Tài liệu Báo cáo khoa học: Inhibitory effects of nontoxic protein volvatoxin A1 on pore-forming cardiotoxic protein volvatoxin A2 by interaction with amphipathic a-helix doc

Inhibitory effects of nontoxic protein volvatoxin A1 on pore-forming cardiotoxic protein volvatoxin A2 by interaction with amphipathic a-helix Pei-Tzu Wu1, Su-Chang Lin2, Chyong-Ing Hsu1, Yen-Chywan Liaw2 and Jung-Yaw Lin1 Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan Institute of Molecular Biology Academia Sinica, Taipei, Taiwan Keywords amphipathic a-helix; co-pull-down experiment; tandem repeat protein; volvatoxin A1; volvatoxin A2 Correspondence J.-Y Lin, Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, F9, no 1, Section 1, Jen-Ai Road, Taipei 10051, Taiwan Fax: +886 23415334 Tel: +886 23123456 (ext 8206 ⁄ 8207) E-mail: jylin@ha.mc.ntu.edu.tw Database The nucleotide sequence reported in this paper has been submitted to the DDBJ ⁄ EMBL ⁄ GenBank databases under the accession number AY952461 (Received 22 March 2006, revised May 2006, accepted 17 May 2006) doi:10.1111/j.1742-4658.2006.05325.x 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 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 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 a-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 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 a-helices, with two molecules of volvatoxin A2, each of which contains one amphipathic a-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 Volvatoxin A (VVA) has been isolated from Volvariella volvacea, and consists of volvatoxin A2 (VVA2) and volvatoxin A1 (VVA1) [1] VVA has several biological activities, such as: (a) lysis of human red blood cells; (b) swelling tumor cells and the mitochondria of liver cells; (c) inhibition of protein biosynthesis; and (d) causing cardiac arrest via activation of the Ca2+-dependent ATPase enzyme in the ventricular microsomal fraction [1–3] The hemolytic activity of VVA2 is totally inhibited by VVA1 at a molar ratio of [4,5] Previous studies have shown that VVA2 is a b-pore-forming toxin, with a heparin-binding site (HBS) encoded within the C-terminal b-strands (b6, b7 and b8) This HBS structure is indispensable for the Abbreviations FITC, fluorescein isothiocyanate; GSH, glutathione; GSP, gene-specific primer; HBS, heparin-binding site; RBC, red blood cell; VVA, volvatoxin A; VVA1, volvatoxin A1; VVA2, volvatoxin A2; VVA1-CTD, volvatoxin A1 C-terminal domain (198–391 residues); VVA1-NTD, volvatoxin A1 N-terminal domain (1–197 residues) 3160 FEBS Journal 273 (2006) 3160–3171 ª 2006 The Authors Journal compilation ª 2006 FEBS P.-T Wu et al membrane interaction of VVA2 [6] Furthermore, the VVA2-binding receptor on the cell membrane has been shown to be a sulfated glycosaminoglycan, as demonstrated by affinity column chromatography [6] Binding of VVA2 to the cell membrane induced a protein conformational change of the VVA2 amphipathic a-helices to form a prepore complex [7–10] Therefore, the amphipathic a-helices play an important role in VVA2 oligomerization and pore formation [6] Pore-forming toxins are essentially naturally occurring biological weapons produced by both prokaryotes and eukaryotes, and include well-known toxins such as diphtheria and anthrax toxins, as well as the less wellknown a-hemolysin and equinatoxin II [11–16] An important objective is to provide an effective inhibitor of these virulence factors, and naturally occurring substances represent one potential source Recently, there has been much interest in the potential application of these toxins to chemotherapy and the delivery of drugs [17,18] In an attempt to determine the inhibitory mechanism of VVA1 on VVA2, we deduced the amino acid sequence of VVA1 from the cDNA nucleotide sequence The primary structure of VVA1 is similar to that of a tandem repeat form of VVA2, and the predicted secondary structure showed that it contains two pairs of amphipathic a-helices VVA1 completely inhibited the hemolytic and cytotoxic