Structural characterization of native mouse zona pellucida proteins using mass spectrometry.
The zona pellucida is an extracellular matrix consisting of three glycoproteins that surrounds mammalian eggs and mediates fertilization. The primary structures of mouse ZP1, ZP2, and ZP3 have been deduced from cDNA. Each has a predicted signal peptide and a transmembrane domain from which an ectodomain must be released. All three zona proteins undergo extensive co- and post-translational modifications important for secretion and assembly of the zona matrix. In this report, native zonae pellucidae were isolated and structural features of individual zona proteins within the mixture were determined by high resolution electrospray mass spectrometry. Complete coverage of the primary structure of native ZP3, 96% of ZP2, and 56% of ZP1, the least abundant zona protein, was obtained. Partial disulfide bond assignments were made for each zona protein, and the size of the processed, native protein was determined. The N termini of ZP1 and ZP3, but not ZP2, were blocked by cyclization of glutamine to pyroglutamate. The C termini of ZP1, ZP2, and ZP3 lie upstream of a dibasic motif, which is part of, but distinct from, a proprotein convertase cleavage site. The zona proteins are highly glycosylated and 4/4 potential N-linkage sites on ZP1, 6/6 on ZP2, and 5/6 on ZP3 are occupied. Potential O-linked carbohydrate sites are more ubiquitous, but less utilized.
Boja
Emily S
ES
Laboratory of Biophysical Chemistry, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. bojae@nhlbi.nih.gov
Hoodbhoy
Tanya
T
Fales
Henry M
HM
Dean
Jurrien
J
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002241:Carbohydrates / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D002853:Chromatography, Liquid; D018076:DNA, Complementary / Q000378:metabolism; D004220:Disulfides; D004527:Egg Proteins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface; D021241:Spectrometry, Mass, Electrospray Ionization; D015044:Zona Pellucida / Q000378:metabolism*; D000072216:Zona Pellucida Glycoproteins
D002241:Carbohydrates; D018076:DNA, Complementary; D004220:Disulfides; D004527:Egg Proteins; D008562:Membrane Glycoproteins; D010455:Peptides; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; D000072216:Zona Pellucida Glycoproteins; C000606519:Zp1 protein, mouse; C000606520:Zp2 protein, mouse; C000606521:Zp3 protein, mouse