Conservation of the extended substrate specificity profiles among homologous granzymes across species

ABSTRACT: 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

INSTRUMENT(S): Q-Tof micro, Waters instrument model

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Pieter-Jan De Bock  

LAB HEAD: Pieter-Jan De Bock

PROVIDER: PXD000152 | Pride | 2013-11-27

REPOSITORIES: Pride