title: Maturation of human tripeptidyl-peptidase I in vitro.
abstract: Tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal aminopeptidase that cleaves off tripeptides from the free N termini of oligopeptides and also shows minor endopeptidase activity. TPP I is synthesized as a preproenzyme. Its proenzyme autoactivates under acidic conditions in vitro, resulting in a rapid conversion into the mature form. In this study, we examined the process of maturation in vitro of recombinant latent human TPP I purified to homogeneity from secretions of Chinese hamster ovary cells overexpressing TPP I cDNA. Autoprocessing of TPP I proenzyme was carried out at a wide pH range, from approximately 2.0 to 6.0, albeit with different efficiencies depending on the pH and the type of buffer. However, the acquisition of enzymatic activity in the same buffer took place in a narrower pH "window," usually in the range of 3.6-4.2. N-terminal sequencing revealed that mature, inactive enzyme generated during autoactivation at higher pH contained N-terminal extensions (starting at 6 and 14 amino acid residues upstream of the prosegment/mature enzyme junction), which could contribute to the lack of activity of TPP I generated in this manner. Autoprocessing was not associated with any major changes of the secondary structure of the proenzyme, as revealed by CD spectroscopy. Both the activation and proteolytic processing of the recombinant TPP I precursor were primarily concentration-independent. The addition of the mature enzyme did not accelerate the processing of the proenzyme. In addition, the maturation of the proenzyme was not affected by the presence of glycerol. Finally, the proenzyme with the active site mutated (S475L) was not processed in the presence of the wild-type enzyme. All of these findings indicate a primarily intramolecular (unimolecular) mechanism of TPP I activation and autoprocessing and suggest that in vivo mature enzyme does not significantly participate in its own generation from the precursor.
authors.affiliation: Department of Developmental Neurobiology, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314, USA. a.golabek@att.net
authors.forename: Adam A
authors.initials: AA
authors.lastname: Golabek
authors.forename: Peter
authors.initials: P
authors.lastname: Wujek
authors.forename: Marius
authors.initials: M
authors.lastname: Walus
authors.forename: Sylvain
authors.initials: S
authors.lastname: Bieler
authors.forename: Claudio
authors.initials: C
authors.lastname: Soto
authors.forename: Krystyna E
authors.initials: KE
authors.lastname: Wisniewski
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Kida
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases; D000818:Animals; D002021:Buffers; D016466:CHO Cells; D020134:Catalytic Domain / Q000235:genetics; D006224:Cricetinae; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057057:Serine Proteases; D015533:Transcriptional Activation; D000091346:Tripeptidyl-Peptidase 1
chemical_list: D002021:Buffers; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D000091346:Tripeptidyl-Peptidase 1; D010450:Endopeptidases; D057057:Serine Proteases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C000717267:TPP1 protein, human