Inhibitory effects of nontoxic protein volvatoxin A1 on pore-forming cardiotoxic protein volvatoxin A2 by interaction with amphipathic alpha-helix.
Volvatoxin A2, a pore-forming cardiotoxic protein, was isolated from the edible mushroom Volvariella volvacea. Previous studies have demonstrated that volvatoxin A consists of volvatoxin A2 and volvatoxin A1, and the hemolytic activity of volvatoxin A2 is completely abolished by volvatoxin A1 at a volvatoxin A2/volvatoxin A1 molar ratio of 2. In this study, we investigated the molecular mechanism by which volvatoxin A1 inhibits the cytotoxicity of volvatoxin A2. Volvatoxin A1 by itself was found to be nontoxic, and furthermore, it inhibited the hemolytic and cytotoxic activities of volvatoxin A2 at molar ratios of 2 or lower. Interestingly, volvatoxin A1 contains 393 amino acid residues that closely resemble a tandem repeat of volvatoxin A2. Volvatoxin A1 contains two pairs of amphipathic alpha-helices but it lacks a heparin-binding site. This suggests that volvatoxin A1 may interact with volvatoxin A2 but not with the cell membrane. By using confocal microscopy, it was demonstrated that volvatoxin A1 could not bind to the cell membrane; however, volvatoxin A1 could inhibit binding of volvatoxin A2 to the cell membrane at a molar ratio of 2. Via peptide competition assay and in conjunction with pull-down and co-pull-down experiments, we demonstrated that volvatoxin A1 and volvatoxin A2 may form a complex. Our results suggest that this occurs via the interaction of one molecule of volvatoxin A1, which contains two amphipathic alpha-helices, with two molecules of volvatoxin A2, each of which contains one amphipathic alpha-helix. Taken together, the results of this study reveal a novel mechanism by which volvatoxin A1 regulates the cytotoxicity of volvatoxin A2 via direct interaction, and potentially provide an exciting new strategy for chemotherapy.
Wu
Pei-Tzu
PT
Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Lin
Su-Chang
SC
Hsu
Chyong-Ing
CI
Liaw
Yen-Chywan
YC
Lin
Jung-Yaw
JY
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000187:drug effects; D005656:Fungal Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009183:Mycotoxins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D005656:Fungal Proteins; D008081:Liposomes; D009183:Mycotoxins; D011506:Proteins; D011994:Recombinant Proteins; C099459:volvatoxin A2 protein, Volvariella volvacea; D005982:Glutathione Transferase