Nagai, Y.; Nakao, H.; Kojima, A.; Komatsubara, Y.; Ohta, Y.; Kawasaki, N.; Kawasaki, N.; Toyoda, H.; Kawasaki, T. Glycan Epitopes on 201B7 Human-Induced Pluripotent Stem Cells Using R-10G and R-17F Marker Antibodies. Biomolecules2021, 11, 508.
Nagai, Y.; Nakao, H.; Kojima, A.; Komatsubara, Y.; Ohta, Y.; Kawasaki, N.; Kawasaki, N.; Toyoda, H.; Kawasaki, T. Glycan Epitopes on 201B7 Human-Induced Pluripotent Stem Cells Using R-10G and R-17F Marker Antibodies. Biomolecules 2021, 11, 508.
We developed two human induced pluripotent stem cell (hiPSC)/human embryonic stem cell-specific glycan-recognizing mouse antibodies, R-10G and R-17F, using the Tic (JCRB1331) hiPSC line as an antigen. R-10G recognizes a low-sulfate keratan sulfate, and R-17F recognizes lacto-N-fucopentaose-1. To evaluate the general characteristics of stem cell glycans, we used the hiPSC line 201B7 (HPS0063), a prototype iPSC line. Using an R-10G affinity column, an R-10G-binding protein was isolated. The protein yielded a single but very broad band from 480 to 1,236 kDa by blue native gel electrophoresis. After trypsin digestion, the protein was identified as podocalyxin by liquid chromatography/mass spectrometry. According to Western blotting, the protein reacted with R-10G and R-17F. The R-10G positive band was resistant to digestion with glycan-degrading enzymes, including peptide N-glycanase, but the intensity of the band was decreased significantly by digestion with keratanase, keratanase II, and endo-β-galactosidase, suggesting the R-10G epitope to be a keratan sulfate. These results suggest that keratan sulfate-type epitopes are shared by hiPSCs. However, the keratan sulfate from 201B7 cells contained a polylactosamine disaccharide unit (Galβ1-4GlcNAc) at a significant frequency, whereas that from Tic cells consisted mostly of keratan sulfate disaccharide units (Galβ1-4GlcNAc(6S)). In addition, the abundance of the R-10G epitope was significantly lower in 201B7 cells than in Tic cells.
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