SERPINB12 Is a Slow-Binding Inhibitor of Granzyme A and Hepsin

Abstract

The clade B/intracellular serpins protect cells from peptidase-mediated injury by forming covalent complexes with their targets. SERPINB12 is expressed in most tissues, especially at cellular interfaces with the external environment. This wide tissue distribution pattern is similar to that of granzyme A (GZMA). Because SERPINB12 inhibits trypsin-like serine peptidases, we determined whether it might also neutralize GZMA. SERPINB12 formed a covalent complex with GZMA and inhibited the enzyme with typical serpin slow-binding kinetics. SERPINB12 also inhibited Hepsin. SERPINB12 may function as an endogenous inhibitor of these peptidases.

Figure 1

SERPINB12 (B12) forms SDS-stable complexes with granzyme GZMA. SERPINB12 and/or GZMA were incubated at a ∼5:1 serpin:peptidase ratio, separated by SDS-PAGE, and probed with a SERPINB12 (A) or GZMA (B) specific Mab. Higher molecular mass SERPINB12-GZMA complexes (arrowheads) were detected by both Mab.

Figure 2

Representative analyses of the SERPINB12 stoichiometry of inhibition (SI) with GZMA and HPN (A) and the k_ass determination for GZMA (B) and HPN (C). The SI for the SERPINB12-GZMA or -HPN interaction was 2 and 1.3, respectively. The k_ass for the SERPINB12 inhibition of GZMA (0.9 × 10⁴ M^-1 s^-1) or HPN is (1.5 × 10⁴ M^-1 s^-1) were from representative experiments. The k_ass in the text were the means ± SD from at least three experiments.

Figure 3

Mapping of the GZMA (A) or HPN (B) meidatedSERPINB12 RSL cleavage sites (P1-P1′) by mass spectrometry of the released C-terminal RSL peptide. (C) Schematic of the of SERPINB12 RSL (P5-P5′) with predicted peptidase cleavage sites and expected masses of the respective C-terminal peptides.