The carboxyl-terminal 90 residues of porcine submaxillary mucin are sufficient for forming disulfide-bonded dimers

Abstract

COS-7 cells transfected with expression vectors encoding 90 and 154 amino acid residues, respectively, from the carboxyl terminus of the disulfide-rich domain (240 residues) of porcine submaxillary mucin were shown to form disulfide-bonded dimers. Cells with expression vectors that encoded the disulfide-rich domain lacking the last 90 and 150 carboxyl-terminal residues, respectively, from the carboxyl terminus of the disulfide-rich domain were unable to secrete truncated domains. These results indicate that the information required to form disulfide-bonded dimers resides in only 90 residues, including 11 half-cystines. Site-specific mutagenesis was employed to change, one at a time, each codon for the 11 half-cystines to serine. Eight of the 11 mutants formed disulfide-bonded dimers indistinguishable from those produced by unmutated vector, although 6 of the 8 mutants also produced aggregates thought to be misfolded protein with scrambled disulfide bonds. Two additional mutant vectors encoding serine instead of half-cystine at residues 13244 and 13246 in submaxillary mucin expressed both monomers and dimers of the disulfide-rich domain but no aggregates. The final mutant vector, C13223S, expressed protein aggregates that were poorly secreted from transfected cells. A mutant vector with two codon changes, C13244A/C13246A, expressed both monomers and dimers, just like the single mutants at these half-cystines. These results suggest that three half-cystine residues (Cys13223, Cys13244, and Cys13246) may be involved in forming interchain disulfide bonds in mucin dimers. Two of these half-cystines, Cys13244 and Cys13246, are in the highly conserved sequence C13244LC13246C in the disulfide-rich domain of several other human mucins and in prepro-von Willebrand factor and norrin, a protein that in mutant forms gives rise to Norrie disease. Support for the involvement of these half-cystines in formation of disulfide-bonded dimers of these molecules is also provided by known mutations in prepro-von Willebrand factor and norrin.