Rapid and sensitive GC/MS characterization of glycolipid released Galalpha1,3Gal-terminated oligosaccharides from small organ specimens of a single pig

Pig to human xenotransplantation is considered a possible solution to theprevailing chronic lack of human donor organs for allotransplantation. TheGalalpha1,3Gal determinant is the major porcine xenogeneic epitope causinghyperacute rejection following human antibody binding and complementactivation. In order to characterize the tissue distribution ofGalalpha1,3Gal-containing and blood group- type glycosphingolipids in pig,acid and nonacid glycosphingolipids were isolated from the kidney, smallintestine, spleen, salivary gland, liver, and heart of a single pigobtained from a semi-inbred strain homozygous at the SLA locus. Glycolipidswere analyzed by thin-layer immunostaining using monoclonal antibodies, andfollowing ceramide glycanase cleavage as permethylated oligosaccharides bygas chromatography, gas chromatography-mass spectrometry, and matrix-assisted laser desorption/ionization mass spectrometry. The kidneycontained large amounts of Galalpha1,3Gal-containing penta- andhexasaccharides having carbohydrate sequences consistent with theGalalpha1,3nLc4and Galalpha1,3Lexstructures, respectively. The formerstructure was tentatively identified in all organs by GC/MS. The presenceof extended Galalpha1,3Gal-terminated structures in the kidney and heartwas suggested by antibody binding, and GC/MS indicated the presence of aGalalpha1,3nLc6structure in the heart. The kidney, spleen, and heartcontained blood group H pentaglycosylceramides based on type 1 (H-5-1) andtype 2 (H-5-2) chains, and H hexaglycosylceramides based on the type 4chain (H-6-4). In the intestine H-5-1 and H-6-4 were expressed, in thesalivary gland H-5-1 and H-5-2, whereas only the H-5-1 structure wasidentified in the liver. Blood group A structures were identified in thesalivary gland and the heart by antibody binding and GC/MS, indicating anorgan- specific expression of blood group AH antigens in the pig.