clinically important extent in viv o. The most common PPT nonsense mutation, R151X, was associated with an abs ence of PPT mRNA. Mannose 6-phosphate modification of wild-type and mutant PPT enzymes was grossly normal at the level of the phosphotransferase react ion. However, mutant PPT enzymes did not bind to mannose 6-phosphate recept ors in a blotting assay. This observation was related to the failure of the mutant expressed enzymes to gain access to 'uncovering enzyme' (N-acetylgl ucosamine-1-phosphodiester alpha -N-acetyl glucosaminidase), presumably due to a block in transit out of the endoplasmic reticulum, where mutant enzym es are degraded.