Purification and characterization of bovine brain lysosomal pepstatin-insensitive proteinase, the gene product deficient in the human late-infantile neuronal ceroid lipofuscinosis

A lysosomal pepstatin-insensitive proteinase (CLN2p) deficiency is the unde rlying defect in the classical late-infantile neuronal ceroid lipofuscinosi s (LINCL, CLN2). The natural substrates for CLN2p and the causative factors for the neurodegeneration in this disorder are still not understood. We ha ve now purified the CLN2p from bovine brain to apparent homogeneity, The pr oteinase has a molecular mass of 46 kDa and an aminoterminal sequence, L-H- L-G-V-T-P-S-V-I-R-K, that is identical to the human enzyme, Peptide: N-glyc osidase F and endoglycosidase H treatment of the CLN2p reduced its molecula r mass to 39.5 and 40.5 kDa, respectively, suggesting the presence of as ma ny as five N-glycosylated residues. The CLN2p activity was not affected by common protease inhibitors, and thiol reagents, metal chelators, and divale nt metal ions had no significant effect on the proteolytic activity of the CLN2p, Among the naturally occurring neuropeptides, angiotensin II, substan ce P, and beta-amyloid were substrates for the CLN2p, whereas angiotensin I , Leu-enkephalin, and gamma-endorphin were not. Peptide cleavage sites indi cated that the CLN2p is a tripeptidyl peptidase that cleaves peptides havin g free amino-termini, Synthetic amino- and carboxyl-terminal peptides from the subunit c sequence, which is the major storage material in LINCL, are h ydrolyzed by the CLN2p, suggesting that the subunit c may be one of the nat ural substrates for this proteinase and its accumulation in LINCL is the di rect result of the proteinase deficiency.