activities of VVA2 at VVA2 ⁄ VVA1 molar ratios of or lower Taken together, our results provide evidence that VVA1 interacts with VVA2 and regulates the cytotoxic pore-forming activity of VVA2 Results and Discussion Characteristics of VVA1 structure To study the structure of VVA1, we cloned VVA1 cDNA by the RACE method, as described previously (supplementary Table S1) [19] The coding region of the cloned VVA1 cDNA contained 1179 nucleotides, and the deduced amino acid sequence was identical to that determined previously by protein sequencing (supplementary Fig S1) [5] Interestingly, the amino acid sequence of VVA1 was very similar to that of a tandem repeat of VVA2 The N-terminal half fragment of VVA1, designated volvatoxin A1 N-terminal domain (VVA1-NTD) (1–197 residues), had 46.3% similarity with VVA2 (Fig 1A), whereas the C-terminal fragment, designated volvatoxin A1 C-terminal domain (VVA1-CTD) (198–391 residues), displayed 49.2% similarity to VVA2 (Fig 1A) The similarity between VVA1-NTD and VVA1-CTD is 42.6% The tertiary VVA1 is a novel toxin regulator of VVA2 structure of VVA2 shows that it has a pair of amphipathic a-helices, denoted a-helix-C and a-helix-D [24], which are essential for VVA2 dimerization [6] Interestingly, VVA1 also contains a pair of amphipathic a-helices similar to VVA2 (Fig 1A) (supplementary Fig S2) It has been shown that the amphiphilicity of the amphipathic a-helix of VVA2 is indispensable for protein interaction and oligomerization [6] Secondary structure analysis of VVA1 suggests that there might be two pairs of amphipathic a-helices in both the N-terminal and C-terminal domains The hydrophobic moments of amphipathic a-helix-C and a-helix-D of VVA1-NTD were calculated to be 0.4 and 0.57, respectively, while those of amphipathic a-helix-D¢ and a-helix-E¢ of VVA1-CTD were 0.49 and 0.57 [25] VVA2 has a basic HBS at its C-terminus that is located within its b-strand, is indispensable for binding to cell membranes, and has a pI value of 9.6, similar to that of the snake venom cardiotoxin [6,20–22] Neither VVA1-NTD nor VVA1-CTD has a basic HBS at their C-terminus as VVA2 does Additionally, the pI values of the corresponding C-terminal regions of VVA1-NTD and VVA1-CTD were found to be 4.3 and 4.6, respectively, suggesting that VVA1 has very weak, if any, affinity for the anionic surface of cell membranes [23] Furthermore, we demonstrate here that VVA1 has no noticeable affinity for simple lipid membranes (Fig 1B) When VVA1 was incubated with liposomes and then centrifuged and electrophoresed, analysis of the supernatant and pellet fractions showed that VVA1 cannot bind to these simple membranes (Fig 1B, lanes and 4) Additionally, VVA2 binds liposome-containing membranes, as demonstrated by the exclusive presence of VVA2 in the pellet fraction (Fig 1B, lanes and 6) Intriguingly, the presence of VVA1 inhibited the oligomerization of VVA2 and thus its binding to simple lipid membranes (Fig 1B, lanes and 2) Therefore, this investigation of the structural and binding characteristics of VVA1 indicates that VVA1 may have the capacity for protein–protein interaction with VVA2 via its amphipathic a-helices, but it is unlikely that VVA1 would be able to bind to the membrane of cells Inhibitory effects of VVA1 on the hemolytic and cytotoxic activity of VVA2 The effects of VVA1 on the hemolytic activity of VVA2 were studied by incubation of human red blood cells (RBCs) with the purified proteins VVA1 itself had no hemolytic activity when incubated with human RBCs (Fig 2A, column 2) Strikingly, VVA1 completely abolished the hemolytic activity of VVA2 at FEBS Journal 273 (2006) 3160–3171 ª 2006 The Authors Journal compilation ª 2006 FEBS 3161 VVA1 is a novel toxin regulator of VVA2 P.