Role of the cystine-knot motif at the C-terminus of rat mucin protein Muc2 in 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 (35)S-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 45 kDa 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.
Bell
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SL
Division of Structural Biology and Biochemistry, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8.
Xu
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Forstner
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JF
D000818:Animals; D001483:Base Sequence; D003545:Cysteine; D003553:Cystine; D017931:DNA Primers; D019281:Dimerization; D004220:Disulfides / Q000032:analysis; D055262:Mucin-2; D009077:Mucins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine
D017931:DNA Primers; D004220:Disulfides; C524628:Muc2 protein, rat; D055262:Mucin-2; D009077:Mucins; D011994:Recombinant Proteins; D012694:Serine; D003553:Cystine; D003545:Cysteine