earticle

영어

The α-Gal epitope (Galα1,3Galα1,4GlcNAc-R), created by alpha-1,3-glycosyltransferase-1 (GT), is recognized by natural anti-α-1,3-galactose antibodies in human serum and responsible for hyperacute rejection (HAR) in pig-to-human/primate xenotransplantation. Although, the generation of GT knockout (KO) pigs has overcome hyperacute rejection, an isoglobotriosylceramide synthase (iGb3s) may produce additional alpha-1,3-terminated galactose residues on glycosphingolipid. This study was performed to investigate effects of iGb3s KO on the glycoproteomic characterization in the transgenic pig cells. We established and used 2 different transgenic cells such as GT-MCP/-MCP (GT/MCP) and GT-MCP/-MCP+iGb3s—/— (GT/MCP/iGb3s) cells. WT pig cells were used as a control. To characterize the expression of α-Gal epitope, we analyzed the cells by FACS using BS-I-B4 lectin antibody. The α-Gal epitope expression in all both TG cells was significantly down regulated as compared with WT (p<0.05). The iGb3s protein level was decreased in GT/MCP/iGb3s cells compared with GT/MCP cells. To analyze changes of glycoproteome level, LC-MS/MS analysis was performed in WT, GT/MCP, and GT/MCP/iGb3s cells. A total amount of glycopeptide in GT/MCP/iGb3s was increased as compared with that of GT/MCP. However, glycopeptide forms of gal-gal were reduced in GT/MCP/ iGb3s compared to both WT and GT/MCP. Our results demonstrated that additional KO of iGb3s gene reduced the expressions of α-Gal epitope and glycopeptide forms of gal-gal in GT/MCP. Further studies are needed to evaluate synergic effects of double gene knock out in order to minimize a HAR response after xenotransplantation.