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Identification of a Novel Human Granzyme B Inhibitor Secreted by Cultured Sertoli Cells¹

Simonetta Sipione^2,, Katia C. Simmen^2,, Sarah J. Lord^2,*, Bruce Motyka, Catherine Ewen, Irene Shostak, Gina R. Rayat^*, Jannette M. Dufour^*, Greg S. Korbutt^*, Ray V. Rajotte^3,* and R. Chris Bleackley^3,

^* Department of Surgery, Department of Biochemistry, and Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada

Sertoli cells have long since been recognized for their ability to suppress the immune system and protect themselves as well as other cell types from harmful immune reaction. However, the exact mechanism or product produced by Sertoli cells that affords this immunoprotection has never been fully elucidated. We examined the effect of mouse Sertoli cell-conditioned medium on human granzyme B-mediated killing and found that there was an inhibitory effect. We subsequently found that a factor secreted by Sertoli cells inhibited killing through the inhibition of granzyme B enzymatic activity. SDS-PAGE analysis revealed that this factor formed an SDS-insoluble complex with granzyme B. Immunoprecipitation and mass spectroscopic analysis of the complex identified a proteinase inhibitor, serpina3n, as a novel inhibitor of human granzyme B. We cloned serpina3n cDNA, expressed it in Jurkat cells, and confirmed its inhibitory action on granzyme B activity. Our studies have led to the discovery of a new inhibitor of granzyme B and have uncovered a new mechanism used by Sertoli cells for immunoprotection.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from the Canadian Institute of Health Research (CIHR), the Edmonton Civic Employees’ Charitable Assistance Fund, the C. F. "Curly" and Gladys MacLachlan Fund, and Peter A. Allard. R.C.B. is a CIHR Distinguished Scientist, a Medical Scientist of the Alberta Heritage Foundation for Medical Research (AHFMR), a Howard Hughes International Scholar, and a Canada Research Chair. S.J.L. was supported by an AHFMR studentship; S.S. was supported by an AHFMR research fellowship.

² S.S., K.C.S., and S.J.L. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. R. Chris Bleackley, Department of Biochemistry, University of Alberta, 467A Medical Sciences Building, Edmonton, Alberta, T6G 2H7, Canada; E-mail address: Chris.Bleackley{at}ualberta.ca or Dr. Ray V. Rajotte, Department of Surgery, University of Alberta, 1074 Dentistry/Pharmacy Building, Edmonton, Alberta, T6G 2N8, Canada; E-mail address: rrajotte{at}ualberta.ca

⁴ Abbreviations used in this paper: M6P, mannose-6 phosphorylated; IGF-II, insulin-like growth factor-II; PI-9, proteinase inhibitor-9; SPI-6, serine proteinase inhibitor-6; Adv, adenovirus; SCCM, Sertoli cell-conditioned medium; PVDF, polyvinylidene difluoride; HA, hemagglutinin; RCL, reactive-center loop.

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