DNA constructs based on the 534-amino-acid C-terminus of rat mucin protein Muc2 (RMC), were transfected into COS cells and the resultant 35S-labelled dimers and monomers were detected by SDS/PAGE of immunoprecipitates. The cystine-knot construct, encoding the C-terminal 115 amino acids, appeared in cell lysates as a 45kDa dimer, but was not secreted. A construct, devoid of the cystine knot, failed to form dimers. Site-specific mutagenesis within the cystine knot was performed on a conserved unpaired cysteine (designated Cys-X), which has been implicated in some cystine-knot-containing growth factors as being important for intermolecular disulphide-bond formation. Dimerization of RMC was effectively abolished. Each cysteine (Cys-1–Cys-6) comprising the three intramolecular disulphide bonds of the cystine knot was then mutated. Dimer formation was impaired in each case, although much less so for the Cys-3 mutant than the others. Abnormal high-molecular-mass, disulphide-dependent aggregates formed with mutations Cys-1, Cys-2, Cys-4 and Cys-5, and were poorly secreted. It is concluded that the intact cystine-knot domain is essential for dimerization of the C-terminal domain of rat Muc2, and that residue Cys-X in the knot plays a key role. The structural integrity of the cystine knot, maintained by intramolecular bonds Cys-1–Cys-4, Cys-2–Cys-5 and Cys-3–Cys-6, also appears to be important for dimerization, probably by allowing correct positioning of the unpaired Cys-X residue for stable intermolecular cystine-bond formation.
Abbreviations used: Cys-X, conserved unpaired cysteine within the cystine knot; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; ER, endoplasmic reticulum; PDGF, platelet-derived growth factor; PSM, porcine submaxillary mucin; RMC, 534-amino-acid C-terminus of rat Muc2; pCK, construct plasmid containing the cystine knot; pRMC-CK, construct plasmid of RMC lacking the cystine knot; pRMC-CXA, construct plasmid of RMC with Cys778 mutated to Ala; pRMC-C1A, pRMC-C2A, pRMC-C3A, pRMC-C4A, pRMC-C5A and pRMC-C6A, construct plasmids of RMC with the Cys residue mutated to Ala at positions 732, 755, 759, 779, 809 and 811, respectively; rMuc2, rat intestinal mucin, Muc2; TGF-β2, transforming growth factor-β2; vWF, von Willebrand factor.