Partial purification and characterization of an ovarian tripeptidyl peptidase: A lysosomal exopeptidase that sequentially releases collagen-related (Gly-Pro-X) triplets

McDonald, J. Ken; Hoisington, Anne R.; Eisenhauer, Donald A.

doi:10.1016/0006-291x(85)90571-6

Cited by 50 publications

(23 citation statements)

References 14 publications

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“…They found that Ala-Ala-PhepNA was ϳ60 times more specific than Ala-Ala-Ala-pNA, and no hydrolysis was detected for Ala-Ala-Pro-pNA. In another study, McDonald et al (24) tested partially purified porcine TPP I using several fluorogenic Gly-Pro-Xaa tripeptides and found that the relative substrate specificity of the P1 position was Met Ͼ Leu ϳ Ala Ͼ Arg. Considering that the overall geometry of Met is very similar to Nle, these earlier results are consistent with our findings that Leu, Phe, and Nle are optimal at P1 and that peptides with Pro at P1 are extremely poor substrates.…”

Section: Discussionmentioning

confidence: 99%

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

Tian¹,

Sohár²,

Taylor

et al. 2006

Journal of Biological Chemistry

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Classical late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease caused by mutations in CLN2, the gene encoding the lysosomal protease tripeptidyl-peptidase I (TPP I). The natural substrates for TPP I and the pathophysiological processes associated with lysosomal storage and disease progression are not well understood. Detailed characterization of TPP I substrate specificity should provide insights into these issues and also aid in the development of improved clinical and biochemical assays. To this end, we constructed fluorogenic and standard combinatorial peptide libraries and analyzed them using fluorescence and mass spectrometry-based activity assays. The fluorogenic group 7-amino-4-carbamoylmethylcoumarin was incorporated into a series of 7-amino-4-carbamoylmethylcoumarin tripeptide libraries using a design strategy that allowed systematic evaluation of the P1, P2, and P3 positions. TPP I digestion of these substrates liberates the fluorescence group and results in a large increase in fluorescence that can be used to calculate kinetic parameters and to derive the substrate specificity constant k cat /K M . In addition, we implemented a mass spectrometry-based assay to measure the hydrolysis of individual peptides in peptide pools and thus expand the scope of the analysis. Nonfluorogenic tetrapeptide and pentapeptide libraries were synthesized and analyzed to evaluate P1 and P2 residues. Together, this analysis allowed us to predict the relative specificity of TPP I toward a wide range of potential biological substrates. In addition, we evaluated a variety of new fluorogenic peptides with a P3 Arg residue, and we demonstrated their superiority compared with the widely used substrate Ala-Ala-Phe-AMC for selectively measuring TPP I activity in biological specimens.

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Section: Discussionmentioning

confidence: 99%

“…23), no comprehensive study has been performed to date. Originally, TPP I was found to release Gly-Pro-Xaa tripeptides from collagen-related synthetic polypeptides (24). Pro was not tolerated at P1 and P1Ј (25)(26)(27), and the substrate specificity was related to peptide length (27).…”

mentioning

confidence: 99%

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

Tian¹,

Sohár²,

Taylor

et al. 2006

Journal of Biological Chemistry

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“…2 However, GAGs also exerted a potentially unfavorable biological effect on human TPP I by inhibiting its activity in vitro and in vivo. Inhibition of the activity of numerous lysosomal hydrolases by GAGs in vitro is well documented (50 -56).…”

Section: Discussionmentioning

confidence: 99%

Glycosaminoglycans Modulate Activation, Activity, and Stability of Tripeptidyl-peptidase I in Vitro and in Vivo

Golabek

Walus

Wisniewski

et al. 2005

Journal of Biological Chemistry

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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 (t1 ⁄2 of mature enzyme at pH 7.4 increased by ϳ8-fold in the presence of heparin) and in vivo (ϳ2-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.

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“…DPP II from porcine ovary had a selective action on collagen tissues and has been postulated to be involved in collagen degradation (17,18). On the other hand, since many peptide hormones and proteins are synthesized as inactive precursors and are activated by cleavage at specific internal sites followed by further removal of Nterminal peptides (19,20), DPP II could be involved in prohormone activation and peptide processing.…”

Section: Introductionmentioning

confidence: 99%

Purification and characterization of a novel dipeptidyl peptidase from Dictyostelium discoideum

Choi

Jones

1999

IUBMB Life

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SUMMARY:A dipeptidyl peptidase (DPP) was purified to homogeneity using lys-ala-J~-naphthylamide, the standard substrate for DPP I1. The enzyme is a monomer with a Mr of 70kDa, pl 5.2, and Km 5.0t1M. Its terminal amino acid sequence was XXLLYA|QKRLF and was not identical to that of any known protein. Although initially considered to be a DPP II, the enzyme differed in some properties from classical DPP IIs. It had a pH optimum of 7.9, was not active on X-pro-naphthylamides, the usual substrates of mammalian DPP II, but was active on arg-arg-and asp-arg-naphthylamides, substrates acted on by the DPP III class of enzymes. This enzyme therefore combines properties typical of both DPP II and I11 and differs from all previously described DPPs. Activity on lys-ala-[~-naphthylamide was most abundant during aggregation and its activity is consistent with processing specific peptides during development.

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Partial purification and characterization of an ovarian tripeptidyl peptidase: A lysosomal exopeptidase that sequentially releases collagen-related (Gly-Pro-X) triplets

Cited by 50 publications

References 14 publications

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

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

Glycosaminoglycans Modulate Activation, Activity, and Stability of Tripeptidyl-peptidase I in Vitro and in Vivo

Purification and characterization of a novel dipeptidyl peptidase from Dictyostelium discoideum

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