B4GALT5-deficient CHO-Lec2 cells expressing human α1,4-galactosyltransferase: A glycoengineered cell model for studying Shiga toxin receptors

Human alpha 1,4-galactosyltransferase (A4galt) is a glycosyltransferase existing in humans as two isoforms, widespread A4galt (named A4G) and its rare variant with p.Q211E substitution (A4Gmut). Both isoforms produce Gb3 (Gal alpha 1 -> 4Gal beta 1 -> 4Glc-Cer) on glycosphingolipids and P1 glycotope (Gal alpha 1 -> 4Gal beta 1 -> 4GlcNAc-R) on glycoproteins, which serve as receptors for Shiga toxin types 1 and 2 (Stx1 and Stx2). Stx1 is bound by Gb3 and P1 glycotope, while Stx2 is recognized solely by Gb3. To elucidate the role of these receptors, CHO-Lec2 cells expressing human A4G and A4Gmut were modified by disrupting the hamster B4GALT5 gene using CRISPR/Cas9 technology. The B4GALT5 gene encodes beta 1,4-galactosyltransferase 5 (B4galt5), synthesizing lactosylceramide, the key substrate for Gb3 synthesis. Consequently, B4GALT5-deficient CHO-Lec2-expressing A4G and A4Gmut cells lacked Gb3 glycosphingolipid but retained the ability to synthesize glycoprotein-based P1 glycotope. Both B4GALT5-deficient CHO-Lec2 cells expressing A4G and A4Gmut demonstrated no binding of Stx1B and Stx2B. The cytotoxicity assay showed that B4GALT5-deficient CHO-Lec2 cells expressing A4G were completely resistant to Stx1 holotoxin while A4Gmut-expressing cells revealed reduced sensitivity to Stx2. The glycoengineered CHOLec2 cells obtained in this study provide a valuable model for studying receptors for Stxs, enabling a detailed assessment of their roles in toxin binding and cytotoxicity.