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Journal of Biochemistry Advance Access originally published online on February 12, 2008
Journal of Biochemistry 2008 143(5):641-647; doi:10.1093/jb/mvn015
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© 2008 The Japanese Biochemical Society.

Identification of Amino Acid Positions Involved in HLA-E Expression

Katsuyoshi Matsunami1,2,3, Eiji Okura1, Hiroko Uchida1, Hideaki Otsuka3, Masahiro Fukuzawa1 and Shuji Miyagawa1,*

1Division of Organ Transplantation, Department of Molecular Therapeutics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871; 2The Animal Engineering Research Institute (AERI), 3-3 Midorigahara, Tsukuba, Ibaraki 300-2646; and 3Department of Pharmacognosy, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan

*To whom correspondence should be addressed. Tel: +81-6-6879-3067, Fax: +81-6-6879-3069, E-mail: miyagawa{at}orgtrp.med.osaka-u.ac.jp

Received October 6, 2007; Accepted January 27, 2008


  Abstract

The cell surface expression of HLA-E molecules by transfection is faint in xenogeneic cells. Therefore, this study was done for the aim of better expression of HLA-E molecules on the surface of pig cells in order to overcome xenograft rejection mediated by human natural killer (NK) cells. The importance of the loading peptide sequence for HLA-E expression has been studied extensively, but much less information is available concerning the HLA-E heavy chain sequence. In our previous study, we developed the S147C substitution of HLA-E as a useful gene tool for xenotransplantation. In this study, a more extensive substitution analysis throughout the entire region led to the identification of nine amino acid positions, positions-9, 11, 25, 40, 66, 67, 74, 99 and 174, that are significantly involved in the cell surface expression of HLA-E molecules. In view of xenotransplantation usage, double and triple point substitutions, HLA-Ev(11,147) and HLA-Ev(11,66,147), were constructed. These constructs led to a high expression on the xenogeneic cell surface and possessed inhibitory functions against human NK cell-mediated cytolysis in an in vitro pig to human xenotransplantation model system.

Key Words: acute rejection, HLA-E, NK, porcine endothelial cell, xenotransplantation

Abbreviations: NK, natural killer cell; PEC, porcine endothelial cell; HLA, human leucocyte antigen; SLA, swine leucocyte antigen; CHO, Chinese hamster ovary tumor; DMEM, Dulbecco's modified eagle's medium; FBS, fetal bovine serum; mAb, monoclonal antibody; TM, transmembrane; β2m, β2 microglobulin


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