Dataset Information

Substrate specificity divergence of granzyme tryptases.

ABSTRACT: Elucidating the true physiological functions of granzymes has been challenging since the origin of the granzyme studied, the use of non-physiological granzyme concentrations and the interspecies use of granzymes, with extrapolation of all these data to orthologous granzymes has led to inconsistencies. This is highly relevant for granzymes A and K where a debate is ongoing concerning their involvement in either cytotoxic or inflammatory processes, or in both. In order to tackle such contradictions, detailed knowledge of the substrate repertoires and substrate specificities of granzymes as well as their cleavage efficiencies may add in unraveling the primary signaling pathways these granzymes are involved in. Therefore, a degradome analysis using N-terminal COFRADIC was performed to unravel the substrate repertoires and subsite determinants for the closely related homologous human tryptases. We furthermore also profiled the uncharacterized mouse granzyme K in a comparative analysis with its human ortholog. Despite the subtle differences observed in the primary specificity profiles of these granzymes, for each granzyme distinguishing substrate subsite features could be elucidated.

INSTRUMENT(S): LTQ Orbitrap, instrument model

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Pieter-Jan De Bock

LAB HEAD: Kris Gevaert

PROVIDER: PXD000570 | Pride | 2015-09-30

REPOSITORIES: Pride

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Publications

Substrate specificities of the granzyme tryptases A and K.

Plasman Kim K Demol Hans H Bird Philip I PI Gevaert Kris K Van Damme Petra P

Journal of proteome research 20141119 12

The physiological roles of the granzymes A and K have been debated, especially concerning their involvement in cytotoxic and inflammatory processes. By performing N-terminal COFRADIC assisted N-terminomics on the homologous human granzymes A and K, we here provide detailed data on their substrate repertoires, their specificities, and differences in efficiency by which they cleave their substrates, all of which may aid in elucidating their key substrates. In addition, the so far uncharacterized m ...[more]

PMID: 25383893

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Project description:Granzymes are a family of structurally related serine proteases involved in immunity. To date four out of five human granzymes have been assigned orthologues in mice; however for granzyme H, no clear murine orthologue has been suggested and its role in cytotoxicity remains controversial. In this study, we demonstrate that granzyme H is an inefficient cytotoxin. Besides analyzing the substrate specificity profile of granzyme H by means of substrate phage display, we assessed human granzyme H and mouse granzyme C cleavage susceptibility on a proteome-wide level. The extended specificity profiles of granzyme C and granzyme H (i.e. beyond P4-P4') match those previously observed for granzyme B. We demonstrate conservation of these extended specificity profiles among various granzymes since granzyme B cleavage susceptibility of an otherwise granzyme H/C specific cleavage site can be conferred simply by altering the P1-residue to aspartate, the preferred P1-residue of granzyme B. Our results thus indicate a conserved, but hitherto generally underappreciated specificity-determining role of extended protease-substrate contacts in steering cleavage susceptibility. Bio-IT: The spectra were searched with Mascot Daemon 2.2. The following search parameters were used. Peptide mass tolerance was set at 0.2 Da and peptide fragment mass tolerance at 0.1 Da; with the ESI-QUAD-TOF as selected instrument for peptide fragmentation rules for the Q-TOF Premier data. Endoproteinase semiArg-C/P (i.e., no restriction towards arginine-proline cleavage) was set as enzyme allowing one missed cleavage. Variable modifications were pyroglutamate formation of N-terminal glutamine, pyrocarbamidomethyl formation of N-terminal alkylated cysteine, deamidation of asparagine, acetylation or tri-deuteroacetylation of the alpha-N-terminus. Fixed modifications were methionine oxidation (sulfoxide), carbamidomethyl for cysteine, tri-deuteroacetylation of lysine and for identifying heavy labelled peptides, [13C6] or [13C615N4]-arginine were additionally set as fixed modifications. The spectra and identifications were stored in ms_lims

Dataset Information

Substrate specificity divergence of granzyme tryptases.

Publications

Substrate specificities of the granzyme tryptases A and K.

Similar Datasets

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