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.