Dataset Information

A Panel of Recombinant Mucins Carrying a Repertoire of Sialylated O-Glycans Based on Different Core Chains for Studies of Glycan Binding Proteins.

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 ?2,3- or ?2,6-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 (Galb3GlcNAc) or type 2 (Galb4GlcNAc) outer chains with or without ?2,6-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.

SUBMITTER: Cherian RM

PROVIDER: S-EPMC4598776 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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A Panel of Recombinant Mucins Carrying a Repertoire of Sialylated O-Glycans Based on Different Core Chains for Studies of Glycan Binding Proteins.

Cherian Reeja Maria RM Jin Chunsheng C Liu Jining J Karlsson Niclas G NG Holgersson Jan J

Biomolecules 20150812 3

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 ...[more]

PMID: 26274979

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Project description:FUT8 is an essential α-1,6-fucosyltransferase that fucosylates the innermost GlcNAc of N-glycans, a process called core fucosylation. In vitro, FUT8 exhibits substrate preference for the biantennary complex N-glycan oligosaccharide (G0), but the role of the underlying protein/peptide to which N-glycans are attached remains unclear. Here, we explored the FUT8 enzyme with a series of N-glycan oligosaccharides, N-glycopeptides, and an Asn-linked oligosaccharide. We found that the underlying peptide plays a role in fucosylation of paucimannose (low mannose) and high-mannose N-glycans but not for complex-type N-glycans. Using saturation transfer difference (STD) NMR spectroscopy, we demonstrate that FUT8 recognizes all sugar units of the G0 N-glycan and most of the amino acid residues (Asn-X-Thr) that serve as a recognition sequon for the oligosaccharyltransferase (OST). The largest STD signals were observed in the presence of GDP, suggesting that prior FUT8 binding to GDP-β-l-fucose (GDP-Fuc) is required for an optimal recognition of N-glycans. We applied genetic engineering of glycosylation capacities in CHO cells to evaluate FUT8 core fucosylation of high-mannose and complex-type N-glycans in cells with a panel of well-characterized therapeutic N-glycoproteins. This confirmed that core fucosylation mainly occurs on complex-type N-glycans, although clearly only at selected glycosites. Eliminating the capacity for complex-type glycosylation in cells (KO mgat1) revealed that glycosites with complex-type N-glycans when converted to high mannose lost the core Fuc. Interestingly, however, for erythropoietin that is uncommon among the tested glycoproteins in efficiently acquiring tetra-antennary N-glycans, two out of three N-glycosites obtained Fuc on the high-mannose N-glycans. An examination of the N-glycosylation sites of several protein crystal structures indicates that core fucosylation is mostly affected by the accessibility and nature of the N-glycan and not by the nature of the underlying peptide sequence. These data have further elucidated the different FUT8 acceptor substrate specificities both in vitro and in vivo in cells, revealing different mechanisms for promoting core fucosylation.

Dataset Information

A Panel of Recombinant Mucins Carrying a Repertoire of Sialylated O-Glycans Based on Different Core Chains for Studies of Glycan Binding Proteins.

Publications

A Panel of Recombinant Mucins Carrying a Repertoire of Sialylated O-Glycans Based on Different Core Chains for Studies of Glycan Binding Proteins.

Similar Datasets

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