Abstract
Tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal exopeptidase that sequentially removes tripeptides from the N termini of polypeptides and shows a minor endoprotease activity. Mutations in TPP I lead to classic late-infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disease. TPP I proenzyme is converted in lysosomes into a mature enzyme with the assistance of another protease and is able to autoactivate in acidic pH in vitro via a unimolecular mechanism. Because autoactivation in vitro at the pH values reported for lysosomes generated inactive enzyme, we intended to determine whether physiologically relevant factors can modify this process to also make it plausible in vivo. Here, we report that high ionic strength and glycosaminoglycans (GAGs) increase yields (ionic strength) or yields and rates (GAGs) of activation, enhance degradation of liberated TPP I prosegment fragments, and switch effective autoactivation of TPP I proenzyme toward less acidic pH values (up to pH 6.0). Although ionic strength and GAGs also inhibited TPP I activity in vitro and in living cells, the degree of inhibition (from 20 to 60%) appears to be of rather limited functional significance. Importantly, binding to GAGs improved thermal stability of TPP I and protected the enzyme against alkaline pH-induced denaturation in vitro (t((1/2)) of mature enzyme at pH 7.4 increased by approximately 8-fold in the presence of heparin) and in vivo ( approximately 2-fold higher loss of TPP I in cells deficient in GAGs than in control cells after bafilomycin A1 treatment). These findings elucidate a potent physiologically relevant mechanism of TPP I regulation by GAGs and suggest that generation of the active enzyme via autoactivation can be accomplished not only in vitro but in vivo as well.
Highlights
Tripeptidyl-peptidase I (TPP I,1 CLN2 protein) (EC 3.4.14.9) is the first mammalian representative of a growing family of serine carboxyl peptidases or sedolisins [1]
We report that high ionic strength and glycosaminoglycans (GAGs) increase yields or yields and rates (GAGs) of activation, enhance degradation of liberated TPP I prosegment fragments, and switch effective autoactivation of TPP I proenzyme toward less acidic pH values
Given that the values of the lysosomal pH most commonly reported are in the range of 4.5–5.5 (26 –30), our previous studies suggested that lysosomal milieu could not favor effective autoactivation of pro-TPP I and its conversion to an active enzyme in vivo or that this process could be restricted to those cells that are able to acidify lysosomal milieu more prominently [47, 48]
Summary
Materials—Heparin (sodium salt, Mr 6,000 –30,000 and 3,000), dextran sulfate (Mr 8,000, 60,000, and 500,000), heparan sulfate (from bovine kidney), dermatan sulfate (chondroitin sulfate B from porcine intestinal mucosa), and poly-L-lysine (Mr 14,400) were purchased from Sigma. Time-course Analysis of Pro-TPP I Activation and Processing—Unless otherwise indicated, 2ϫ concentrated stock solutions of purified pro-TPP I at the concentration specified in the figure legends (final concentrations) were prepared in 2 mM Tris, pH 8.0, 0.2% Triton X-100, mixed at 0 time with an equal volume of 200 mM sodium acetate (activation buffer), and maintained either at room temperature (25 °C) or 37 °C. 72 h after transfections, bafilomycin A1 was added to the culture media for 3 and 6 h (final concentration 100 nM) and cells were lysed in 1% Triton X-100, 50 mM Tris, pH 7.4, and protease inhibitor mixture (Complete) supplemented with pepstatin A (lysis buffer) and TPP I was assessed by using Western blotting and enzymatic activity assay. Cells were washed in PBS, harvested with trypsin/EDTA solution to remove cell membrane-associated pro-TPP I, centrifuged twice at 2,000 ϫ g for 2 min (first in full medium and in PBS), and lysed as above
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