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
J. Perez-vilar et Rl. Hill, 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, J BIOL CHEM, 273(51), 1998, pp. 34527-34534
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.