Identification of the Half-cystine Residues in Porcine Submaxillary Mucin Critical for Multimerization through the D-domains

doi:10.1074/jbc.273.51.34527

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*

Juan Perez-Vilar and
Robert L. Hill‡

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Abstract

Plasmids encoding the amino-terminal region of porcine submaxillary mucin were modified by site-specific mutagenesis to assess the roles of individual half-cystine residues in the assembly of disulfide-linked multimers of mucin. COS-7 cells with the plasmid containing C1199A expressed primarily monomers, suggesting that half-cystine 1199 in the D3-domain is involved in forming mucin multimers. This residue is in the sequence C¹¹⁹⁹SWRYEPCG, which is highly conserved in the D3-domain of other secreted mucins and human prepro-von Willebrand factor. In contrast, cells with the plasmid containing C1276A expressed trimers like those with unmutated plasmid, suggesting that half-cystine 1276 is not involved in formation of disulfide-bonded multimers. The roles of the half-cystines in the CGLCG motifs in the assembly of disulfide-bonded multimers of mucin were also assessed. Cells with plasmids in which both half-cystines in the motif in the D1- or D3-domain of mucin are replaced by alanine expressed proteins that were poorly secreted, suggesting that these mutations impair normal folding of the expressed proteins. 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 non-acidic compartments, as judged by expression studies in the presence of monensin, which inhibits trimer formation by unmutated plasmid, but not by the mutant plasmid. These results suggest that the CGLCG motif in the D1-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-acidic compartments of the cell. Plasmids encoding the D1- and D2-domains, the D1- and D3-domains, or only the D3-domain also expressed oligomers in the presence of monensin, suggesting that the three D-domains must be contiguous to avoid multimerization in non-acidic compartments. It is possible that these motifs in mucins are engaged in the thiol-disulfide interchange reactions during the assembly of disulfide-bonded multimers of mucin.

Footnotes

↵* This work was supported in part by NIGMS Grant 25766 from the National Institutes of Health (to R. L. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed: Dept. of Biochemistry, P. O. Box 3711, Duke University Medical Center, Durham, NC 17710. Tel.: 919-681-8805; Fax: 919-684-5040; E-mail:hill{at}biochem.duke.edu.
↵1 GenBank^TM accession numberAF005273.
↵2 GenBank^TM accession numberY08296.
↵3 GenBank^TM accession numberL21998.
↵4 GenBank^TM accession numberM76740.
↵5 GenBank^TM accession numberAF015521.
↵6 GenBank^TM accession numberX04385.
↵7 GenBank^TM accession numberD29738.
↵8 GenBank^TM accession numberX99805.
↵9 GenBank^TM accession numberU97068.
↵10 GenBank^TM accession numberU40024.
↵11 Genbank^TM accession numberU96411.
↵12 GenBank^TM accession numberJ05016.
↵13 GenBank^TM accession numberAE001274.
↵14 GenBank^TM accession numberU32800.
- Received July 27, 1998.
- Revision received September 28, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.