Characterization of novel antibodies that recognize sialylated keratan sulfate and lacto-N-fucopentaose I on human induced pluripotent cells: comparison with existing antibodies
Characterization of novel antibodies that recognize sialylated keratan sulfate and lacto-N-fucopentaose I on human induced pluripotent cells: comparison with existing antibodies
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识别人诱导多能细胞上唾液酸化角质素硫酸盐和乳糖-N-岩藻五糖 I 的新型抗体的表征：与现有抗体的比较
DOI：
10.1093/glycob/cwac074
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发表时间：
2023
期刊：
影响因子：
4.3
通讯作者：
Toshisuke Kawasaki
中科院分区：
文献类型：
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作者：
Hiromi Nakao;Tomoko Yamaguchi;Kenji Kawabata;Katsuaki Higashi;Motohiro Nonaka;Makoto Tsuiji;Yuko Nagai;Hidenao Toyoda;Yoshiki Yamaguchi;Nobuko Kawasaki;Toshisuke Kawasaki
本报告描述了使用Tic（JCRB 1331）人诱导多能细胞（hiPSC）系作为抗原在C57 BL/6小鼠（小家鼠）中产生的两种新抗体R-6C（IgM）和R-13 E（IgM）的分离和表征，以及它们与两种现有抗体R-10 G（IgG 1）和R-17 F（IgG 1）的比较。它们的表位进行了研究后，各种糖苷酶消化，结合分析，使用酶联免疫吸附试验（ELISA）和微阵列与各种合成寡糖的蛋白质印迹。鉴定的最小表位结构为：Siaα2-3Galβ1- 3GlcNAc（6S）β1-3Galβ1-4GlcNAc（6S）β1（R-6C）、Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1（R-1 - 3 E）、Galβ1-4GlcNAc（6S）β1-3Galβ1- 4GlcNAc（6S）β1（R-10 G）和Fucα1-2Galβ1 - 3GlcNAc β1 -3Galβ1-4Glc（乳糖-N-岩藻五糖I）（R-17 F）。大多数糖蛋白表位表达为0-聚糖。这些表位的共同特征是在其非还原末端存在1型N-乙酰乳糖胺结构（Galβ1-3GlcNAc），随后是2型结构（Galβ1-4GlcNAc）;这种排列包含1型-2型基序。TRA-1-60是一种传统的癌胎儿抗原，也有这种基序。相反，R-10 G表位具有2型-2型基序。在这些抗体中，R-17 F和R-13 E表现出对hiPSC的细胞毒性活性。R-17 F和R-13 E在其互补决定区（CDR）的氨基酸序列中表现出极高的相似性，这与其高度相似的聚糖识别一致。这些抗体是用于研究hiPSC/hESC中糖缀合物的生物学功能的极好工具;它们可用于选择、分离和选择性杀伤此类未分化的多能干细胞。
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.