Role of the cystine-knot motif at the C-terminus of rat mucin protein Muc2 in dimer formation and secretion

被引:29
作者
Bell, SL [1 ]
Xu, GQ [1 ]
Forstner, JF [1 ]
机构
[1] Hosp Sick Children, Res Inst, Div Struct Biol & Biochem, Toronto, ON M5G 1X8, Canada
关键词
cysteine mutation; disulphide bond;
D O I
10.1042/0264-6021:3570203
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 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-1; not 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.
引用
收藏
页码:203 / 209
页数:7
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