Survey of glycoantigens in cells from alpha 1-3galactosyltransferase knockout pig using a lectin microarray

Background: Glycoantigens represent major obstacles to successful xenotransplantation. Even after the alpha 1-3galactosyltransferase (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 alpha 2-6 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 Gal beta 1-2GalNAc, indicates that the Thomsen-Friedenreich (T)-antigen is likely present on pig fibroblasts. Conclusion: 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.