Abstract
COS-7 cells transfected with three different expression vectors encoding the 240-amino acid residue, disulfide-rich domain at the carboxyl terminus of porcine submaxillary mucin have been used to determine the possible function of the domain in forming higher oligomers of the mucin polypeptide chain. The domain is expressed as a disulfide-bonded dimer, as shown by SDS-gel electrophoretic analysis of the immunoprecipitated domain in the presence and absence of reducing agent and the cross-linking agent bis(sulfosuccinimidyl) suberate. Molecular weight determination by gel filtration on agarose columns in 6 M guanidine HCl confirmed dimer formation. However, the domain expressed is heterogeneous as the result of different extents of glycosylation. Pulse-chase studies with the 35S-labeled domain show that dimer formation and secretion from cells occur very rapidly. Moreover, dimer formation is not dependent on the N-linked oligosaccharides on the domain. Evidence is presented that dimer formation most likely occurs in the endoplasmic reticulum before complex-type oligosaccharide synthesis is completed. Neither brefeldin A nor tunicamycin interferes with the rate of dimer formation. These studies suggest that the disulfide-rich domain acts to form dimers of the polypeptide chain of mucin. This role of the domain is consistent with its amino acid sequence similarity to the disulfide-rich domain of human prepro-von Willebrand factor, which also serves to form dimers of this blood coagulation factor.
Highlights
Porcine submaxillary mucin has a disulfide-rich domain of ϳ240 residues at its carboxyl terminus [1]
Expression and Secretion of Mucin Disulfide-rich Domains in COS-7 Cells—Fig. 1A shows the SDS-gel electrophoretic patterns under reducing conditions of the proteins isolated from the medium of COS-7 cells transfected with three different expression plasmids encoding the carboxyl-terminal disulfiderich domain of porcine submaxillary mucin
In the presence of tunicamycin, the cells secreted a single species with an Mr of 33,000, which is consistent with the conclusion that the five species differ in the extent of N-glycosylation
Summary
Porcine submaxillary mucin has a disulfide-rich domain of ϳ240 residues at its carboxyl terminus [1]. This domain contains 30 half-cystine residues and no free thiol groups, and unlike the highly O-glycosylated portions of the polypeptide backbone, it appears to be a globular structure Such disulfiderich domains are characteristic of many mucins and have been found in a bovine submaxillary gland mucin-like protein [2]; human intestinal mucin, designated MUC2 [3]; human tracheobroncheal mucin [4]; rat intestinal mucin [5]; and frog integumentary mucin [6]. We report here studies showing that porcine submaxillary mucin can very likely form dimers between its carboxyl-terminal domains This was determined by expression of the disulfide-rich domain in mammalian cells transfected with cDNA encoding the domain, but devoid of the other structures of mucin that interfere with gel electrophoretic analysis. It is shown that the disulfide-rich monomer is synthesized and converted rapidly to a disulfide-linked dimer in the endoplasmic reticulum before secretion of the dimer into the medium
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