-T Wu et al A B + + + – + + S P S P – – + (4µg) – + – (4µg) S P – – liposomes VVA2 VVA1 (MkDa) + + 200 116 97 66 45 VVA1 31 VVA2 (monomer) 21 S:supernatant P:pellet Fig Characteristics of volvatoxin A1 (VVA1) structure (A) Alignment of the deduced amino acid sequence of VVA1 N-terminal domain (VVA1-NTD) and VVA1 C-terminal domain (VVA1-CTD) with that of VVA2 (GenBank accession number AY362729) The secondary structural elements of VVA1-NTD predicted by the PROFSEC program are illustrated at the top of the sequence (orange), and those of VVA2 (green) from the X-ray crystallographic analysis are shown below; the arrows represent b-strands, and the rods represent a-helices Secondary structural elements of VVA1-NTD with PROF scores below are shown in light orange The completely conserved residues are shaded in dark green, and similar aligned residues are shaded in pink The residue numbers are indicated on the right The ‘+’ symbol represents the amino acid residues of the heparin-binding site (HBS) of VVA2 (166–194) (B) Inhibitory effects of VVA1 on the binding of VVA2 to liposomes After incubation of VVA1, VVA2 or the mixture of VVA2 and VVA1 at a molar ratio of with liposomes (5 mM) at 37 °C for 30 min, the reaction mixtures were subjected to centrifugation at 100 000 g at °C for h The presence of VVA1 or VVA2 in the supernatant and pellet were analyzed by 10% SDS ⁄ PAGE and visualized by Coomassie Blue staining 3162 FEBS Journal 273 (2006) 3160–3171 ª 2006 The Authors Journal compilation ª 2006 FEBS P.-T Wu et al A VVA1 is a novel toxin regulator of VVA2 molar ratio of when it was incubated with HeLa cells (Fig 2B, column 6) Additionally, confocal microscopy was employed to study the inhibitory effects of VVA1 on VVA2 The results showed that VVA1 by itself was unable to bind to cell membranes (Fig 3, panel FITC-VVA1 and panels a–d) Moreover, preincubation of VVA2 and VVA1 (at a molar ratio of 2) inhibited VVA2 binding to the cell membrane (Fig 3, panels e–h) These results strongly suggest that VVA1 inhibits the cytotoxicity of VVA2 by preventing the binding of VVA2 to the cell membrane Interactions between VVA1 and VVA2 B Fig Effects of volvatoxin A1 (VVA1) on the hemolytic and cytotoxic activity of volvatoxin A2 (VVA2) (A) The hemolytic activity of VVA2 regulated by VVA1 VVA2 (45 nM) and various concentrations of VVA1 were preincubated as indicated, and the percentage of hemolysis was calculated as described in Experimental procedures Each value represents the mean ± SD of three independent experiments (B) VVA2 cytotoxicity was affected by VVA1 HeLa cells were treated with VVA2 (17 nM) and various amounts of VVA1 at 37 °C for 24 h Cell death was assayed by using a trypan blue exclusion assay [41] Means ± SD are shown for three independent experiments VVA2 ⁄ VVA1 molar ratios of or lower (Fig 2A, columns 3–5), while at a molar ratio of the hemolytic activity of VVA2 was reactivated (Fig 2A, column 6) To examine whether VVA1 affects the cytotoxicity of VVA2, HeLa cells were treated with VVA2 (17 nm, IC50 of VVA2) and various amounts of VVA1 at 37° C for 24 h Similar to the results obtained in hemolytic experiments, VVA1 itself had no cytotoxicity (Fig 2B, column 2), but was able to inhibit the cytotoxicity of VVA2 completely at a VVA2 ⁄ VVA1 molar ratios of or lower (Fig 2B, columns 3–5) Furthermore, the cytotoxicity of VVA2 was reactivated at a To find whether direct interaction between VVA1 and VVA2 is required for the inhibitory effects of VVA1 on the pore-forming activity of VVA2, pull-down experiments were performed As a preliminary experiment, we investigated the effects of different buffer constituents on the oligomerization of VVA2 Only Triton X-100, and not deoxycholate as had been reported for Bcl-2 family members, was able to induce the oligomerization of VVA2 (supplementary Fig S3A) [26] Interestingly, not even the harsh, denaturing environment of electrophoresis through the SDS ⁄ PAGE system could affect VVA2 oligomer formation Furthermore, the Triton X-100 induction of oligomerization of VVA2 was able to mimic the amphipathic environment of an artificial cell membrane of liposomes (supplementary Fig S3B) [26–29] Additionally, incubation of VVA2 without liposomes inhibited their oligomerization (supplementary Fig