Characterization of novel antibodies that recognize sialylated keratan sulfate and lacto-N-fucopentaose I on human induced pluripotent cells: comparison with existing antibodies. (14th November 2022)

• Record Type: Journal Article
• Authors: Nakao, Hiromi; Yamaguchi, Tomoko; Kawabata, Kenji; Higashi, Katsuaki; Nonaka, Motohiro; Tuiji, Makoto; Nagai, Yuko; Toyoda, Hidenao; Yamaguchi, Yoshiki; Kawasaki, Nobuko; Kawasaki, Toshisuke
• Is Part Of: Glycobiology. Volume 33:Number 2(2023)
• Page Start: 150
• Page End: 164
• Publication Date: 2022-11-14
• Subjects: cytotoxic antibody -- hiPSCs -- lacto-N-fucopentaose I -- podocalyxin -- poly-N-acetyllactosamine; Glycoproteins -- Periodicals; Glycolipids -- Periodicals; Glycoconjugates -- Periodicals; 572.567
• DOI: 10.1093/glycob/cwac074
• Languages: English
• ISSNs: 0959-6658

Abstract

Abstract: 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: Siaα2–3Galβ1–3GlcNAc(6S)β1–3Galβ1–4GlcNAc(6S)β1 (R-6C), Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1 (R-13E), Galβ1–4GlcNAc(6S)β1–3Galβ1–4GlcNAc(6S)β1 (R-10G), and Fucα1–2Galβ1–3GlcNAβ1–3Galβ1–4Glc (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 (Galβ1–3GlcNAc) at their nonreducing termini, followed by a type 2 structure (Galβ1–4GlcNAc); 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.