Role of the cystine-knot motif at the C-terminus of rat mucin protein Muc2in dimer formation and secretion

DNA constructs based on the 534-amino-acid C-terminus of rat mucin protein Muc2 (RMC), were transfected into COS cells and the resultant S-35-labelled dimers and monomers were detected by SDS/PAGE of immunoprecipitates. The c ystine-knot construct, encoding the C-terminal 115 amino acids, appeared in cell lysates as a 45 kDa dimer, but was not secreted. A construct, devoid of the cystine knot, failed to form dimers. Site-specific mutagenesis withi n the cystine knot was performed on a conserved unpaired cysteine (designat ed Cys-X), which has been implicated in some cystine-knot-containing growth factors as being important for intermolecular disulphide-bond formation. D imerization of RMC was effectively abolished. Each cysteine (Cys-1-Cys-6) c omprising the three intramolecular disulphide bonds of the cystine knot was then mutated. Dimer formation was impaired in each case, although much les s so for the Cys-3 mutant than the others. Abnormal high-molecular-mass, di sulphide-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-1; not domain is essential for dimerization of the C-terminal domain of rat M uc2, and that residue Cys-X in the knot plays a key role. The structural in tegrity 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 dimerizatio n, probably by allowing correct positioning of the unpaired Cys-X residue f or stable intermolecular cystine-bond Formation.