S3B, lane 4) Thus the environment of detergent micelles set up by buffering with Triton X-100 was very similar to the natural environment and was used for further experiments to determine interactions between VVA2 and VVA1 The input controls for the representative pull-down experiment shown in Fig 4A are lanes 11 and 12, where the oligomerization of VVA2 in lane 11 is clearly shown Intriguingly, when VVA2 and VVA1 at a molar ratio of : were preincubated together and then electrophoresed, no oligomerization of VVA2 was evident (Fig 4A, lane 12) This result seemed to suggest that there was indeed some form of interaction between VVA1 and VVA2 Furthermore, when beads linked to VVA2 were incubated with VVA1 and then washed, eluted and run on a 10% SDS ⁄ PAGE gel, VVA1 had clearly bound to VVA2 (Fig 4A, lane 1) Furthermore, when a : molar mixture of VVA2 and VVA1 was incubated with VVA2-linked beads, both proteins were adsorbed, and after elution both proteins were detected FEBS Journal 273 (2006) 3160–3171 ª 2006 The Authors Journal compilation ª 2006 FEBS 3163 VVA1 is a novel toxin regulator of VVA2 P.-T Wu et al a b c d e f g h Fig Volvatoxin A1 (VVA1) inhibited volvatoxin A2 (VVA2) binding to the cell membrane Binding of VVA2 to the cell membrane was abolished by the presence of VVA1 HeLa cells were treated with fluorescein isothiocyanate (FITC)–VVA1, VVA2 and both at a molar ratio of 2, as described in Experimental procedures VVA1 was conjugated with FITC (green fluorescence, panel FITC–VVA1), and VVA2 was stained with Alexa-568 (red fluorescence, panels a and e), while the nucleus was stained with Hoechst 33258 (blue color, panels b and f) The overlay of both images is shown in panels c and g The phase-contrast image shows cellular morphology (phase panel) Bar, 40 lm via SDS ⁄ PAGE analysis (Fig 4A, lane 2) Interestingly, again no oligomers of VVA2 were detected after incubation of VVA2 with VVA1 at a molar ratio of (Fig 4A, lane 2) When VVA1 beads were incubated with VVA2, VVA2 oligomers were adsorbed and eluted (Fig 4A, lane 4) Additionally, when VVA1 beads were incubated with the mixture of VVA2 and VVA1 (molar ratio : 1), VVA2 and VVA1 were detected, but again, no VVA2 oligomer was found (Fig 4A, lane 5) Taken together, these results strongly support the notion that there is a direct interaction between VVA1 and VVA2, and that at a VVA2 ⁄ VVA1 molar ratio of 2, VVA1 is able to inhibit the oligomerization of VVA2 Extending 3164 these results, we hypothesized that the inhibition of VVA2 cytotoxic pore formation by VVA1 can only occur at the ideal ratio of : or lower, due to the ability of one molecule of VVA1 to interact with two molecules of VVA2 At a higher ratio of VVA2 to VVA1, the latter is not able to prevent VVA2 oligomerization and thus cannot inhibit VVA2 cytotoxicity Number of VVA1-binding sites for VVA2 To further identify the binding characteristics and to investigate the dynamic interaction between VVA1 and VVA2, we carried out co-pull-down experiments The amphipathic a-helix of VVA2 had previously been FEBS Journal 273 (2006) 3160–3171 ª 2006 The Authors Journal compilation ª 2006 FEBS P.-T Wu et al VVA1 is a novel toxin regulator of VVA2 A B Fig Interaction between volvatoxin A1 (VVA1) and volvatoxin A2 (VVA2) in the presence of Triton X-100 (A) Pull-down experiments VVA1 (45 nM), VVA2 (45 nM) or the mixture (VVA2, 45 nM, and VVA1, 22.5 nM) were incubated with VVA2 beads, VVA1 beads or BSA beads at 37 °C for 30 in 0.02% Triton X-100 The beads were washed, and the bound proteins were eluted The protein eluents were identified by 10% SDS ⁄ PAGE and visualized by silver staining (B) Co-pull-down experiments Linear oligomeric VVA2 (VVA1 beads) was prepared from VVA1 beads, which were treated with VVA2 in 0.02% Triton X-100 buffer, and VVA1 (VVA2 beads) was prepared from VVA2 beads, which were treated with VVA1 in the same buffer as described in Experimental procedures The linear oligomeric VVA2 (VVA1 beads) was then incubated with various amounts of VVA1, while the VVA1 (VVA2 beads) was incubated with various amounts of VVA2 The reaction products were eluted with 0.5% SDS loading buffer, and the proteins in the eluents were analyzed by 10% SDS ⁄ PAGE and visualized by silver staining FEBS Journal 273 (2006) 3160–3171 ª 2006 The Authors Journal compilation ª 2006 FEBS 3165 VVA1 is a novel toxin regulator of VVA2 P.