The Molecular Basis for Galα(1,3)Gal Expression in Animals with a Deletion of the α1,3Galactosyltransferase Gene

Abstract

The production of homozygous pigs with a disruption in the GGTA1 gene, which encodes α1,3galactosyltransferase (α1,3GT), represented a critical step toward the clinical reality of xenotransplantation. Unexpectedly, the predicted complete elimination of the immunogenic Galα(1,3)Gal carbohydrate epitope was not observed as Galα(1,3)Gal staining was still present in tissues from GGTA1−/− animals. This shows that, contrary to previous dogma, α1,3GT is not the only enzyme able to synthesize Galα(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 Galα(1,3)Gal synthesized via the isoglobo series pathway, thus demonstrating that mouse iGb3S is an additional enzyme capable of synthesizing the xenoreactive Galα(1,3)Gal epitope. Galα(1,3)Gal synthesized by iGb3S, in contrast to α1,3GT, was resistant to down-regulation by competition with α1,2fucosyltransferase. Moreover, Galα(1,3)Gal synthesized by iGb3S was immunogenic and elicited Abs in GGTA1 −/− mice. Galα(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.

Modulation of human natural killer T cell ligands on TLR-mediated antigen-presenting cell activation

“…We did not detect any amplification of iGb3 synthase, consistent with reports of it being a pseudogene in humans (data not shown and ref. 22). Similar results were obtained by using LPS matured DCs (data not shown).…”

Identification of a GH110 Subfamily of α1,3-Galactosidases

“…40). Importantly, while the use of animals deficient in the ␣1,3-galactosyltransferase may appear to be the most attractive option for animal to human xenotransplantation, studies have suggested that some tissues, including the vascular endothelium of deficient animals, may in fact still express minor quantities of Gal␣1-3Gal epitopes (41,42). The most likely explanation for this has been identified as expression of the isogloboside glycolipid (Gal␣1-3Gal␤1-4Glc␤1-Cer) (42), which is controlled by another homologous glycosyltransferase of the CAZy GT6 family, the iGb3 synthase (UDP-Gal:lactosylceramide ␣1,3-galactosyltransferase) (43).…”

Progress and prospects: genetic engineering in xenotransplantation

“…Thus, there is a possibility that iGb3S may also be present in other species and may therefore account for the residual aGal expression in GalT-KO animals. 3 Along these lines, low levels of iGb3S mRNA have been detected both in the GalT-KO mouse and the pig thymus and lung, as well as in pECs. The results of immunization of GalT-KO mice with cells transfected with iGb3S, in comparison to GalT, suggest that aGal synthesized by iGb3S is as immunogenic as GalT.…”

“…Milland et al 3 Gene inactivation GalT GalT-KO pig Complementarity breeding and SCNT in GalT-KO pig production Nottle et al 4 Gene inactivation GalT GalT-KO pig heart Baboon No improvement in coagulation problems in the GalT-KO context Ezzelarab et al 5 Gene inactivation GalT PBMC from WT vs GalT-KO pig…”

“…The targets of these antibodies are potentially other forms of Gal epitopes and a myriad of xenoantigens that generate induced antibodies. The deletion of the iGb3S gene 3,33 or the expression of EndoGalC 1 in the GalT À/À background could result in further reduction in Gal epitope expression.…”