A kinetic model of intermediate formation during assembly of cholera toxin B-subunit pentamers

被引:30
作者
Lesieur, C
Cliff, MJ
Carter, R
James, RFL
Clarke, AR
Hirst, TR [1 ]
机构
[1] Univ Bristol, Sch Med Sci, Dept Pathol, Bristol BS8 1TD, Avon, England
[2] Univ Leicester, Dept Surg, Leicester LE2 7LX, Leics, England
[3] Univ Bristol, Sch Med Sci, Dept Microbiol, Bristol BS8 1TD, Avon, England
[4] Univ Bristol, Sch Med Sci, Dept Biochem, Bristol BS8 1TD, Avon, England
关键词
D O I
10.1074/jbc.M110561200
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
Cholera toxin is the most important virulence factor produced by Vibrio cholerae. The pentameric B-subunit of the toxin can bind to GM1-ganglioside receptors, leading to toxin entry into mammalian cells. Here, the in vitro disassembly and reassembly of CtxB(5) (the B subunit pentamer of cholera toxin) is investigated. When CtxB(5) was acidified at pH 1.0 and then neutralized, the B-subunits disassembled and could no longer migrate as SDS-stable pentamers on polyacrylamide gels or be captured by GM1. However, continued incubation at neutral pH resulted in the B-subunits regaining the capacity to be detected by GM1 enzyme-linked immunosorbent assay (t(1/2) similar to 8 min) and to migrate as SDS-stable pentamers (t(1/2) similar to 15 min). Time-dependent changes in Trp fluorescence intensity during B-subunit reassembly occurred with a half-time of similar to8 min, similar to that detected by GM1 enzyme-linked immunosorbent assay, suggesting that both methods monitor earlier events than B-pentamer formation alone. Based on the Trp fluorescence intensity measurements, a kinetic model of the pathway of CtxB(5) reassembly was generated that depended on trans to cis isomerization of Pro-93 to give an interface capable of subunit-subunit interaction. The model suggests formation of intermediates in the reaction, and these were successfully detected by glutaraldehyde cross-linking.
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页码:16697 / 16704
页数:8
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