Abstract
Human tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal serine protease that removes tripeptides from the free N termini of small polypeptides and also shows a minor endoprotease activity. Due to various naturally occurring mutations, an inherited deficiency of TPP I activity causes a fatal lysosomal storage disorder, classic late infantile neuronal ceroid lipofuscinosis (CLN2). In the present study, we analyzed biosynthesis, glycosylation, transport, and proteolytic processing of this enzyme in stably transfected Chinese hamster ovary cells as well as maturation of the endocytosed proenzyme in CLN2 lymphoblasts, fibroblasts, and N2a cells. Human TPP I was initially identified as a single precursor polypeptide of approximately 68 kDa, which, within a few hours, was converted to the mature enzyme of approximately 48 kDa. Compounds affecting the pH of intracellular acidic compartments, those interfering with the intracellular vesicular transport as well as inhibition of the fusion between late endosomes and lysosomes by temperature block or 3-methyladenine, hampered the conversion of TPP I proenzyme into the mature form, suggesting that this process takes place in lysosomal compartments. Digestion of immunoprecipitated TPP I proenzyme with both N-glycosidase F and endoglycosidase H as well as treatment of the cells with tunicamycin reduced the molecular mass of TPP I proenzyme by approximately 10 kDa, which indicates that all five potential N-glycosylation sites in TPP I are utilized. Mature TPP I was found to be partially resistant to endo H treatment; thus, some of its N-linked oligosaccharides are of the complex/hybrid type. Analysis of the effect of various classes of protease inhibitors and mutation of the active site Ser(475) on human TPP I maturation in cultured cells demonstrated that although TPP I zymogen is capable of autoactivation in vitro, a serine protease that is sensitive to AEBSF participates in processing of the proenzyme to the mature, active form in vivo.
Highlights
Degradation of polypeptides requires the collective action of various endo- and exopeptidases, releasing free amino acids and dipeptides reused in the cell cytoplasm according to the metabolic needs of the cell
Biosynthesis of hTPP I—hTPP I expressed in Chinese hamster ovary (CHO) cells under the control of the cytomegalovirus promoter was present in the lysosomes, as revealed by double immunostaining and laser-scanning confocal microscopy (Fig. 1A), similar to endogenous enzyme in human cells [5, 11]
On immunoblots of cell lysates, the majority of hTPP I appeared as a 48-kDa species representing a mature form of the enzyme and as a minor species with a mass of ϳ68 kDa, corresponding to the proenzyme (Fig. 1B, lane 1). hTPP I proenzyme was found on immunoblots of conditioned media (Fig. 1B, lane 2), but its mass was about 2 kDa higher than that of its cellular counterpart
Summary
Degradation of polypeptides requires the collective action of various endo- and exopeptidases, releasing free amino acids and dipeptides reused in the cell cytoplasm according to the metabolic needs of the cell. Analysis of the effect of various classes of protease inhibitors and mutation of the active site Ser475 on human TPP I maturation in cultured cells demonstrated that TPP I zymogen is capable of autoactivation in vitro, a serine protease that is sensitive to AEBSF participates in processing of the proenzyme to the mature, active form in vivo.
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