Expression of human alpha-galactosidase leads to reduction of major xenoepitope Galalpha(1,3) Gal in NIH 3T3 cell
Abstract
AIM: To examine the effects of the expression of alpha-galactosidase on the expression of the major xenoepitope Galalpha(1,3) Gal (G antigen) in NIH 3T3 cell.
METHODS: The expression levels of G antigen and H antigen and binding of human natural antibodies (IgG and IgM) and complement (C3c) to NIH 3T3 cells were analyzed by flow cytometry. Western blot was employed to further determine the expression of glycoproteins of G antigen. Cytolysis assay with normal human serum was performed by MTT assay.
RESULTS: In transfectants, Western blot showed that the binding of human IgG to glycosylated proteins located on the cell membrane was decreased, even abrogated totally. Together with the reduced binding of Gs-IB4 (Griffonia simplicifolia) to transfectants, the stable expression of human alpha-galactosidase effectively inhibited Galalpha(1,3) Gal, Gal epitope synthesis in NIH 3T3 cell. As a result, the xenoreactivities of human IgG, IgM, and C3c were reduced by 73.4 %, 22.3 % and 47.9 %, respectively, while the cell lysis mediated by human XNA and complements was decreased by 42.4 %.
CONCLUSION: The stable expression of human alpha-galactosidase in NIH 3T3 cell strongly inhibits the expression of Gal epitopes, resulting in abrupt reduction in xenorejection induced by human serum.
Keywords:
METHODS: The expression levels of G antigen and H antigen and binding of human natural antibodies (IgG and IgM) and complement (C3c) to NIH 3T3 cells were analyzed by flow cytometry. Western blot was employed to further determine the expression of glycoproteins of G antigen. Cytolysis assay with normal human serum was performed by MTT assay.
RESULTS: In transfectants, Western blot showed that the binding of human IgG to glycosylated proteins located on the cell membrane was decreased, even abrogated totally. Together with the reduced binding of Gs-IB4 (Griffonia simplicifolia) to transfectants, the stable expression of human alpha-galactosidase effectively inhibited Galalpha(1,3) Gal, Gal epitope synthesis in NIH 3T3 cell. As a result, the xenoreactivities of human IgG, IgM, and C3c were reduced by 73.4 %, 22.3 % and 47.9 %, respectively, while the cell lysis mediated by human XNA and complements was decreased by 42.4 %.
CONCLUSION: The stable expression of human alpha-galactosidase in NIH 3T3 cell strongly inhibits the expression of Gal epitopes, resulting in abrupt reduction in xenorejection induced by human serum.