Intracellular Serine Protease Inhibitor SERPINB4 Inhibits Granzyme M-Induced Cell Death

Abstract

Granzyme-mediated cell death is the major pathway for cytotoxic lymphocytes to kill virus-infected and tumor cells. In humans, five different granzymes (i.e. GrA, GrB, GrH, GrK, and GrM) are known that all induce cell death. Expression of intracellular serine protease inhibitors (serpins) is one of the mechanisms by which tumor cells evade cytotoxic lymphocyte-mediated killing. Intracellular expression of SERPINB9 by tumor cells renders them resistant to GrB-induced apoptosis. In contrast to GrB, however, no physiological intracellular inhibitors are known for the other four human granzymes. In the present study, we show that SERPINB4 formed a typical serpin-protease SDS-stable complex with both recombinant and native human GrM. Mutation of the P2-P1-P1′ triplet in the SERPINB4 reactive center loop completely abolished complex formation with GrM and N-terminal sequencing revealed that GrM cleaves SERPINB4 after P1-Leu. SERPINB4 inhibited GrM activity with a stoichiometry of inhibition of 1.6 and an apparent second order rate constant of 1.3×104 M−1s−1. SERPINB4 abolished cleavage of the macromolecular GrM substrates α-tubulin and nucleophosmin. Overexpression of SERPINB4 in tumor cells inhibited recombinant GrM-induced as well as NK cell-mediated cell death and this inhibition depended on the reactive center loop of the serpin. As SERPINB4 is highly expressed by squamous cell carcinomas, our results may represent a novel mechanism by which these tumor cells evade cytotoxic lymphocyte-induced GrM-mediated cell death.

Identification of SERPINB1 As a Physiological Inhibitor of Human Granzyme H

“…SERPINB9 was reported to be a physiological inhibitor for human GzmB (12), which protects lymphocytes from GzmB-induced injury. A recent study showed that SERPINB4 is an intracellular inhibitor for human GzmM that suppresses GzmM-mediated cell death (13). We showed that SERPINB1 and SERPINB8 resided in YTS cells; however, it is unknown whether they inhibit Gzm.…”

“…SERPINB9 was identified as an intracellular inhibitor of Gzm B (GzmB) (12). SERPINB4 is an inhibitor of Gzm M (GzmM) and suppresses GzmM-mediated cell death (13). However, no physiological intracellular inhibitors have been defined for human GzmH.…”

Granzymes Regulate Proinflammatory Cytokine Responses

“…The three major extracellular protease inhibitors present in normal lung fluid (a 1 -antitrypsin, elafin, and secretory leukocyte protease inhibitor) do not inhibit GrA or GrB, and GrA is active in bronchoalveolar lavage fluid (20). GrH is inhibited by serpin B1 (89), and GrM is inhibited by serpin B4 (90). Furthermore, GrM is inhibited by a1-antichymotrypsin and a 1 -proteinase inhibitor, although weakly (91).…”

Granzyme M: behind enemy lines

“…72 In addition, GrM can stimulate LPS/TLR-4-mediated signaling via yet to be identified mechanisms. 90 GrM intracellular functioning can be inhibited in tumor cells by human SERPINB4 92 and mouse SPI-CI. 38 Next to these intracellular effects, GrM has also been found to cleave VWF, and may in that way regulate FVIII levels and coagulation 91 in humans: as mentioned previously, mGrM and hGrM display species-specific substrate specificities.…”

“…92 SPI-CI has been initially identified in mouse cytotoxic T lymphocytes 93 and has later been found to inhibit purified rat GrM 38 and -to a lesser extent -hGrM. 94 SPI-CI forms the classical SDS-stable serpin-protease complex with rat GrM, and is expressed by purified murine CD8 þ T cells and even more by murine LAK cells.…”