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 ϳ68 kDa, which, within a few hours, was converted to the mature enzyme of ϳ48 kDa. Degradation of polypeptides requires the collective action of various endo-and exopeptidases, finally releasing free amino acids and dipeptides reused in the cell cytoplasm according to the metabolic needs of the cell. Two tripeptidyl peptidases identified to date in mammalian cells sequentially cleave tripeptides from the N termini of oligopeptides: tripeptidyl peptidase I (TPP I, 1 CLN2 protein) and tripeptidyl peptidase II (TPP II) (for a recent review, see Ref.

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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.

Tripeptidyl peptidase I (TPP I) is a lysosomal exopeptidase that cleaves tripeptides from the free N-termini of oligopeptides. Mutations in this enzyme are associated with the classic late-infantile form of neuronal ceroid lipofuscinosis (CLN2), an autosomal recessive disorder leading to severe brain damage. To gain more insight into CLN2 pathogenesis and the role of TPP I in human tissues in general, we analyzed the temporal and spatial distribution of TPP I in the brain and its localization in internal organs under normal and pathological conditions. We report that TPP I immunoreactivity appears in neurons late in gestation and increases gradually in the postnatal period, matching significantly the final differentiation and maturation of neural tissue. Endothelial cells, choroid plexus, microglial cells, and ependyma showed TPP I immunostaining distinctly earlier than neurons. Acquisition of the adult pattern of TPP I distribution in the brain at around the age of 2 years correlates with the onset of clinical signs in CLN2 subjects. In adults, TPP I was found in all types of cells in the brain and internal organs we studied, although the intensity of TPP I labeling varied among several types of cells and showed a noticeable predilection for cells and/or organs associated with peptide hormone and neuropeptide production. In addition, TPP I immunoreactivity was increased in aging brain, neurodegenerative and lysosomal storage disorders, and some differentiated neoplasms and was reduced in ischemic/anoxic areas and undifferentiated tumors. These findings suggest that TPP I is involved in general protein turnover and that its expression may be controlled by various regulatory mechanisms, which highlights the importance of this enzyme for normal function of cells and organs in humans.

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