Structural characterization of alpha1,3-galactosyltransferase knockout pig heart and kidney glycolipids and their reactivity with human and baboon antibodies.
BACKGROUND
alpha1,3-galactosyltranferase knockout (GalT-KO) pigs have been established to avoid hyperacute rejection in GalT-KO pig-to-human xenotransplantation. GalT-KO pig heart and kidney glycolipids were studied focusing on elimination of Gal-antigens and whether novel antigens would appear. Non-human primates are used as pre-clinical transplantation experimental models. Therefore, sera from baboons transplanted with GalT-KO hearts were compared with human serum regarding reactivity with pig glycolipids.


METHODS
Neutral and acidic glycolipids were isolated from GalT-KO and WT pig hearts and kidneys. Glycolipid immune reactivity was tested on TLC plates using human affinity-purified anti-Gal Ig, anti-blood group monoclonal antibodies, lectins, and human serum as well as baboon serum collected before and after GalT-KO pig heart transplantations. Selected glycolipid fractions, isolated by HPLC, were structurally characterized by mass spectrometry and proton NMR spectroscopy.


RESULTS
GalT-KO heart and kidney lacked alpha3Gal-terminated glycolipids completely. Levels of uncapped N-acetyllactosamine precursor compounds, blood group H type 2 core chain compounds, the P1 antigen and the x(2) antigen were increased. Human serum antibodies reacted with Gal-antigens and N-glycolylneuraminic acid (NeuGc) in WT organs of which only the NeuGc reactivity remained in the GalT-KO tissues. A clear difference in reactivity between baboon and human antibodies with pig glycolipids was found. This was most pronounced for acidic, not yet identified, compounds in GalT-KO organs which were less abundant or lacking in the corresponding WT tissues.


CONCLUSIONS
GalT-KO pig heart and kidney completely lacked Gal glycolipid antigens whilst glycolipids synthesized by competing pathways were increased. Baboon and human serum antibodies showed a different reactivity pattern to pig glycolipid antigens indicating that non-human primates have limitations as a human pre-clinical model for immune rejection studies.
Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.
Mette
M
Diswall
Jonas
J
Angström
Hasse
H
Karlsson
Carol J
CJ
Phelps
David
D
Ayares
Susann
S
Teneberg
Michael E
ME
Breimer
D000818:Animals; D030801:Animals, Genetically Modified; D000906:Antibodies / Q000097:blood / Q000276:immunology*; D000941:Antigens / Q000276:immunology; D002240:Carbohydrate Sequence; D002855:Chromatography, Thin Layer; D005700:Galactosyltransferases / Q000235:genetics* / Q000378:metabolism; D006017:Glycolipids / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007668:Kidney / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010215:Papio / Q000276:immunology*; D013552:Swine / Q000235:genetics / Q000276:immunology
D000906:Antibodies; D000941:Antigens; D006017:Glycolipids; D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase