A Novel Serpin Regulatory Mechanism: SerpinB9 IS REVERSIBLY INHIBITED BY VICINAL DISULFIDE BOND FORMATION IN THE REACTIVE CENTER LOOP

Matthew S.J Mangan,Catherina H Bird,Dion Kaiserman,Anthony Y Matthews,Corinne Hitchen,David L Steer,Philip E Thompson,Phillip I Bird

doi:10.1074/jbc.m115.699298

Abstract

The intracellular protease inhibitor Sb9 (SerpinB9) is a regulator of the cytotoxic lymphocyte protease GzmB (granzyme B). Although GzmB is primarily involved in the destruction of compromised cells, recent evidence suggests that it is also involved in lysosome-mediated death of the cytotoxic lymphocyte itself. Sb9 protects the cell from GzmB released from lysosomes into the cytosol. Here we show that reactive oxygen species (ROS) generated within cytotoxic lymphocytes by receptor stimulation are required for lyososomal permeabilization and release of GzmB into the cytosol. Importantly, ROS also inactivate Sb9 by oxidizing a highly conserved cysteine pair (P1-P1' in rodents and P1'-P2' in other mammals) in the reactive center loop to form a vicinal disulfide bond. Replacement of the P4-P3' reactive center loop residues of the prototype serpin, SERPINA1, with the P4-P5' residues of Sb9 containing the cysteine pair is sufficient to convert SERPINA1 into a ROS-sensitive GzmB inhibitor. Conversion of the cysteine pair to serines in either human or mouse Sb9 results in a functional serpin that inhibits GzmB and resists ROS inactivation. We conclude that ROS sensitivity of Sb9 allows the threshold for GzmB-mediated suicide to be lowered, as part of a conserved post-translational homeostatic mechanism regulating lymphocyte numbers or activity. It follows, for example, that antioxidants may improve NK cell viability in adoptive immunotherapy applications by stabilizing Sb9.

Highlights

The intracellular protease inhibitor Sb9 (SerpinB9) is a regulator of the cytotoxic lymphocyte protease GzmB
reactive oxygen species (ROS) Promote lysosomal membrane permeabilization (LMP) and GzmB-mediated cytotoxic lymphocyte (CL) Death—We have previously shown that LMP is associated with activation-induced cell death (AICD) of CTL following CD3 restimulation or AICD of natural killer (NK) cells following CD2 or CD16 ligation
As previously described [19], treatment of NK cells with either anti-CD2 or anti-CD16 leads to cell death (Fig. 1A) and appearance of GzmB in the cytosol as indicated by the formation of a complex with cytoplasmic Sb9 (Fig. 1B). (The cells were lysed in SDS, which prevents postlysis binding of GzmB and Sb9; complex formation is a direct indicator of GzmB release from lysosomes into the cytoplasm [8].) Strikingly, both complex formation and cell death were abrogated by pretreatment with a mixture of ROS inhibitors (Fig. 1, A and B), strongly implicating ROS as a second messenger in AICD

Summary

Introduction

The intracellular protease inhibitor Sb9 (SerpinB9) is a regulator of the cytotoxic lymphocyte protease GzmB (granzyme B). The pathophysiological importance of GzmB in immune cell homeostasis is exemplified by decreased death of GzmB-null Th2 CD4ϩ T helper cells [14] These cells have longer life spans than normal, resulting in a skewed cytokine response in vivo and an increase in the allergic immune response [14]. For GzmB to cause apoptosis of CLs during an immune response, it must access the CL cytosol It could be delivered from a neighboring cell (fratricide), as observed in Sendai virus infection where regulatory T cells limit effector CD8ϩ T cell life span by killing these cells in a GzmB- and perforin-dependent manner [15, 16]. ROS can come from the external environment, generated by neighboring neutrophils and macrophages [25]

Results

Discussion

Conclusion