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 ϳ2.0 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.

Prosegment of Tripeptidyl Peptidase I Is a Potent, Slow-binding Inhibitor of Its Cognate Enzyme

“…As demonstrated in Fig. 2A, TPP I alone displayed a broad peak of maximum activity between pH 4.5 and 5.0, as previously shown (10,20). The presence of the prosegment (at 1 M) led from a significant to complete inhibition of TPP I activity at pH between 3.5 and 6.0.…”

“…Efficient in vitro processing and autoactivation of the proenzyme is triggered by lowering the pH of the proenzyme solution to below 4.5. When pro-TPP I is activated at pH 4.5 and above, it is processed slowly and generates additional 6-and 14-aa N-terminal extensions in the newly formed polypeptide, rendering it enzymatically inactive (10). However, as we showed earlier, the presence of polyanionic compounds, such as glycosaminoglycans (GAGs), allows for an efficient activation of TPP I proenzyme at higher pH, up to pH 5.5 (11).…”

“…The prepro-TPP I consists of a 19-amino acid (aa) signal peptide cleaved off when the newly forming polypeptide chain is inserted into the endoplasmic reticulum lumen, the 176-aa prosegment (or prodomain), and a 368-aa catalytic domain. As we demonstrated earlier, the removal of the prosegment from a purified TPP I proenzyme (pro-TPP I) occurs via an autocatalytic, intramolecular mechanism, whereby the mature enzyme does not significantly participate in its own generation (10). Efficient in vitro processing and autoactivation of the proenzyme is triggered by lowering the pH of the proenzyme solution to below 4.5.…”

“…Proenzyme Purification-Pro-TPP I was purified, as described earlier (10), with the following modifications: the Bio-Rad Bio-Logic system was used instead of the Amersham Biosciences Akta Purifier, and, as the last step, heparin affinity chromatography was introduced as follows. Pro-TPP I in 10 mM Tris, pH 7.4, was diluted 10-fold to 50 mM sodium acetate, pH 5.7 (buffer A), and injected onto 1 ml of heparin-agarose (BioRad) cartridge, washed, and eluted with a gradient of buffer A and 1 M NaCl in 20 mM Tris, pH 8.0 (buffer B), over 20 min at 1 ml/min.…”

Determination of the Substrate Specificity of Tripeptidyl-peptidase I Using Combinatorial Peptide Libraries and Development of Improved Fluorogenic Substrates

“…This enzyme is a serine protease (7) that, based on the structure of the bacterial homologs, has an unusual Ser, Asp, and Glu catalytic triad, allowing activity at acidic pH (8,9). TPP I has two proteolytic activities, a weak endopeptidase activity with a pH optimum of 3.0 (10) that may be important for the low pH-triggered intramolecular autoactivation and autoprocessing of the 66-kDa inactive proenzyme to the 46-kDa mature enzyme (7,11), and a stronger exopeptidase activity with a pH optimum of 4.5. The exopeptidase activity of TPP I enables the cleavage of tripeptides sequentially from unsubstituted N termini of polypeptides or proteins.…”