Identification of Serpinb6b as a species-specific mouse granzyme A inhibitor suggests functional divergence between human and mouse granzyme A [Enzymology]

February 6th, 2014 by Kaiserman, D., Stewart, S. E., Plasman, K., Gevaert, K., van Damme, P., Bird, P. I.

The granzyme family of serine proteases are key effector molecules expressed by cytotoxic lymphocytes. The physiological role of granzyme A (GzmA) is controversial, with significant debate over its ability to induce death in target cells. Here we investigate natural inhibitors of GzmA. We employed substrate phage display and positional proteomics to compare substrate specificities of mouse and human GzmA at the peptide and proteome-wide level respectively, and used the resulting substrate specificity profiles to search for potential inhibitors from the intracellular serpin family. We identified Serpinb6b as a potent inhibitor of mGzmA. Serpinb6b interacts with mGzmA, but not hGzmA, with an association constant of 1.9 ± 0.8 × 105 M-1s-1 and a stoichiometry of inhibition of 1.8. Mouse GzmA is over 5 times more cytotoxic than hGzmA when delivered into P815 target cells with streptolysin O, while transfection of target cells with a Serpinb6b cDNA increases the EC50 of mGzmA 13-fold, without affecting hGzmA cytotoxicity. Unexpectedly, we also found that Serpinb6b employs an exosite to specifically inhibit dimeric, but not monomeric mGzmA. The identification of an intracellular inhibitor specific for mGzmA only indicates that a lineage-specific increase in GzmA cytotoxic potential has driven cognate inhibitor evolution.
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