Structures of asparagine-linked oligosaccharides from hen egg-yolk antibody (IgY). Occurrence of unusual glucosylated oligo-mannose type oligosaccharides in a mature glycoprotein

Abstract

Asparagine-linked oligosaccharides present on hen egg-yolk immunoglobulin, termed IgY, were liberated from the protein by hydrazinolysis. After N-acetylation, the oligosaccharides were labelled with a UV-absorbing compound, p-aminobenzoic acid ethyl ester (ABEE). The ABEE-derivatized oligosaccharides were fractionated by anion exchange, normal phase and reversed phase HPLC, and their structures were determined by a combination of sugar composition analysis, methylation analysis, negative ion FAB-MS, 500 MHz 1H-NMR and sequential exoglycosidase digestions. IgY contained monoglucosylated oligomannose type oligosaccharides with structures of Glc alpha 1-3Man7-9-GlcNAc-GlcNAc, oligomannose type oligosaccharides with the size range of Man5-9GlcNAc-GlcNAc, and biantennary complex type oligosaccharides with core region structure of Man alpha 1-6(+/- GlcNAc beta 1-4)(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(+/- Fuc alpha 1-6)GlcNAc. The glucosylated oligosaccharides, Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2, have not previously been reported in mature glycoproteins from any source.

Glycan-dependent and -independent Interactions Contribute to Cellular Substrate Recruitment by Calreticulin

“…IgY, the avian homolog of mammalian IgG, was used as a substrate protein because it is one of the few mature secreted proteins that contains an appreciable level of monoglucosylated glycans (ϳ25%) (32). Gel filtration purified IgY was enriched on a calreticulin column (IgY-EN) and used in BLI assays, which revealed the rapid association and dissociation kinetics of the calreticulin-IgY-EN interaction ( Fig.…”

N-Glycan Structures of Pigeon IgG

“…It should be pointed out that 61.7% of the high mannose-type glycans in pigeon IgG are mono-glucosylated as found in chicken (serum, egg yolk) (41) and quail (egg yolk) IgGs (43). Specifically, we demonstrated that pigeon IgG-CH3 glycopeptides contain only high mannosetype oligosaccharides.…”

“…The data are therefore consistent with the presence of a Glc residue at one of the non-reducing termini of a Man 9 GlcNAc 2 that would block the digestion of the Man residues (later to be shown to be on the ␣3-arm), allowing a removal of only 4 or 5 Man residues from the ␣6-arm. The presence of a monoglucosylated Man 9 GlcNAc 2 along with Man 9 GlcNAc 2 as the major glycoforms has been reported for chicken and quail IgG (41)(42)(43) and may be a characteristic of avian IgG. Further characterization based on two-dimensional HPLC mapping of the PA-derivatives confirmed this structure (see below) and has further resolved Hex 9 HexNAc 2 into Man 9 GlcNAc 2 (n-6, major component) and Glc 1 Man 8 GlcNAc 2 (n-7, minor component), along with Glc 1 Man 9 GlcNAc 2 (n-8), as the predominant high mannose-type structures.…”

“…This might be explained that the sites for complextype N-glycans on these avian IgG are more exposed and accessible by ''processing'' enzymes, which may form more branched, longer N-glycans. The presence of triantennary Nglycans is unique in pigeon IgG, as it has not been seen in chicken and quail IgGs (41)(42)(43).…”

Synthesis and characterization of biotin derivatives as multifunctional oligosaccharide tags

“…2,3 Because carbohydrates lack chromophores, they have been labeled in the past with UV-active or fluorescent [4][5][6][7][8] tags to improve detectability during chromatography. [9][10][11][12][13] The label is most commonly introduced by reductive amination. 14,15 Our group has previously synthesized a prototypical multifunctional small-molecule tag ( Figure 1) for covalent introduction into oligosaccharides which helps to increase the UV sensitivity, separation and isolation of the labeled oligosaccharides.…”