Humans Lack iGb3 Due to the Absence of Functional iGb3-Synthase: Implications for NKT Cell Development and Transplantation

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Abstract
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 Galα(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 Galα(1,3)Gal xenoantigen, with obvious significance in the field of xenotransplantation. Identification of endogenous antigens that regulate natural killer T (NKT) cell development and function is a major goal in immunology. Originally the glycosphingolipid, iGb3, was suggested to be the main endogenous ligand in both mice and humans. However, recent studies have challenged this hypothesis. From a xenotransplantation (animal to human transplants) perspective, iGb3 expression is also important as it represents another form of the major xenoantigen Galα(1,3)Gal. In this study, we assessed whether humans expressed a functional iGb3 synthase (iGb3S), the enzyme responsible for lipid synthesis. We showed that spliced iGb3S mRNA was not detected in any human tissue analysed. Furthermore, chimeric molecules composed of the catalytic domain of human iGb3S were unable to synthesize iGb3 lipid, due to at least one amino acid substitution. We also demonstrated that purified human anti-Gal antibodies bound iGb3 lipid and mediated destruction of cells transfected to express iGb3. A nonfunctional iGb3S in humans has two major consequences: (1) iGb3 is unlikely to be a natural human NKT ligand and (2) natural human anti-Gal antibodies in human serum could react with iGb3 on the surface of organs from pigs, marking these tissues for immunological destruction.