The alpha1,3GalT knockout/alpha1,2FucT transgenic pig does not appear to have an advantage over the alpha1,3GalT knockout pig with respect to glycolipid reactivity with human serum antibodies.
BACKGROUND
The human H-transferase (α2FucT) was introduced in Gal-negative pigs to produce pig organs not only free from Gal-antigens, but also in which the uncapped N-acetyllactosamine precursor had been transformed into non-xenogenic blood group H type 2 compounds. This work is the first descriptive analysis of glycolipids from the GalT-KO/FucT-TG pig. The aim was to investigate the cell membrane antigens in GalT-KO/FucT-TG tissues to explore its efficacy as an organ donor. Also, detailed knowledge on the correlation between the cellular glycosyltransferase configuration and the resulting carbohydrate phenotype expression is valuable from a basic glycobiological perspective.


METHODS
Neutral and acidic glycolipids from GalT-KO/FucT-TG small intestine were compared with glycolipids from two wildtype and two GalT-KO pig intestines. Glycolipid reactivity was tested on thin layer chromatography plates using chemical reagents, antibodies, lectins, and human serum. Structural characterization of neutral glycolipids was performed by LC-ESI/MS and proton NMR spectroscopy.


RESULTS
Characterization of the glycolipid expression in GalT-KO/FucT-TG intestine showed absence of Gal antigens and decreased/unchanged levels of the N-acetyllactosamine precursor and the blood group H type 2 expression, when compared with the wildtype. The reactivity of human serum antibodies to GalT-KO/FucT-TG derived glycolipids was similar or slightly elevated when compared with GalT-KO glycolipids. Results from LC-ESI/MS and proton NMR spectroscopy revealed no established neutral xenogenic antigens in the GalT-KO/FucT-TG pig, and could thus not explain the immunologic reactivity to human serum antibodies. The antibody binding to acidic glycolipids is most likely to be explained by the abundance of N-glycolylneuraminic acid epitopes in pig tissues. Six neutral complex biantennary glycolipids with blood group H type 1, 2, Lewis(x) and Lewis(y) determinants were found, of which three were identified in this work for the first time. One of these was a nonaglycosylceramide with blood group H type 2 and lactosyl determinants linked to a lactotetraosyl core, and the other two were decaglycosylceramides with blood group H type 1 and H type 2 determinants linked to a neolactotetraosyl core, and Lewis(x) and blood group H type 1 determinants on a lactotetraosyl core, respectively.


CONCLUSIONS
Lipid-linked carbohydrate antigens in the GalT-KO/FucT-TG pig intestine showed no or minor qualitative difference when compared with GalT-KO pigs. The GalT-KO/FucT-TG pig did not appear to have an advantage over the GalT-KO pig with respect to reactivity with human antibodies from a xenotransplantation perspective.
Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy/Gothenburg University, Gothenburg, Sweden; Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.
Mette
M
Diswall
John
J
Benktander
Jonas
J
Ångström
Susann
S
Teneberg
Michael E
ME
Breimer
D000818:Animals; D030801:Animals, Genetically Modified; D000906:Antibodies / Q000097:blood* / Q000276:immunology; D000941:Antigens / Q000276:immunology; D015478:Antigens, Heterophile / Q000235:genetics / Q000276:immunology*; D005700:Galactosyltransferases / Q000172:deficiency / Q000276:immunology*; D006017:Glycolipids / Q000276:immunology*; D006801:Humans; D007136:Immunoglobulins / Q000378:metabolism*; D007421:Intestine, Small / Q000276:immunology; D034421:Sus scrofa; D013552:Swine; D014183:Transplantation, Heterologous
D000906:Antibodies; D000941:Antigens; D015478:Antigens, Heterophile; D006017:Glycolipids; D007136:Immunoglobulins; D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase
blood group ABO antigens; genetic modification; glycosphingolipids; pig; small intestine; xenotransplantation; α1,2fucosyltransferase transgenic; α1,3galactosyltransferase knockout