The human CLN2 protein/tripeptidyl-peptidase I is a serine protease that autoactivates at acidic pH

The CLN2 gene mutated in the fatal hereditary neurodegenerative disease lat e infantile neuronal ceroid lipofuscinosis encodes a lysosomal protease wit h tripeptidyl-peptidase I activity. To understand the enzymological propert ies of the protein, we purified and characterized C-terminal hexahistidine- tagged human CLN2p/tripeptidyl-peptidase I produced from insect cells trans fected with a baculovirus vector. The N terminus of the secreted 66-kDa pro tein corresponds to residue 20 of the primary CLN2 gene translation product , indicating removal of a 19-pesidue signal peptide. The purified protein i s enzymatically inactive; however, upon acidification, it is proteolyticall y processed and concomitantly acquires enzymatic activity. The N terminus o f the final 46-kDa processed form (Leu(196)) corresponds to that of mature CLN2p/tripeptidyl-peptidase I purified from human brain. The activity of th e mature enzyme is irreversibly inhibited by the serine esterase inhibitor diisopropyl fluorophosphate, which specifically and stoichiometrically reac ts with CLN2p/tripeptidyl-peptidase I at Ser(475), demonstrating that this residue represents the active site nucleophile, Expression of wild type and mutant proteins in CHO cells indicate that Ser(475), Asp(360), Asp(517), b ut not His(236) are essential for activity. These data indicate that the CL N2 gene product is synthesized as an inactive proenzyme that is autocatalyt ically converted to an active serine protease.