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

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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.

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Abbreviations

2-AB:

2-aminobenzamide

ABS:

α-sialidase from Arthrobacter ureafaciens

Ara:

Arabinose

BTG:

β-galactosidase from bovine testes

CD:

Circular dichroism

Fuc:

Fucose

Gal:

Galactose

GalN:

Galactosamine

GalNAc:

N-acetylgalactosamine

Glc:

Glucose

GlcN:

Glucosamine

GlcNAc:

N-acetylglucosamine

HPAEC:

High-pH anion-exchange chromatography

Man:

Mannose

MALDI-TOF/MS:

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

NeuAc:

N-acetylneuraminic acid

NeuGc:

N-glycolylneuraminic acid

NP-HPLC:

Normal-phase high-performance liquid chromatography

OM:

Ovomucoid

OT:

Ovotransferrin

PAD:

Pulsed amperometric detector

SDS-PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SPH:

Hexosaminidase from Streptococcus pneumonia

TFA:

Trifluoroacetic acid

TFMS:

Trifluoromethanesulfonic acid

Xyl:

Xylose

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Acknowledgments

This research was supported by the Korea Food Research Institute and the Chung-Ang University Excellent Student Scholarship.

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Authors and Affiliations

  1. Physical Pharmacy Laboratory, College of Pharmacy, Chung-Ang University, 221 Huksuk-dong, Dongjak-ku, Seoul, 156-756, Korea

    Hye Seong Hwang, Byung Sun Kim, Heajin Park & Ha Hyung Kim

  2. Functional Materials Research Group, Korea Food Research Institute, Seongnam, Kyonggi, 463-746, Korea

    Ho-Young Park & Hee-Don Choi

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  1. Hye Seong Hwang
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Correspondence to Ha Hyung Kim.

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Hwang, H.S., Kim, B.S., Park, H. et al. Type and branched pattern of N-glycans and their structural effect on the chicken egg allergen ovotransferrin: a comparison with ovomucoid. Glycoconj J 31, 41–50 (2014). https://doi.org/10.1007/s10719-013-9498-2

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