Expression and binding analysis of GST-VacA fusions reveals that the C-terminal approximately 100-residue segment of exotoxin is crucial for binding in HeLa cells.
The Helicobacter pylori toxin VacA induces intracellular vacuolation and plays an essential role in H. pylori-related diseases. The mature exotoxin is divided into two domains, P37 and P58. A soluble form of VacA fused with GST was expressed in Escherichia coli. Although the soluble fusion lacked vacuolating activity after cleavage by thrombin, it had a binding affinity similar to that of the native VacA. Moreover, it blocked the vacuolating activity induced by the native toxin. Different C-terminal truncated fusions were generated (GST-P72, GST-P53, and GST-P37) and were also produced in a soluble form. A significantly reduced binding activity was seen for GST-P72 and nearly no specific association was detected for GST-P37. Our results suggested that the whole P58 fragment contributed to the cell binding activity in HeLa cells, particularly in the C-terminal approximately 100-residue region.
Wang
H J
HJ
Department of Life Science, National Tsing Hua University, Hsinchu, 30013, Republic of China.
Wang
W C
WC
D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D005982:Glutathione Transferase / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; C074689:VacA protein, Helicobacter pylori; D005982:Glutathione Transferase