The long-term goal of this proposed project is to characterize the factors and mechanisms underlying maturation, stability, and catalytic activity of tripeptidyl-peptidase I (TPP I). Naturally occurring mutations in TPP I lead to a fatal neurodegenerative lysosomal storage disorder- classic late-infantile neuronal ceroid lipofuscinosis (CLN2). TPP I is a lysosomal aminopeptidase that releases tripeptides from a free N-terminus of proteins. TPP I is extensively glycosylated. Our preliminary data suggest that N-glycan attached to Asn286 is critical for lysosomal targeting and activity of TPP I. We propose to further elucidate the role of N-glycosylation in the trafficking of TPP I as well as to determine whether and how N-glycosylation affects folding, activity, and stability of the enzyme. These experiments will be performed by expressing and studying N-glycosylation defective TPP I mutants in mammalian cells by means of laser-scanning confocal microscopy, Western blotting, in vivo labeling, immunoprecipitation and enzymatic assay. TPP I is a serine-type protease, although it is atypical in that instead of classical Ser/His/Asp catalytic triad in the active site, it appears to contain Ser/Asp/Asp triad. Although TPP I is able to autoactivate in vitro, according to our data, autoactivation of TPP I in vitro is an intramolecular process that is efficient in a very narrow pH range. Furthermore, our studies suggest that in vivo, another serine protease is involved in proteolytic processing of TPP I. Experiments we are planning to perform will assess the effect of various factors (e.g., ionic strenght, charged compounds) on autoactivation of the proenzyme; will elucidate the role of the prodomain in the activation, activity, and stability of the enzyme and will identify the protease responsible for in vivo processing of TPP I. For these studies, we will use human TPP I purified from conditioned media of overexpressing CHO cells. TPP I prodomain will be expressed and purified from yeast, Pichia pastoris. The activity and stability of the enzyme in vitro will be analyzed by using SDS-PAGE, Western blotting, gel filtration chromatography, and spectrofluorometry. Protease involved in processing of TPP I in vivo will be identified by using the biochemical purification approach and active-site mutant proenzyme as a substrate. Finally, to elucidate the catalytic mechanism of TPP I, we intend to analyze the secondary and three-dimensional structure of the enzyme by means of CD spectroscopy and high-resolution crystallography of wild-type TPP I proenzyme and mature enzyme as well as active-site mutants. Analysis of the localization of naturally occurring TPP I mutations on three-dimensional structure of the wild-type TPP I precursor will give important insights into the structural basis of the CLN2 disease process. We also anticipate that completion of these studies will allow us to better understand the molecular biology of TPP I and shed new light on the pathogenesis of the CLN2 and thus may be useful for development in the future of therapy for this devastating human disorder.
| Golabek, Adam A; Kida, Elizabeth (2006) Tripeptidyl-peptidase I in health and disease. Biol Chem 387:1091-9 |
| Walus, Mariusz; Kida, Elizabeth; Wisniewski, Krystyna E et al. (2005) Ser475, Glu272, Asp276, Asp327, and Asp360 are involved in catalytic activity of human tripeptidyl-peptidase I. FEBS Lett 579:1383-8 |
| Wujek, Peter; Kida, Elizabeth; Walus, Marius et al. (2004) N-glycosylation is crucial for folding, trafficking, and stability of human tripeptidyl-peptidase I. J Biol Chem 279:12827-39 |
