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.
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.
Wang
W C
WC
Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan. lswwc@life.nthu.edu.tw
Wang
H J
HJ
Kuo
C H
CH
D000483:Alleles; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D003603:Cytotoxins / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D005434:Flow Cytometry; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding
D001426:Bacterial Proteins; D003603:Cytotoxins; D017931:DNA Primers; C074689:VacA protein, Helicobacter pylori; D005982:Glutathione Transferase