The molecular basis for Galα(1,3)Gal expression in animals with a deletion of the α1,3galactosyltransferase gene

The production of homozygous pigs with a disruption in the GGTA1 gene, which encodes alpha 1,3galactosyltransferase (alpha 1,3GT), represented a critical step toward the clinical reality of xenotransplantation. Unexpectedly, the predicted complete elimination of the immunogenic Gal alpha(1,3)Gal carbohydrate epitope was not observed as Gal alpha(1,3)Gal staining was still present in tissues from GGTA1(-/-) animals. This shows that, contrary to previous dogma, alpha 1,3GT is not the only enzyme able to synthesize Gal alpha(1,3)Gal. As iGb3 synthase (iGb3S) is a candidate glycosyltransferase, we cloned iGb3S cDNA from GGTA1(-/-) mouse thymus and confirmed mRNA expression in both mouse and pig tissues. The mouse iGb3S gene exhibits alternative splicing of exons that results in a markedly different cytoplasmic tail compared with the rat gene. Transfection of iGb3S cDNA resulted in high levels of cell surface Gal alpha(1,3)Gal synthesized via the isoglobo series pathway, thus demonstrating that mouse iGb3S is an additional enzyme capable of synthesizing the xenoreactive Gal alpha(1,3)Gal epitope. Gal alpha(1,3)Gal synthesized by iGb3S, in contrast to alpha 1,3GT, was resistant to down-regulation by competition with alpha 1,2fucosyltransferase. Moreover, Gal alpha(1,3)Gal synthesized by iGb3S was immunogenic and elicited Abs in GGTA1(-/-) mice. Gal alpha(1,3)Gal synthesized by iGb3S may affect survival of pig transplants in humans, and deletion of this gene, or modification of its product, warrants consideration.