title: The human CLN2 protein/tripeptidyl-peptidase I is a serine protease that autoactivates at acidic pH.
abstract: The CLN2 gene mutated in the fatal hereditary neurodegenerative disease late infantile neuronal ceroid lipofuscinosis encodes a lysosomal protease with tripeptidyl-peptidase I activity. To understand the enzymological properties of the protein, we purified and characterized C-terminal hexahistidine-tagged human CLN2p/tripeptidyl-peptidase I produced from insect cells transfected with a baculovirus vector. The N terminus of the secreted 66-kDa protein corresponds to residue 20 of the primary CLN2 gene translation product, indicating removal of a 19-residue signal peptide. The purified protein is enzymatically inactive; however, upon acidification, it is proteolytically processed and concomitantly acquires enzymatic activity. The N terminus of the final 46-kDa processed form (Leu196) corresponds to that of mature CLN2p/tripeptidyl-peptidase I purified from human brain. The activity of the mature enzyme is irreversibly inhibited by the serine esterase inhibitor diisopropyl fluorophosphate, which specifically and stoichiometrically reacts with CLN2p/tripeptidyl-peptidase I at Ser475, demonstrating that this residue represents the active site nucleophile. Expression of wild type and mutant proteins in CHO cells indicate that Ser475, Asp360, Asp517, but not His236 are essential for activity. These data indicate that the CLN2 gene product is synthesized as an inactive proenzyme that is autocatalytically converted to an active serine protease.
authors.affiliation: Graduate Program in Cell and Developmental Biology, Rutgers University, Piscataway, New Jersey 08854, USA.
authors.forename: L
authors.initials: L
authors.lastname: Lin
authors.forename: I
authors.initials: I
authors.lastname: Sohar
authors.forename: H
authors.initials: H
authors.lastname: Lackland
authors.forename: P
authors.initials: P
authors.lastname: Lobel
mesh_terms: D000143:Acids; D000595:Amino Acid Sequence; D000626:Aminopeptidases; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002384:Catalysis; D006224:Cricetinae; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D010450:Endopeptidases; D004789:Enzyme Activation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D057057:Serine Proteases; D000091346:Tripeptidyl-Peptidase 1
chemical_list: D000143:Acids; D000091346:Tripeptidyl-Peptidase 1; D010450:Endopeptidases; D010447:Peptide Hydrolases; D057057:Serine Proteases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C000717267:TPP1 protein, human; D012697:Serine Endopeptidases