-T Wu et al identified as necessary for the oligomerization of VVA2 [6] Furthermore, we had determined that VVA1 encoded two regions that displayed a reasonably high degree of conservation to the VVA2 oligomerization domain Thus, we hypothesized that VVA1 may contain two binding sites for complex formation with VVA2 To address this issue, co-pulldown experiments were carried out First, VVA1linked beads (VVA1 beads) were incubated with 45 nm VVA2 [referred to as linear oligomeric VVA2 (VVA1 beads)] This mixture was then incubated with various amounts of VVA1 in the presence of 0.02% Triton X-100, and eluted with 0.5% SDS buffer (Fig 4B) The results demonstrated that no VVA1 could be bound to a VVA1 bead that was saturated with VVA2 oligomers (Fig 4B, lanes 1–4), which may imply that one molecule of VVA2 has one binding site for interacting with either VVA1 or VVA2 Additional investigation of the characteristics of binding of VVA2 to VVA1 will be necessary to further understand this important interaction Next, we utilized VVA2-linked beads (VVA2 beads) and incubated them with nm VVA1 protein [referred to as VVA1 (VVA2) beads] (Fig 4B) VVA1 (VVA2 beads) was incubated with increasing amounts of VVA2, and visualization on an SDS ⁄ PAGE gel showed that the adsorbed VVA2 had oligomerized As the amount of VVA2 in the reaction was increased, more VVA2 was bound to the VVA1 (VVA2 beads) in the form of oligomers (Fig 4B, lanes 7–10) Collectively, these data indicate that one molecule of VVA1 has two binding sites for interaction with two molecules of VVA2, and that large amounts of free VVA2 can use VVA1 as a basis for the formation of VVA2 oligomers Interaction of VVA1 and VVA2 by amphipathic a-helix To identify the binding sites in VVA1 responsible for direct interaction with VVA2, peptide competition assays, pull-down experiments and western blots were carried out For the peptide competition assays, the amphipathic a-helices of VVA1 were generated as recombinant peptide fragments denoted as reVVA1NTD-aH-C-D (amino acids 72–109) and reVVA1CTD-aH-D¢-E¢ (amino acids 260–302) (Fig 5A) These fragments were then used to compete with bead-linked VVA1 for binding to VVA2 The effectiveness of competition was interpreted via the amount of VVA2 binding to the bead-linked VVA1 after pull-down and SDS ⁄ PAGE electrophoresis The results showed that the interaction of VVA2 with VVA1 beads was subject to competition by the N-terminal helix pair (reVVA13166 NTD-aH-C-D) at a reVVA1-NTD-aH-C-D ⁄ VVA2 molar ratio of 10 (Fig 5B, lanes 1–3) Interestingly, the peptide fragment containing the C-terminal helix pair (reVVA1-CTD-aH-D¢-E¢) was able to efficiently compete with binding of VVA2 to the bead-linked VVA1 at a reVVA1-CTD-aH-D¢-E¢ ⁄ VVA2 molar ratio of 2.5 (Fig 5B, lanes 4–6) Furthermore, the reHBSF peptide fragment could not compete with the interaction of VVA2 with VVA1 beads (Fig 5B, lanes 7–9) This was an expected result, as the HBS fragment in VVA2 was identified as the membrane-binding domain [6] These results suggest that the N-terminal and the C-terminal pair of a-helices of VVA1 can bind to VVA2 independently of each other and thus enable the direct binding by one molecule of VVA1 of two molecules of VVA2 This further complements our previous results suggesting an optimal molar ratio of for binding of VVA2 to VVA1 The anti-VVA2 IgG used in this experiment only detects VVA2 and does not crossreact with VVA1 (supplementary Fig S4) In the present study, we have shown that VVA1 completely inhibits the biological activity of VVA2 in vitro at VVA2 ⁄ VVA1 molar ratios or lower This begs the question of why a mushroom would