Biochemical characterization of a lysosomal protease deficient in classical late infantile neuronal ceroid lipofuscinosis (LINCL) and development of an enzyme-based assay for diagnosis and exclusion of LINCL in human specimens and animal models

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL), a progress ive and fatal neurodegenerative disease of childhood, results from mutation s in a gene (CLN2) that encodes a protein with significant sequence similar ity to prokaryotic pepstatin-insensitive acid proteases, We have developed a sensitive protease activity assay that allows biochemical characterizatio n of the CLN2 gene product in various human biological samples, including s olid tissues (brain and chorionic villi), blood (buffy coat leukocytes, pla telets, granulocytes, and mononuclear cells), and cultured cells (lymphobla sts, fibroblasts, and amniocytes), The enzyme has a pH optimum of 3.5 and i s rapidly inactivated at neutral pH, A survey of fibroblasts and lymphoblas ts demonstrated that lack of activity was associated with LINCL arising fro m mutations in the CLN2 gene but not other neuronal ceroid lipofuscinoses ( NCLs), including the CLN6 variant LINCL, classical infantile NCL, classical juvenile NCL, and adult NCL (Kufs' disease), A study conducted using blood samples collected from classical LINCL families whose affliction was confi rmed by genetic analysis indicates that the assay can distinguish homozygot es, heterozygotes, and normal controls and thus is useful for diagnosis and carrier testing, Analysis of archival specimens indicates that several spe cimens previously classified as LINCL have enzyme activity and thus disease is unlikely to arise from mutations in CLN2, Conversely, a specimen previo usly classified as juvenile NCL lacks pepinase activity and is associated w ith mutations in CLN2, In addition, several animals with NCL-like neurodege nerative symptoms [mutant strains of mice (nclf and mnd), English setter, b order collie, and Tibetan terrier dogs, sheep, and cattle] were found to co ntain enzyme activity and are thus unlikely to represent models for classic al LINCL, Subcellular fractionation experiments indicate that the CLN2 prot ein is located in lysosomes, which is consistent with its acidic pH optimum for activity and the presence of mannose 6-phosphate, Taken together, thes e findings indicate that LINCL represents a lysosomal storage disorder that is characterized by the absence of a specific protease activity.