Type and branched pattern of N-glycans and their structural effect on the chicken egg allergen ovotransferrin: a comparison with ovomucoid

Abstract

Ovotransferrin (OT), a multifunctional glycoprotein with defensive and protective activities, accounts for approximately 13% of chicken egg white proteins and is known as a major egg-associated allergen along with ovomucoid (OM). In contrast to the well-characterized N-glycans of OM, the N-glycan structure of OT has not been reported. Here, using HPLC equipped with a fluorescence detector and mass spectrometric analysis in combination with exoglycosidase digestion, we investigated the N-glycan type and branched pattern of OT, and compared them with those of OM. The HPLC peak area was used to calculate the relative quantity (%) of each glycan. Seventeen N-glycans, including 11 glycans (1 core structure and 10 complex-type oligosaccharides), that commonly exist in ovotransferrin and ovomucoid were identified. Six characteristic glycans (2 truncated structures, 1 complex-type, and 3 hybrid-type oligosaccharides) in OT and eight characteristic glycans in OM were classified. OT contains the following branched complex-type structures: mono-(13.2%), bi-(23.9%), tri-(9.0%), tetra-(2.7%), and penta-(2.8%) antennary oligosaccharides. However, OM contained mostly tri-(33.5%) and penta-(31.2%) antennary oligosaccharides. The N-glycan-containing bisecting N-acetylglucosamine comprised 43.4% and 79.8% of the total glycans in OT and OM, respectively. Moreover, using circular dichroism analysis, we observed that the secondary structure of the deglycosylated OT is quite different from that of the intact protein. To our knowledge, this is the first study to analyze N-glycans in OT in comparison with those of OM.

Rapid screening for specific glycosylation and pathogen interactions on a 78 species avian egg white glycoprotein microarray

“…2). Furthermore, the presence of α-(2,3)-linked sialic acid on OVM was indicated by MAA binding, in agreement with previous reports 13, 33 . Also, WGA binding, which recognises GlcNAc and sialic acid (Table 1), was observed only for OVM and OVT.…”

“…Also, WGA binding, which recognises GlcNAc and sialic acid (Table 1), was observed only for OVM and OVT. The results for OVT and OVM correlated well with structures previously elucidated by MS and HPLC 13 . OVT N -linked oligosaccharides were mainly composed of GlcNAc (54%) and Man (43%) and some Gal residues (3.5%), while the structures reported for OVM were mostly complex-type, with dominating tri-, tetra- and penta-antennary structures 13 .…”

Structure characterization and IgE-binding of soybean 7S globulin after enzymatic deglycosylation

“…Our results were consistent with the changes reported by Shivanjali, who discovered that α-helix content of deglycosylated carboxypeptidase is lower than that of the native form. [30] Hwang [31] also noted that secondary structures of deglycosylated ovotransferrin were fairly different from native forms, showing that α-helix and random coil transformed into β-sheets, and this finding completely coincides with our results.…”

Glycan structure and serum half-life of recombinant CTLA4Ig, an immunosuppressive agent, expressed in suspension-cultured rice cells with coexpression of human β1,4-galactosyltransferase and human CTLA4Ig

“…Glycan analyses of hCTLA4Igs (100 μg) were performed according to a method that is described in detail in our papers [24][25][26]. Briefly, the samples were incubated in the presence of trypsin (Sigma, USA) and chymotrypsin (Sigma) in 10 mM Tris-HCl (pH 8.0) at 37°C for overnight; glycans were released after reaction with 0.1 mU of glycoamidase A (Seikagaku Kogyo, Japan) in 0.5 M citrate-phosphate buffer (pH 5.0) at 37°C overnight.…”