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Glycobiology Advance Access published online on December 15, 2004
Glycobiology, doi:10.1093/glycob/cwi022
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© Oxford University Press 2004; all rights reserved.
Received August 27, 2004
Revised November 30, 2004
Accepted December 4, 2004

Article

Analysis of N-linked glycans of porcine zona pellucida glycoprotein ZPA by MALDI-ToF mass spectrometry: a contribution to understanding zona pellucida structure

Dorothee von Witzendorff 1, Mahnaz Ekhlasi-Hundrieser 1, Zuzanna Dostalova 1, Martin Resch 2, Detlef Rath 3, Hans-Wilhelm Michelmann 4, and Edda Töpfer-Petersen 1*

1 Institute for Reproductive Medicine, School of Veterinary Medicine Hannover, Foundation, Bünteweg 15, 30559 Hannover, Germany
2 Shimadzu Deutschland GmbH, Albert-Hahn-Str. 6-10, 47269 Duisburg, Germany
3 Institute for Animal Breeding, Hoeltystr. 10, 31535 Neustadt, Germany
4 Department of Gynaecology and Obstetrics, Georg August University of Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany

* To whom correspondence should be addressed.
Edda Töpfer-Petersen, E-mail: Edda.Toepfer-Petersen{at}tiho-hannover.de


  Abstract

The mammalian oocyte is encased by a transparent extracellular matrix, the zona pellucida, which consists of three glycoproteins ZPA, ZPB and ZPC. The glycan structures of the porcine zona pellucida and the complete N-glycosylation pattern of the ZPB/ZPC oligomer has been recently described. Here we report the N-glycan pattern and N-glycosylation sites of the porcine zona pellucida glycoprotein ZPA of an immature oocyte population as determined by a mass spectrometric approach. Ingel deglycosylation of the electrophoretically separated ZPA protein and comparison of the pattern obtained from the native, the desialylated and the endo-ßgalactosidase-treated glycoprotein allowed the assignment of the glycan structures by MALDI-ToF-MS by considering the reported oligosaccharide structures. The major N-glycans are neutral bi-antennary complex structures containing one or two terminal galactose residues. Complex N-glycans carrying N-acetyllactosamine repeats are minor components and are mostly sialylated. A significant signal corresponding to a high-mannose type chain appeared in the three glycan maps. MS-MS analysis confirmed its identity as a pentamannosyl N-glycan. By the combination of tryptic digestion of the endo-ß-galactosidase-treated ZP glycoprotein mixture and in-gel digestion of ZPA with lectin-affinity chromatography and reverse-phase HPLC five of six N-glycosylation sites at Asn84/93, Asn268, Asn316, Asn323 and Asn530 were identified by MS. Only one sites was found to be glycosylated in the N-terminal tryptic glycopeptide with Asn84/93. N-glycosidase F treatment of the isolated glycopeptides and MS analysis resulted in the identification of the corresponding deglycosylated peptides. By in-gel deglycosylation of the electrophoretically separated large tryptic glycopeptides the high-mannose type chain was mapped to the position at Asn268.

Keywords: glycosylation sites/MALDI-ToF-MS/N-linked glycan chains/zonapellucida.
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