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 thr ee glycoproteins, called mZP1-3. The glycoproteins are synthesized and secr eted concomitantly by growing oocytes during their 2-3-week growth phase. E ach of the glycoproteins has a consensus furin cleavage site (-Arg-X-Lys/Ar g-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, pr ior to secretion. Molecular mass determinations of deglycosylated mZP2 and mZP3 suggest that their polypeptides are similar to 9 and similar to 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, A rg residues were released by carboxypeptidase B, consistent with processing at the consensus furin cleavage site. Furthermore, an antiserum raised aga inst an mZP3 peptide, located downstream of the consensus furin cleavage si te, failed to label purified mZP3 on Western immunoblots. The antiserum als o 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 sec retion and incorporation into the zona pellucida.