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A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl
Published on: November 28, 2014
Intracellular serine protease inhibitor SERPINB4 inhibits granzyme M-induced cell death
Pieter J A de Koning1, J Alain Kummer, Stefanie A H de Poot
1Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.
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 1.3×10(4) M(-1) s(-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.
Insights
Serine protease inhibitor SERPINB4 inhibits Granzyme M (GrM), a key player in cytotoxic lymphocyte-mediated cell death. This finding reveals a novel mechanism for tumor cells, particularly squamous cell carcinomas, to evade immune attack.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Cytotoxic lymphocytes eliminate virus-infected and tumor cells via granzyme-mediated cell death.
- Tumor cells can evade this killing through intracellular serine protease inhibitors (serpins).
- While SERPINB9 inhibits Granzyme B (GrB), inhibitors for other granzymes remain largely unknown.
Purpose of the Study:
- To investigate the inhibitory potential of SERPINB4 against human Granzyme M (GrM).
- To elucidate the mechanism of SERPINB4-GrM interaction and its functional consequences.
- To explore the role of SERPINB4 in tumor cell evasion of cytotoxic lymphocyte-mediated killing.
Main Methods:
- Formation of SDS-stable complexes between SERPINB4 and GrM (recombinant and native).
- Mutation analysis of the SERPINB4 reactive center loop.
- Kinetic analysis of SERPINB4 inhibition of GrM activity.
- Assessment of SERPINB4's effect on GrM-induced cell death in tumor cells and NK cell-mediated cytotoxicity.
Main Results:
- SERPINB4 formed stable complexes with GrM, indicating a typical serpin-protease interaction.
- GrM cleaves SERPINB4 at the P1-Leu residue, and mutations abolished complex formation.
- SERPINB4 potently inhibited GrM activity and its cleavage of macromolecular substrates.
- Overexpression of SERPINB4 in tumor cells reduced GrM-induced and NK cell-mediated cell death.
Conclusions:
- SERPINB4 is a physiological inhibitor of human Granzyme M.
- SERPINB4-mediated inhibition of GrM represents a novel mechanism for tumor cells to evade cytotoxic lymphocyte attack.
- High SERPINB4 expression in squamous cell carcinomas suggests its role in immune evasion in these cancers.
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