Complete absence of the aGal xenoantigen and isoglobotrihexosylceramide in a1,3galactosyltransferase knock-out pigs

BACKGROUND Anti Gala13Galß R natural antibodies are responsible for hyperacute rejection in pig to primate xenotransplantation. Although the generation of pigs lacking the a13galactosyltransferase (GalT) has overcome hyperacute rejection antibody mediated rejection is still a problem. It is possible that other enzymes synthesize antigens similar to Gala13Gal epitopes that are recognized by xenoreactive antibodies. The glycosphingolipid isoglobotrihexosylceramide (iGb3) represents such a candidate expressing an alternative Gala13Gal epitope. The present work determined whether the terminal Gala13Gal disaccharide is completely absent in Immerge pigs lacking the GalT using several different highly sensitive methods. METHODS The expression of Gala13Gal was evaluated using a panel of antibodies and lectins by flow cytometry and fluorescent microscopy; GalT activity was detected by an enzymatic assay; and ion trap mass spectroscopy of neutral cellular membranes extracted from aortic endothelial was used for the detection of sugar structures. Finally the presence of iGb3 synthase mRNA was tested by RT PCR in pig thymus spleen lymph node kidney lung and liver tissue samples. RESULTS Aortic endothelial cells derived from GalT knockout pigs expressed neither Gala13Gal nor iGb3 on their surface and GalT enzymatic activity was also absent. Lectin staining showed an increase in the blood group H type sugar structures present in GalT knockout cells as compared to wild type pig aortic endothelial cells (PAEC). Mass spectroscopic analysis did not reveal Gala13Gal in membranes of GalT knockout PAEC; iGb3 was also totally absent whereas a fucosylated form of iGb3 was detected at low levels in both pig aortic endothelial cell extracts. Isoglobotrihexosylceramide 3 synthase mRNA was expressed in all pig tissues tested whether derived from wild type or GalT knockout animals. CONCLUSIONS These results confirm unequivocally the absence of terminal Gala13Gal disaccharides in GalT knockout endothelial cells. Future work will have to focus on other mechanisms responsible for xenograft rejection in particular non Gala13Gal antibodies and cellular responses.