Rapid and sensitive GC/MS characterization of glycolipid released Galα1,3Gal-terminated oligosaccharides from small organ specimens of a single pig

Pig to human xenotransplantation is considered a possible solution to the prevailing chronic lack of human donor organs for allotransplantation. The Gal alpha 1,3Gal determinant is the major porcine xenogeneic epitope causing hyperacute rejection following human antibody binding and complement activation. In order to characterize the tissue distribution of Gal alpha 1,3Gal-containing and blood group-type glycosphingolipids in pig, acid and nonacid glycosphingolipids were isolated from the kidney, small intestine, spleen, salivary gland, liver, and heart of a single pig obtained from a semi-inbred strain homozygous at the SLA locus. Glycolipids were analyzed by thin-layer immunostaining using monoclonal antibodies, and following ceramide glycanase cleavage as permethylated oligosaccharides by gas chromatography, gas chromatography-mass spectrometry, and matrix-assisted laser desorption/ionization mass spectrometry. The kidney contained large amounts of Gal alpha 1,3Gal-containing penta- and hexasaccharides having carbohydrate sequences consistent with the Gal alpha 1,3nLc(4) and Gal alpha 1,3Le(x) structures, respectively. The former structure was tentatively identified in all organs by GC/MS. The presence of extended Gal alpha 1,3Gal-terminated structures in the kidney and heart was suggested by antibody binding, and GC/MS indicated the presence of a Gal alpha 1,3nLc(6) structure in the heart. The kidney, spleen, and heart contained blood group H pentaglycosylceramides based on type 1 (H-5-1) and type 2 (H-5-2) chains, and H hexaglycosylceramides based on the type 4 chain (H-6-4). In the intestine H-5-1 and H-6-4 were expressed, in the salivary gland H-5-1 and H-5-2, whereas only the H-5-1 structure was identified in the liver. Blood group A structures were identified in the salivary gland and the heart by antibody binding and GC/MS, indicating an organ-specific expression of blood group AH antigens in the pig.