Document Detail

Two distinctive cell binding patterns by vacuolating toxin fused with glutathione S-transferase: one high-affinity m1-specific binding and the other lower-affinity binding for variant m forms.
MedLine Citation:
PMID:  11570889     Owner:  NLM     Status:  MEDLINE    
The Helicobacter pylori VacA causes large intracellular vacuoles in epithelial cells such as HeLa or RK13 cells. Two major VacA forms, m1 and m2, divergent in an approximately 300 amino acid segment within the cell binding domain P58, display distinct cell-type specificity. Sequence analysis of four vacA alleles showed that a m1-like allele (61) and two m2 alleles (62 and v226) mainly differed in the midregion and that v225, a m1m2 chimera, was a natural double crossover from v226 and another allele. Each of these alleles was expressed as a soluble GST-VacA fusion that did not form a large oligomer. The recombinant VacA portion nevertheless assembled into higher ordered structures and possessed biological binding activity similar to that of the native VacA. A direct comparison of fusion-cell binding activity showed that m1 > m1m2 > m2 in HeLa cells, whereas there were more similar activities in RK13 cells. Vacuolating analyses of three forms revealed a positive correlation between cell binding activity and vacuolating activity. Moreover, the m1-type N-terminal half portion of the midregion was crucial for HeLa cell cytotoxicity. Kinetic, Scatchard, and inhibition analyses suggested the presence of at least two receptors: a m1-type specific high-affinity receptor (K(d) = approximately 5 nM) and a common VacA receptor interacting similarly with m1, m1m2, and m2 via a lower affinity (K(d) = 45-67 nM). Expression of mainly the m1-type receptor on HeLa cells whereas both receptors on RK13 cells may account for distinct cell binding activity and therefore for cell-type specificity.
W C Wang; H J Wang; C H Kuo
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biochemistry     Volume:  40     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  2001 Oct 
Date Detail:
Created Date:  2001-09-25     Completed Date:  2001-10-25     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  11887-96     Citation Subset:  IM    
Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan.
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MeSH Terms
Bacterial Proteins / genetics,  metabolism*
Base Sequence
Cell Line
Cytotoxins / genetics,  metabolism*
DNA Primers
Flow Cytometry
Glutathione Transferase / genetics,  metabolism*
Protein Binding
Reg. No./Substance:
0/Bacterial Proteins; 0/Cytotoxins; 0/DNA Primers; 0/VacA protein, Helicobacter pylori; EC Transferase

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