Survey of glycoantigens in cells from alpha1-3galactosyltransferase knockout pig using a lectin microarray.
BACKGROUND
Glycoantigens represent major obstacles to successful xenotransplantation. Even after the alpha1-3galactosyltransferase (GalT) gene knockout (GalT-KO) pigs were produced, non-Gal antigens continue to be present. This study reports on lectin blot analyses for endothelial cells (EC) and fibroblasts from GalT-KO pigs.


METHODS
Differences in glycoantigens that are produced on cell surfaces in humans and pigs were surveyed. Differences between ECs and fibroblasts from wild-type and GalT-KO pigs were also examined. EC and fibroblasts from GalT-KO pigs (heterozygous and homozygous) with N-acetylglucosaminyltransferase-III (GnT-III), a wild-type EC from the sibling, human EC lines, HUVEC (human EC from umbilical veins), & HAOEC (human EC from aortas), and human fibroblast line were used. EC and fibroblasts were cultured in gelatin-coated dishes for several days. After sonication and centrifugation, the supernatant protein from each cell was labeled with Cy3, applied to a lectin array and scanned with an SC Profiler, and analyzed using an Array Pro Analyzer.


RESULTS
The pig EC showed higher signals in Euonymus Europaeus (EEL) & Griffonia simplicifolia I-B(4) (GSI-B4), binds alpha-Gal, and in Wisteria Floribunda (WFA), Helix pomatia (HPA), Glycine max (SBA), & Griffonia simplicifolia I-A(4) (GSI-A4), binds GalNAc including the Thomsen-Friedenreich precursor (Tn)-antigen, while the human EC showed strong signals in Ulex europaeus I (UEA-I), Maackia amurensis (MAL), Erythrina cristagalli (ECA), & Trichosanthes japonica I (TJA-I) instead. The EC from the GalT-KO pig signals for EEL & GSI-B4 disappeared and those for Bauhinia purpurea alba (BPL), HPA, SBA, & GSI-A4 were greatly diminished as well, while it up-regulated signals for Sambucus Nigra (SNA), Sambucus sieboldiana (SSA), & TJA-I, bind alpha2-6 sialic acid, compared to the wild-type pig EC. Concerning fibroblasts, the signals for HPA, SBA, & GSI-A4 were the most intense in the wild-type, and the intensities for homozygous-KO were less, approaching those of humans. In addition, the order of the intensities, as detected by Arachis hypogaea (PNA) & Maclura pomifera (MPA), binding Galbeta1-2GalNAc, indicates that the Thomsen-Friedenreich (T)-antigen is likely present on pig fibroblasts.


CONCLUSIONS
It is possible that the T-antigen and Tn-antigen related to GalNAc are non-Gal antigens, but, fortunately, not only alpha-Gal but also GalNAc were found to be decreased in the KO-pig.
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan. miyagawa@orgtrp.med.osaka-u.ac.jp
Shuji
S
Miyagawa
Shunsaku
S
Takeishi
Aki
A
Yamamoto
Kosuke
K
Ikeda
Hitomi
H
Matsunari
Masao
M
Yamada
Masaru
M
Okabe
Eiji
E
Miyoshi
Masahiro
M
Fukuzawa
Hiroshi
H
Nagashima
D000818:Animals; D030801:Animals, Genetically Modified; D000941:Antigens / Q000737:chemistry* / Q000276:immunology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000276:immunology; D005700:Galactosyltransferases / Q000235:genetics* / Q000378:metabolism; D006084:Graft Rejection / Q000276:immunology; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000276:immunology*; D046228:Microarray Analysis / Q000379:methods*; D037121:Plant Lectins / Q000378:metabolism*; D012794:Sialic Acids / Q000737:chemistry / Q000276:immunology; D013552:Swine
D000941:Antigens; D008562:Membrane Glycoproteins; D037121:Plant Lectins; D012794:Sialic Acids; D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase