Characterization of glycoproteins expressing the blood group H type 1 epitope on human induced pluripotent stem (hiPS) cells

摘要

AbstractRecently, we established two mouse monoclonal antibodies (R-10G and R-17F). The R-17F antibody (IgG1 subtype) exhibited a strong cytotoxic effect on hiPS/ES cells. The R-17F antigen isolated from a total lipid extract of hiPS (Tic) cells was identified as LNFP I (Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4Glc). In the present study, R-17F binding proteins were isolated from hiPS (Tic) cell lysates with an affinity column of R-17F. They gave one major R-17F positive band around 250?kDa, and several minor bands between 150?kDa and 25?kDa. The former band was identified as podocalyxin by LC/MS/MS after SDS-PAGE. Hapten inhibition studies on R-17F binding to R-17F column-purified proteins with various synthetic oligosaccharides revealed that the blood group H type 1 triaose structure (Fucα1–2Galβ1–3GlcNAc) was the predominant epitope on all the R-17F binding proteins. These bands disappeared completely on digestion with α1–2 fucosidase, but not with α1–3/4 fucosidase. Upon PNGase F digestion, the R-17F positive band around and above 250?kDa did not show any change, while the minor bands between 150?kDa and 25?kDa disappeared completely, suggesting that the epitope is expressed onN-glycans in the latter and probably onO-glycans in the former. These results, together with those obtained in our previous studies on R-10G (Kawabeet al.Glycobiology, 23, 322–336 (2013)), indicated that both R-10G and R-17F epitopes are carried on the same podocalyxin molecule. The R-17F epitopes on these glycoproteins expressed on hiPS cells could be associated with the molecular mechanism underlying the carbohydrate-mediated cytotoxic activity of R-17F.]]>