Abstract
Granzymes are structurally related serine proteases involved in cell death and immunity. To date four out of five human granzymes have assigned orthologs in mice; however for granzyme H, no murine ortholog has been suggested and its role in cytotoxicity remains controversial. Here, we demonstrate that, as is the case for granzyme C, human granzyme H is an inefficient cytotoxin that together with their similar pattern of GrB divergence and functional similarity strongly hint to their orthologous relationship. Besides analyzing the substrate specificity profile of granzyme H by substrate phage display, substrate cleavage susceptibility of human granzyme H and mouse granzyme C was assessed on a proteome-wide level. The extended specificity profiles of granzymes C and H (i.e. beyond cleavage positions P4-P4') match those previously observed for granzyme B. We demonstrate conservation of these extended specificity profiles among various granzymes as granzyme B cleavage susceptibility of an otherwise granzyme H/C specific cleavage site can simply be conferred by altering the P1-residue to aspartate, the preferred P1-residue of granzyme B. Our results thus indicate a conserved, but hitherto underappreciated specificity-determining role of extended protease-substrate contacts in steering cleavage susceptibility.
Highlights
Several molecular mechanisms are in place to combat transformed malignant cells and virally infected cells
The over 170-fold greater EC50 value for human granzyme H (GrH) (1.658 M Ϯ 0.457) as compared with human granzyme B (GrB) (9.9 nM Ϯ 4.7) (Fig. 1), suggests that GrH is an inefficient cytotoxin similar to granzyme C (GrC) (EC50 values of 39.6 M and 11.2 M for locked and unlocked GrC respectively) [16], an observation supporting its role in viral clearance rather than cell death [24]
When mutating the P2-P1 residues FM310 to DA to investigate if GrB cleavage can occur at position 309 similar to cleavage after Phe309 by GrH and mouse granzyme C (mut GrC), an IKAA310-like pattern could be observed, whereas analysis of the IKAD310 mutant resulted in 41 and 71% of cleavage by GrB and mut GrC respectively. These results suggest that besides the P1 identity, the extended specificity profile is a key determinant for recognition by GrB, GrH, and mut GrC, and indicates that cleavages are predominantly confined to restricted protein areas
Summary
Several molecular mechanisms are in place to combat transformed malignant cells and virally infected cells.
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