Glycobiology Advance Access originally published online on December 15, 2004
Glycobiology 2005 15(5):475-488; doi:10.1093/glycob/cwi022
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Glycobiology vol. 15 no. 5 © Oxford University Press 2004; all rights reserved.
Analysis of N-linked glycans of porcine zona pellucida glycoprotein ZPA by MALDI-TOF MS: a contribution to understanding zona pellucida structure
2 Institute for Reproductive Medicine, School of Veterinary Medicine Hannover, Foundation, Bünteweg 15, 30559 Hannover, Germany; 3 Shimadzu Deutschland GmbH, Albert-Hahn-Str. 6-10, 47269 Duisburg, Germany; 4 Institute for Animal Breeding, Hoeltystr. 10, 31535 Neustadt, Germany; and 5 Department of Gynaecology and Obstetrics, Georg August University of Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany
1 To whom correspondence should be addressed; e-mail: edda.toepfer-petersen{at}tiho-hannover.de
Received on August 27, 2004; revised on November 30, 2004; accepted on December 4, 2004
The mammalian oocyte is encased by a transparent extracellular matrix, the zona pellucida (ZP), which consists of three glycoproteins, ZPA, ZPB, and ZPC. The glycan structures of the porcine ZP 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 ZP glycoprotein ZPA of an immature oocyte population as determined by a mass spectrometric approach. In-gel deglycosylation of the electrophoretically separated ZPA protein and comparison of the pattern obtained from the native, the desialylated and the endo-ß-galactosidase-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 biantennary 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 site 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.
Key words: glycosylation sites / MALDI-TOF MS / N-linked glycan chains / zona pellucida