produce a toxin and at the same time an antidote We believe that the major reason why Volvariella volvacea produces VVA1 is so that it can associate with and, at the right ratio, enhance the toxicity of VVA2 As shown previously, the LD50 of VVA1 or VVA2 individually is higher than 40 mgỈ(kg body weight))1 At a molar ratio of 2, the LD50 of VVA2 ⁄ VVA1 is reduced to mgỈ(kg body weight))1 However, at a molar ratio of 6, which is similar to that in the mushroom, a still lower LD50 was evident This intriguing phenomenon requires further investigation [1] On the basis of the present findings, we propose that one molecule of VVA1 interacts with two molecules of VVA2 and thus inhibits the formation of the mature pore complex Furthermore, we suggest that manipulation of the levels of VVA1 may be utilized to inhibit VVA2 oligomerization and pore formation until certain conditions are present to make it biologically valuable For example, it has recently been proposed that native or recombinant pore-forming toxin may be used as a biotherapeutic agent [30–32] The novel approach of using pore-forming toxins for the treatment of solid tumors, which have proven to be quite resistant to conventional toxins [33–35], shows great promise One of the major drawbacks of using these toxins is that they must be able to preserve the main characteristics of the toxin during the transport process in vivo [32,36] Therefore, a targeted VVA1– VVA2 complex may be introduced to the host as a FEBS Journal 273 (2006) 3160–3171 ª 2006 The Authors Journal compilation ª 2006 FEBS P.-T Wu et al VVA1 is a novel toxin regulator of VVA2 A B Fig Peptide competition assay (A) Schematic representation of peptide competitors (B) Binding of volvatoxin A2 (VVA2) to volvatoxin A1 (VVA1) was inhibited by the amphipathic a-helices of VVA1 The VVA2 and VVA1 mixture (molar ratio 2) was incubated with VVA1 beads; the interaction was examined in the presence of increasing amounts of competitors The adsorbed protein was analyzed by western blots using anti-VVA2 IgG; this indicated that the two amphipathic a-helices competed for the interaction between VVA2 and VVA1 beads protoxin, thus having no toxicity to the animal, but with the ability to target a tumor Once at the appropriate site, the VVA1 molecule could be dissociated, allowing the VVA2 molecules to oligomerize and reactivate their cytotoxic pore-forming activity This has been demonstrated previously, when a mutated anthrax protoxin was cleaved by urokinase plasminogen activator and selectively killed a subset of cancer cells that highly expressed plasminogen activator [31,37–39] Thus, this description of a naturally occurring inhibitor of VVA2 represents a significant discovery, although its importance in a clinical setting remains to be investigated Experimental procedures Materials Taq DNA polymerase and the pGEM-T vector were obtained from Promega (Madison, WI) Restriction endo- nucleases and T4 DNA ligase were from New England Biolabs Inc (Beverly, MA) The Marathon cDNA amplification kit was from Clontech (Palo Alto, CA) Fluorescent Alexa-568-labeled goat anti-rabbit and fluorescein isothiocyanate (FITC) were purchased from Chemicon International (Temecula, CA) CNBr-activated Sepharose 4B, glutathione (GSH)-agarose-4B column and pGEX-2T vector were from Amersham Biosciences (Uppsala, Sweden) All other chemicals were of analytical grade Purification, and cDNA cloning of VVA1 VVA1 was purified from mushroom, V volvacea, and the amino acid sequence of VVA1 was determined by protein techniques as reported previously [1,19] The peptides of VVA1 generated by N-tosyl-l-phenylalanine chloromethylketone treated-trypsin, Streptococcus aureus V8 endoproteinase or Lys-C endoproteinase digestion were fractionated by HPLC with a C18 reverse-phase column FEBS Journal 273 (2006) 3160–3171 ª 2006 The Authors Journal compilation ª 2006 FEBS 3167 VVA1 is a novel toxin regulator of VVA2 P.