Human alpha galactosidase and alpha 1,2 fucosyltransferase concordantly inhibit xenoreactivity of NIH 3T3 cells with human serum.
OBJECTIVE
To study the influence of the expression of human alpha galactosidase and alpha1,2 fucosyltransferase on Gal alpha 1,3 Gal and consequent xenoreactivity in NIH3T3 cells.


METHODS
The expression levels of G antigen and H antigen and binding of human natural antibodies (IgG and IgM) and complement (C3c) to NIH3T3 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
Western blot showed that glycoproteins with molecular weight of 107 kDa, 98 kDa, 88 kDa, 56 kDa, 40 kDa, and 37 kDa were inhibited and even abrogated totally in alpha galactosidase transfectants and alpha 1,2 fucosyltransferase transfectants. The combined transfection of the two enzymes led to a much stronger inhibition of the glycoproteins. The binding of GS-IB4 was decreased by 57.4 % in alpha galactosidase transfectants, 28.8 % in alpha 1,2 fucosyltransferase transfectants, and 72.1 % in combined transfectants, respectively. In contrast, UEA-1 binding was increased about 6.7-fold, 6.0-fold, and 8.0-fold respectively. The xenoreactivity with human IgG was also reduced by 61.4 %, 67.0 %, and 73.4 %, respectively in the three kinds of transfectants. The resistance to cytolysis mediated by human serum was enhanced by 42.4 % in alpha galactosidase transfectants, 51.9 % in alpha 1,2 fucosyltranferase, and even 65.5 % in the combined transfectants.


CONCLUSIONS
Although alpha galactosidase and alpha 1,2 fucosyltransferase had different biochemical properties, they could inhibit the expression of Gal alpha 1,3 Gal synergistically, leading to stronger resistance of xenograft against cytolysis.
Yan
Jing-Lian
JL
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. lhguo@sunm.shcnc.ac.cn
Yu
Lu-Yang
LY
Guo
Li-He
LH
D016475:3T3 Cells; D000818:Animals; D015932:Complement C3c / Q000378:metabolism; D004187:Disaccharides / Q000378:metabolism*; D005647:Fucosyltransferases / Q000096:biosynthesis / Q000235:genetics*; D007074:Immunoglobulin G / Q000378:metabolism; D007075:Immunoglobulin M / Q000378:metabolism; D051379:Mice; D014162:Transfection; D014183:Transplantation, Heterologous; D000519:alpha-Galactosidase / Q000096:biosynthesis / Q000235:genetics*; D000097631:Galactoside 2-alpha-L-fucosyltransferase
D015932:Complement C3c; D004187:Disaccharides; D005647:Fucosyltransferases; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D000519:alpha-Galactosidase; D000097631:Galactoside 2-alpha-L-fucosyltransferase; C055075:galactosyl-(1-3)galactose