Identification of the half-cystine residues in porcine submaxillary mucin critical for multimerization through the D-domains - Roles of the CGLCG motif in the D1- and D3-domains

Plasmids encoding the amino-terminal region of porcine submaxillary mucin w ere modified by site-specific mutagenesis to assess the roles of individual half-cystine residues in the assembly of disulfide-linked multimers of muc in, COS-7 cells with the plasmid containing C1199A expressed primarily mono mers, suggesting that half-cystine 1199 in the D3-domain is involved in for ming mucin multimers, This residue is in the sequence C(1199)SWRYEPCG, whic h is highly conserved in the D3-domain of other secreted mucins and human p repro-von Willebrand factor. In contrast, cells with the plasmid containing C1276A expressed trimers like those: with unmutated plasmid, suggesting th at half-cystine 1276 is not involved in formation of disulfide-bonded multi mers, The roles of the half-cystines in the CGLCG motifs in the assembly of disulfide-bonded multimers of mucin were also assessed. Cells with plasmid is in which both half-cystines in the motif in the D1- or D3-domain of muci n are replaced by alanine expressed proteins that were poorly secreted, sug gesting that these mutations impair normal folding of the expressed protein s, A plasmid with a mutant D1-domain motif expressed monomers, whereas one with a mutant D3-domain motif expressed monomers and trimers, However, the trimers expressed by the latter plasmid were assembled in nonacidic compart ments, as judged by expression studies in the presence of monensin, which i nhibits trimer formation by unmutated plasmid, but not by the mutant plasmi d. These results suggest that the CGLCG motif in the HI-domain is required for multimerization in the trans-Golgi complex. However, the CGLCG motif in the D3-domain appears to prevent formation of mucin multimers in non-acidi c compartments of the-cell, Plasmids encoding the D1- and D2-domains, the D 1- and D3-domains, or only the D3-domain also expressed oligomers in the pr esence of monensin, suggesting that the three D-domains must be contiguous to avoid multimerization in non-acidic compartments. It is possible that th ese motifs in mucins are engaged in the thiol-disulfide interchange reactio ns during the assembly of disulfide-bonded multimers of mucin.