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 (Cys 13223 , Cys 13244 , and Cys 13246 ) may be involved in forming interchain disulfide bonds in mucin dimers. Two of these half-cystines, Cys 13244 and Cys 13246 , are in the highly conserved sequence C 13244 LC 13246 C in the disulfide-rich domain of several other human mucins and in preprovon 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.Porcine submaxillary mucin contains a polypeptide chain of 13,288 residues, 1 which can be divided into domains typical of those found in several other mucins secreted by various human tissues (1). Most of the polypeptide chain is comprised of 81 residue tandem repeats that are rich in serine, threonine, glycine, and alanine, and together account for 75% of the residues in this domain. There are at least three different genes encoding porcine submaxillary mucin that differ from one another in the number (90, 125, and 135) of repeats they encode (1). The tandem repeat domain is flanked on either end by unique sequences that are similar in composition to the repeat domains but exhibit no sequence identity to one another or to the tandem repeat domains. The hydroxyl groups of serine and threonine in the tandem repeat domain (2), and presumably the unique sequences flanking this domain, are in O-glycosidic linkage with oligosaccharides, which account for about 75% of the weight of native mu...

The recombinant C-terminus of the human MUC2 mucin forms dimers in Chinese-hamster ovary cells and heterodimers with full-length MUC2 in LS 174T cells

“…TGFβ2 dimerizes via a cysteine residue located directly at the N-terminus of the fourth cysteine residue in the cystine-knot motif [14]. Perez-Vilar and Hill [16] have performed a thorough mutagenesis study of the cysteine residues in the C-terminus of PSM and analysed these constructs in transfected COS-7 cells. Although they show a relatively high background 'activity' in the stacking/separation gel interface, they found three cysteine residues in the cystineknot motif that were likely to be involved in the dimer formation.…”

“…This proposes that this cysteine might be involved also in the dimerization of mucins. Studies on rat Muc2 [11,12] point out that this cysteine residue is essential for dimer formation, whereas studies on vWF [15], PSM [16] and Norrin [17] indicate that additional cysteine residues may be of importance. The mucin genes MUC2, MUC5AC, MUC5B and MUC6 are clustered on chromosome 11p15.5 [18], all of which contain the cystine-knot motif and show large similarities in the positions of other cysteine residues also outside of the cystine-knot motif, indicating that these mucin genes are part of a family having a common ancestral gene.…”

N-terminal Cleavage of the Salivary MUC5B Mucin

“…Extensive studies on PSM have shown that this mucin forms C-to-C dimers in the endoplasmic reticulum (12,13) followed by N-to-N multimerization through the D-domains in the distal part of the Golgi (10). CGLCG sequences in the D1 and D3 domains have been suggested to be crucial for N-to-N multimerization, and these sequences are highly conserved in other secreted mucins, including MUC5B.…”

“…The human oligomeric mucins have been postulated to be assembled in a manner similar to the vWF (6), and PSM has been shown to form C-to-C dimers in the endoplasmic reticulum (12,13) followed by N-to-N multimerization through the D-domains in the distal part of the Golgi (10). PSM shows homologies to MUC5B, MUC2, and vWF, including the D1, D2, and D3 domains, and it has been suggested that half-cysteine residues in the D1 and D3 domains are needed for multimerization of this mucin (10).…”

The cationic C-terminus of rat Muc2 facilitates dimer formation post translationally and is subsequently removed by furin

“…The CK domain, which includes 85±90 residues near the C-terminal end, has been shown to be important in mediating dimerization of vWF and porcine submandibular mucin [9]. Immediately C-terminal to the CK domain in human intestinal MUC2 and rat Muc2 there is a unique 14-amino-acid cationic domain, which completes the polypeptide chain.…”