-T Wu et al (4.6 · 250 mm) that was eluted with a linear gradient of acetonitrile (0–80%) in 0.1% trifluoroacetic acid The polypeptides obtained by HPLC were subjected to amino acid sequence analysis using an ABI 476 A Applied Biosystems (Foster City, CA) automated amino acid sequencer [19] The amino acid sequence of VVA1 was used for the design of degenerate primers and cloning the cDNA of VVA1 All primers used in this study are reported in supplementary Table S1 Poly(A+) RNA was isolated from the total RNA fraction on an oligo (dT)-cellulose column, and poly(A+)-rich mRNA was reverse-transcribed with a Marathon cDNA amplification kit [19,40] The cDNAs were ligated to Marathon adaptors for 5¢ and 3¢ rapid amplification of cDNA ends (RACE), and the products were used as the template for subsequent PCR In the first PCR, VVA1 cDNA was amplified with the sense degenerate primer A and the antisense degenerate primer B, corresponding to amino acid residues 1–6 and 385–391 of VVA1, respectively The amplified first PCR products were used as template for nested PCR with the sense degenerate primer A and the antisense degenerate primer C, which corresponds to amino acid residues 163–168 of VVA1 The products of this second PCR were sequenced and used to design the specific antisense primers GSP-1, corresponding to amino acid residues 40–47 of VVA1, and GSP-3, corresponding to amino acid residues 24–31 of VVA1 In the third PCR, GSP-1 was used along with the Marathon primer AP-1, and the products were used as the template for the fourth PCR, in which GSP-3 was used along with the sense primer AP-2 to obtain the 5¢-end of the VVA1 cDNA The products of the second PCR were used to design GSP-2 and GSP-4 specific sense primers corresponding to amino acid residues 128–136 and 155–162 of VVA1, respectively The two primers were used along with the Marathon primers AP-1 and AP-2 to yield the 3¢-end of the VVA1 cDNA The full-length VVA1 cDNA was obtained by amplifying V volvacea cDNAs with the sense primer GSP-5, which encodes the start codon and the first eight N-terminal amino acid residues of VVA1, and the antisense primer GSP-6, which encodes the last eight C-terminal amino acid residues and the stop codon of VVA1 (GenBank accession number AY952461) The PCR products were then ligated into the T vector Liposome-binding assay Liposomes were prepared as described previously [6] After incubation of VVA1 (4 lg), VVA2 (4 lg) or the mixture of VVA2 and VVA1 (at a molar ratio 2) with liposomes (5 mm) at 37 °C for 30 min, the reaction mixtures were subjected to centrifugation at 100 000 g at °C for h (Beckman TLA 100.2; Beckman Coulter, 3168 Taipei, Taiwan) Then, the supernatant and pellet were analyzed by 10% SDS ⁄ PAGE and visualized by Coomassie Blue staining Hemolytic activity assay Human RBCs were prepared by washing three times with NaCl ⁄ Pi (137 mm NaCl, 1.5 mm KH2PO4, 2.7 mm KCl, 8.1 mm Na2HPO4, pH 7.4) [6] VVA2 (45 nm, inducing 50% hemolysis) and various amounts of VVA1 were premixed in NaCl ⁄ Pi, and then 0.1 mL of human RBCs (3 · 107 cellsỈmL)1) was added The reaction mixtures were further incubated at 37 °C for 30 min, and the reaction was terminated by centrifuging at 13 000 g for (KUBOTA RA-155; Kubota, Osaka, Japan) The supernatant was measured at 540 nm to determine the degree of hemolysis One hundred per cent hemolysis was defined as the same volume of the human red blood cells in the presence of 0.1% Triton X-100 [6] Cytotoxicity assay HeLa cells were grown in DMEM supplemented with 10% FBS, mm l-glutamine, 100 unitsỈmL)1 penicillin, and 100 lgỈmL)1 streptomycin (Life Technologies, Inc.) under 5% CO2 at 37 °C HeLa cells (3 · 105) were then treated with mixtures of VVA2 (17 nm, causing 50% cytotoxicity) and various amounts of VVA1 for 24 h The cells were then trypsinized, collected, and treated with 2% trypan blue dye in NaCl ⁄ Pi at 37 °C for The surviving cells were counted with a hemocytometer [41,42] Confocal microscope analysis FITC–VVA1 was prepared by coupling 1.5 mgỈmL)1 VVA1 with 40 lgỈmL)1 FITC in 0.1 m NaHCO3 at °C for 16 h The free FITC was