Abstract
The Gal antigen is synthesized by glycoprotein galactosyltransferase alpha 1, 3 (GGTA1) or (and) isoglobotrihexosylceramide 3 synthase (iGb3S). However, whether iGb3S deletion changes Gal epitope expression and immunological properties in animals is still not clear. The objective of this study was to develop iGb3S deficient mice, and characterize their Gal epitope expression and Gal epitope-related immunological properties. iGb3S gene knockout mice were generated on the C57BL/6 background using the bacterial artificial chromosome homology region recombination technique. Gal epitope expression in the iGb3S deficient mice was determined by using a monoclonal anti-Gal antibody. Immunological properties were analyzed by enzyme linked immune sorbent assay. It was found that Gal epitope expression was decreased from 5.19% to 21.74% in the main organs of iGb3S deficient mice, compared with that of C57BL/6 wild type mice, suggesting that the iGb3S gene participated to Gal epitope expression. However, iGb3S deletion alone did not cause significant changes in the immunological properties of iGb3S deficient mice with or without exogenous Gal antigen (Rabbit Red Blood Cell) stimulation. The data from this study suggest that the iGb3S gene likely contributes to Gal epitope expression, but may have a very weak effect on immunological properties of the iGb3S deficient mice.
Highlights
Many studies have shown that the major antigen in pig tissue recognized by primate antibodies is a terminal galalpha1-3gal carbohydrate structure (Gal antigen) present on glycolipids and glycoproteins[1,2,3,4]
Heterozygous F1 progenies were obtained by breeding chimeric isoglobotrihexosylceramide 3 synthase (iGb3S) mice with WT C57BL/6 mice and F1 progenies with pure black color were selected as parents for breeding and expansion
This distribution pattern of iGb3S mRNA in the main organs is similar to GGTA1 mRNA expression (Fig. 2b), albeit at a lower level than GGTA1 mRNA. iGb3S mRNA in iGb3S KO mice completely disappeared as expected, but there were no changes in GGTA1 mRNA compared with that in WT mice (Fig. 2b)
Summary
Many studies have shown that the major antigen in pig tissue recognized by primate antibodies is a terminal galalpha1-3gal carbohydrate structure (Gal antigen) present on glycolipids and glycoproteins[1,2,3,4]. Several studies showed that the Gal epitope is expressed in GGTA1 deficient mice (splenic fibroblasts and tissues including the pancreas, spleen, kidney and liver), and in fetal-pig homozygous GGTA1 knockout (KO) fibroblasts when stained with anti-Gal alpha(1,3)Gal mAb or with sensitized human serum[9,12,13]. The results from Milland et al.[13] verified that GGTA1 KO mice have mRNA for iGb3S and induce an antibody response to Gal antigen synthesized by iGb3S12. Anti-Gal antibody responses were induced in GGTA1 KO mice after immunization with GGTA1-positive cells or iGb3S-positive cells, indicating iGb3S mediates Gal antigen mediated immunologic toxicity[9]. The results from this study provide basic information to help understand whether the iGb3S gene contributes to Gal epitope expression, and the xeno-species anti-Gal antibody-mediated immune response
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