Characterization of novel antibodies that recognize sialylated keratan sulfate and lacto-N-fucopentaose I on human induced pluripotent cells: comparison with existing antibodies

This report describes the isolation and characterization of two new antibodies, R-6C (IgM) and R-13E (IgM), which were generated in C57BL/6 mice (Mus musculus) using the Tic (JCRB1331) human induced pluripotent cell (hiPSC) line as an antigen, and their comparisons with two existing antibodies, R-10G (IgG1) and R-17F (IgG1). Their epitopes were studied by western blotting after various glycosidase digestions, binding analyses using enzyme-linked immunosorbent assays (ELISAs) and microarrays with various synthetic oligosaccharides. The minimum epitope structures identified were: Sia alpha 2-3Gal beta 1-3GlcNAc(6S)beta 1-3Gal beta 1-4GlcNAc(6S)beta 1 (R-6C), Fuc alpha 1-2Gal beta 1-3GlcNAc beta 1-3Gal beta 1 (R-13E), Gal beta 1-4GlcNAc(6S)beta 1-3Gal beta 1-4GlcNAc(6S)beta 1 (R-10G), and Fuc alpha 1-2Gal beta 1-3GlcNA beta 1-3Gal beta 1-4Glc (lacto-N-fucopentaose I) (R-17F). Most glycoprotein epitopes are expressed as O-glycans . The common feature of these epitopes is the presence of an N-acetyllactosamine type 1 structure (Gal beta 1-3GlcNAc) at their nonreducing termini, followed by a type 2 structure (Gal beta 1-4GlcNAc); this arrangement comprises a type 1-type 2 motif. This motif is also shared by TRA-1-60, a traditional onco-fetal antigen. In contrast, the R-10G epitope has a type 2-type 2 motif. Among these antibodies, R-17F and R-13E exhibit cytotoxic activity toward hiPSCs. R-17F and R-13E exhibit extremely high similarity in the amino acid sequences in their complementarity-determining regions (CDRs), which is consistent with their highly similar glycan recognition. These antibodies are excellent tools for investigating the biological functions of glycoconjugates in hiPSCs/hESCs; they could be useful for the selection, isolation and selective killing of such undifferentiated pluripotent stem cells.