isoglobotrihexosylceramide and galactosyl-(1-3)galactose

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.

Topics: Animals; Disaccharides; Epitopes; Erythrocytes; Galactosyltransferases; Globosides; Mice; Mice, Inbred C57BL; Mice, Knockout; Rabbits; Trihexosylceramides

The glycosphingolipid isoglobotrihexosylceramide, or isogloboside 3 (iGb3), is believed to be critical for natural killer T (NKT) cell development and self-recognition in mice and humans. Furthermore, iGb3 may represent an important obstacle in xenotransplantation, in which this lipid represents the only other form of the major xenoepitope Galalpha(1,3)Gal. The role of iGb3 in NKT cell development is controversial, particularly with one study that suggested that NKT cell development is normal in mice that were rendered deficient for the enzyme iGb3 synthase (iGb3S). We demonstrate that spliced iGb3S mRNA was not detected after extensive analysis of human tissues, and furthermore, the iGb3S gene contains several mutations that render this product nonfunctional. We directly tested the potential functional activity of human iGb3S by expressing chimeric molecules containing the catalytic domain of human iGb3S. These hybrid molecules were unable to synthesize iGb3, due to at least one amino acid substitution. We also demonstrate that purified normal human anti-Gal immunoglobulin G can bind iGb3 lipid and mediate complement lysis of transfected human cells expressing iGb3. Collectively, our data suggest that iGb3S is not expressed in humans, and even if it were expressed, this enzyme would be inactive. Consequently, iGb3 is unlikely to represent a primary natural ligand for NKT cells in humans. Furthermore, the absence of iGb3 in humans implies that it is another source of foreign Galalpha(1,3)Gal xenoantigen, with obvious significance in the field of xenotransplantation.

Topics: Amino Acid Substitution; Animals; Antigens, Heterophile; Cell Line; Cell Transplantation; Disaccharides; Galactosyltransferases; Globosides; Humans; Killer Cells, Natural; Mice; RNA Splicing; Transplantation, Heterologous; Trihexosylceramides