Abstract
Sialylated glycans serve as key elements of receptors for many viruses, bacteria, and bacterial toxins. The microbial recognition and their binding specificity can be affected by the linkage of the terminal sugar residue, types of underlying sugar chains, and the nature of the entire glycoconjugate. Owing to the pathobiological significance of sialylated glycans, we have engineered Chinese hamster ovary (CHO) cells to secrete mucin-type immunoglobulin-fused proteins carrying terminal α2,3- or α2,6-linked sialic acid on defined O-glycan core saccharide chains. Besides stably expressing P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b cDNA (PSGL-1/mIgG2b), CHO cells were stably transfected with plasmids encoding glycosyltransferases to synthesize core 2 (GCNT1), core 3 (B3GNT6), core 4 (GCNT1 and B3GNT6), or extended core 1 (B3GNT3) chains with or without the type 1 chain-encoding enzyme B3GALT5 and ST6GAL1. Western blot and liquid chromatography-mass spectrometry analysis confirmed the presence of core 1, 2, 3, 4, and extended core 1 chains carrying either type 1 (Galβ3GlcNAc) or type 2 (Galβ4GlcNAc) outer chains with or without α2,6-linked sialic acids. This panel of recombinant mucins carrying a repertoire of sialylated O-glycans will be important tools in studies aiming at determining the fine O-glycan binding specificity of sialic acid-specific microbial adhesins and mammalian lectins.
Highlights
O-GalNAc glycosylation of proteins contributes to protein structure and stability, and is a crucial element in protein-carbohydrate interactions [1]
Using the mucin-type fusion protein P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b (PSGL-1/mIgG2b) as a probe, we have recently defined the in vivo specificity of O-glycan core chain glycosyltransferases transiently expressed in Chinese hamster ovary (CHO)-K1 cells [18]
CHO cells express -N-acetylgalactosaminide 2,6-sialyltransferase (ST6GALNAC4 and ST6GALNAC6), which was reflected in the O-glycans produced on our recombinant mucin-type fusion protein [31]
Summary
O-GalNAc glycosylation of proteins contributes to protein structure and stability, and is a crucial element in protein-carbohydrate interactions [1]. Using the mucin-type fusion protein P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b (PSGL-1/mIgG2b) as a probe, we have recently defined the in vivo specificity of O-glycan core chain glycosyltransferases transiently expressed in CHO-K1 cells [18]. CHO cells express -N-acetylgalactosaminide 2,6-sialyltransferase (ST6GALNAC4 and ST6GALNAC6), which was reflected in the O-glycans produced on our recombinant mucin-type fusion protein [31] They lack β-galactoside 2,6-sialyltransferase (ST6GAL) activity that transfers sialic acids to the penultimate galactoses with an 2,6-linkage [28]. Competition between endogenous and exogenously expressed glycosyltransferases for the same precursor chain may explain O-glycan heterogeneity We believe that this panel of recombinant mucins carrying a repertoire of sialylated O-glycans will be an important tool of studies aimed at determining the fine O-glycan binding specificity of sialic acid-specific microbial adhesins and mammalian lectins
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