Analysis of N-linked glycans of porcine zona pellucida glycoprotein ZPA by MALDI-TOF MS: a contribution to understanding zona pellucida structure.
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-beta-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-beta-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 Asn(84/93), Asn268, Asn316, Asn323, and Asn530 were identified by MS. Only one site was found to be glycosylated in the N-terminal tryptic glycopeptide with Asn(84/93.) N-glycosidase F treatment of the isolated glycopeptides and MS analysis resulted in the identification of the corresponding deglycosylated peptides.
von Witzendorff
Dorothee
D
Institute for Reproductive Medicine, School of Veterinary Medicine Hannover, Foundation, Bünteweg 15, 30559 Hannover, Germany; Shimadzu Deutschland GmbH, Albert-Hahn-Str. 6-10, 47269 Duisburg, Germany.
Ekhlasi-Hundrieser
Mahnaz
M
Dostalova
Zuzanna
Z
Resch
Martin
M
Rath
Detlef
D
Michelmann
Hans-Wilhelm
HW
Töpfer-Petersen
Edda
E
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004527:Egg Proteins / Q000032:analysis* / Q000737:chemistry*; D046911:Macromolecular Substances / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000737:chemistry*; D009865:Oocytes / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine; D014357:Trypsin / Q000378:metabolism; D015044:Zona Pellucida / Q000737:chemistry*; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D000072216:Zona Pellucida Glycoproteins; D014357:Trypsin