Mouse zona pellucida glycoproteins mZP2 and mZP3 undergo carboxy-terminal proteolytic processing in growing oocytes.
The extracellular coat, or zona pellucida, of the mouse egg consists of three glycoproteins, called mZP1-3. The glycoproteins are synthesized and secreted concomitantly by growing oocytes during their 2-3-week growth phase. Each of the glycoproteins has a consensus furin cleavage site (-Arg-X-Lys/Arg-Arg-) near the C-terminus of their polypeptide. Here, several approaches were employed to determine whether nascent mZP2 and mZP3 are cleaved at the consensus sites, -Arg-Ser-Lys-Arg- and -Arg-Asn-Arg-Arg-, respectively, prior to secretion. Molecular mass determinations of deglycosylated mZP2 and mZP3 suggest that their polypeptides are approximately 9 and approximately 7 kDa smaller, respectively, than predicted from exon sequences. Two-dimensional thin-layer chromatographic analyses were also carried out to identify amino acids released from the C-terminus of mZP2 and mZP3 by carboxypeptidase B. On the basis of exon sequences, there are no Arg residues at the predicted C-terminus of the mature glycoproteins. However, for both mZP2 and mZP3, Arg residues were released by carboxypeptidase B, consistent with processing at the consensus furin cleavage site. Furthermore, an antiserum raised against an mZP3 peptide, located downstream of the consensus furin cleavage site, failed to label purified mZP3 on Western immunoblots. The antiserum also failed to label the zona pellucida of oocytes examined by laser scanning confocal microscopy. Collectively, these results strongly suggest that mZP2 and mZP3 are processed at their consensus furin cleavage site prior to secretion and incorporation into the zona pellucida.
Litscher
E S
ES
Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York 10029-6574, USA.
Qi
H
H
Wassarman
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PM
D000818:Animals; D015153:Blotting, Western; D002236:Carbohydrate Conformation; D004527:Egg Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D006026:Glycoside Hydrolases / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008698:Mesylates / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D009844:Oligosaccharides / Q000378:metabolism; D009865:Oocytes / Q000254:growth & development* / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D011956:Receptors, Cell Surface; D015044:Zona Pellucida / Q000378:metabolism*; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D008562:Membrane Glycoproteins; D008698:Mesylates; D009844:Oligosaccharides; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D000072216:Zona Pellucida Glycoproteins; D006026:Glycoside Hydrolases; C020668:glycanase; C012077:trifluoromethanesulfonic acid