Complete absence of the ?Gal xenoantigen and isoglobotrihexosylceramide in ?1,3galactosyltransferase knock-out pigs.

ABSTRACT: Anti-Gal?1,3Gal?-R natural antibodies are responsible for hyperacute rejection in pig-to-primate xenotransplantation. Although the generation of pigs lacking the ?1,3galactosyltransferase (GalT) has overcome hyperacute rejection, antibody-mediated rejection is still a problem. It is possible that other enzymes synthesize antigens similar to Gal?1,3Gal epitopes that are recognized by xenoreactive antibodies. The glycosphingolipid isoglobotrihexosylceramide (iGb?) represents such a candidate expressing an alternative Gal?1,3Gal epitope. The present work determined whether the terminal Gal?1,3Gal disaccharide is completely absent in Immerge pigs lacking the GalT using several different highly sensitive methods.The expression of Gal?1,3Gal 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 iGb? synthase mRNA was tested by RT-PCR in pig thymus, spleen, lymph node, kidney, lung, and liver tissue samples.Aortic endothelial cells derived from GalT knockout pigs expressed neither Gal?1,3Gal nor iGb? 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 Gal?1,3Gal in membranes of GalT knockout PAEC; iGb? was also totally absent, whereas a fucosylated form of iGb? 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.These results confirm unequivocally the absence of terminal Gal?1,3Gal disaccharides in GalT knockout endothelial cells. Future work will have to focus on other mechanisms responsible for xenograft rejection, in particular non-Gal?1,3Gal antibodies and cellular responses.

SUBMITTER: Puga Yung GL 

PROVIDER: S-EPMC3614413 | biostudies-literature | 2012 May-Jun

REPOSITORIES: biostudies-literature