Structural Analysis of Murine Zona Pellucida Glycans

doi:10.1074/jbc.275.11.7731

Structural Analysis of Murine Zona Pellucida Glycans

EVIDENCE FOR THE EXPRESSION OF CORE 2-TYPE O-GLYCANS AND THE Sd^a ANTIGEN*

From the ^‡Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AY, United Kingdom and ^§Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 23501

Abstract

Murine sperm initiate fertilization by binding to specific oligosaccharides linked to the zona pellucida, the specialized matrix coating the egg. Biophysical analyses have revealed the presence of both high mannose and complex-type N-glycans in murine zona pellucida. The predominant high mannose-type glycan had the composition Man₅GlcNAc₂, but larger oligosaccharides of this type were also detected. Biantennary, triantennary, and tetraantennary complex-type N-glycans were found to be terminated with the following antennae: Galβ1–4GlcNAc, NeuAcα2–3Galβ1–4GlcNAc, NeuGcα2–3Galβ1–4GlcNAc, the Sd^a antigen (NeuAcα2–3[GalNAcβ1–4]Galβ1–4GlcNAc, NeuGcα2–3[GalNAcβ1–4]Galβ1–4GlcNAc), and terminal GlcNAc. Polylactosamine-type sequence was also detected on a subset of the antennae. Analysis of the O-glycans indicated that the majority were core 2-type (Galβ1–4GlcNAcβ1–6[Galβ1–3]GalNAc). The β1–6-linked branches attached to these O-glycans were terminated with the same sequences as the N-glycans, except for terminal GlcNAc. Glycans bearing Galβ1–4GlcNAcβ1–6 branches have previously been suggested to mediate initial murine gamete binding. Oligosaccharides terminated with GalNAcβ1–4Gal have been implicated in the secondary binding interaction that occurs following the acrosome reaction. The significant implications of these observations are discussed.

Footnotes

↵* This work was supported by a grant from the Biotechnology and Biological Sciences Research Council (to H. R. M. and A. D.) and a grant from the Wellcome Trust (to H. R. M. and A. D.). This study was also supported by the Jeffress Trust (to G. F. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ To whom correspondence should be addressed: Tel.: 44 171 594 5219; Fax: 44 171 225 0458: E-mail: a.dell@ic.ac.uk (A. Dell and H. R. Morris) or Tel.: 757 446 5650; Fax: 757 624 2270; E-mail: gfc@ borg.evms.edu (G. F. Clark).
↵2 J. Bleil, personal communication.
↵3 A. Thall, personal communication.
↵4 M. Patankar and G. Clark, unpublished observation.
Abbreviations:

mZP

murine zona pellucida

FAB

fast atom bombardment

GC

gas chromatography

MS

mass spectrometry

ZP

zona pellucida

PNGase F

peptideN-glycosidase F

Hex

hexose

HexNAc

N-acetylhexosamine

Fuc

fucose, NeuAc,N-acetylneuraminic acid

NeuGc

N-glycolylneuraminic acid

Gal

galactose

GalNAcitol

reduced N-acetylgalactosamine

GlcNAc

N-acetylglucosamine

Man

mannose

GalNAc

N-acetylgalactosamine

Sd^a

NeuAcα2–3[GalNAcβ1–4]Galβ1–4GlcNAc

BSA

bovine serum albumin
- Received October 22, 1999.
- Revision received December 18, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.