removed with a Sephadex G25 column, and the effect of FITC–VVA1 on the hemolytic activity of VVA2 was shown to be the same as that of VVA1 HeLa Cells (4 · 105) grown on coverslips were treated with FITCconjugated VVA1 (17 nm), VVA2 (17 nm) or the mixture of VVA2 (17 nm) and VVA1 (8.5 nm) Immunostaining was performed by fixing the cells with 4% paraformaldehyde in · NaCl ⁄ Pi on coverslips To detect VVA2 protein, nonpermeabilized fixed cells were blocked in blocking buffer (10% FBS in NaCl ⁄ Pi) for 30 The cells were then probed with anti-VVA2 (1 : 1000) at room temperature for h After extensive washing in NaCl ⁄ Pi, the washed cells were stained with Alexa-568-conjugated goat anti-rabbit IgG (1 : 1000) for 60 During the last 15 of secondary antibody staining, Hoechst 33258 (5 lgỈmL)1) was applied for observation of the nucleus After washing with NaCl ⁄ Pi, slides were mounted in mounting solution (80% glycerol in NaCl ⁄ Pi), and sealed with nail polish The cells were subjected to FEBS Journal 273 (2006) 3160–3171 ª 2006 The Authors Journal compilation ª 2006 FEBS P.-T Wu et al immunostaining for observation of the VVA1 and VVA2 as described previously [41] Pull-down experiment VVA2, VVA1 or BSA (8 mg) was coupled to CNBr-Sepharose beads (Amersham Pharmacia Biotech) in coupling buffer (100 mm NaHCO3, pH 8.3, and 500 mm NaCl) and incubated at °C overnight Residual active groups were blocked with m ethanolamine, pH 8.0, at room temperature for h The beads were then washed four times with alternating pH buffers Each wash cycle consisted of Tris buffer (0.1 m Tris, pH 8.0, and 500 mm NaCl) and acid wash buffer (0.1 m sodium acetate, pH 4.0, and 500 mm NaCl) [43] Thirty microliters of protein-conjugated beads was incubated with 45 nm VVA1, VVA2 or a mixture of VVA2, 45 nm, and VVA1, 22.5 nm, at 37 °C for 30 in 0.02% Triton X-100, 50 mm Tris, pH 8.0 The beads were washed three times with 50 mm Tris buffer and eluted with 0.5% SDS loading buffer (50 mm Tris, pH 8.0, 0.5% SDS, 10% glycerol and 0.03% bromophenol blue) The eluent was analyzed by 10% SDS ⁄ PAGE and visualized by silver staining Co-pull-down experiment For further study of the protein–protein interaction, we performed a co-pull-down experiment to analyze the number of binding sites of VVA1 and VVA2 VVA1 (VVA2 beads) was prepared by incubating VVA2 beads with 45 nm VVA1 at 37 °C for 30 Adsorbed proteins were analyzed as mentioned above After removal of the unbound VVA1, the VVA1 (VVA2 beads) was incubated with various amounts of VVA2 in 0.02% Triton X-100 at 37 °C for 30 Linear oligomeric VVA2 (VVA1 beads) was prepared by incubating VVA1 beads with 45 nm VVA2 at 37 °C for 30 After removal of the unbound VVA2 by washing with 50 mm Tris buffer, the linear oligomeric VVA2 (VVA1 beads) was incubated with various amounts of VVA1 in 0.02% Triton X-100 at 37 °C for 30 The reaction was terminated by centrifugation, and the beads were washed three times with 50 mm Tris buffer, and eluted with 0.5% SDS loading buffer The eluents were subjected to 10% SDS ⁄ PAGE analysis Peptide competition assay To determine the protein–protein interaction site for VVA1 and VVA2, the amphipathic a-helix regions of VVA1-NTD (72–109 residues of VVA1) and VVA1-CTD (260–302 residues of VVA1) were PCR amplified (supplementary Table S1), and then ligated into the pGEX-2T vector for protein expression The HBS fragment of VVA2 (165–199 residues of VVA2) was constructed as described previously [6] The VVA1 is a novel toxin regulator of VVA2 GST fusion proteins were expressed in Escherichia coli and purified by affinity chromatography on a GSH-agarose-4B column, and this was followed by thrombin digestion to obtain pure peptide fragments of VVA1-NTD-aH-C-D, VVA1-CTD-aH-D¢-E¢ or VVA2 (HBS fragment) For the competition assay, the various amounts of peptide competitor were added to the mixture of VVA2 and VVA1 (at a molar ratio of 2), and the mixture was incubated with VVA1 beads at 37 °C for 30 min, and then washed and eluted as described above The eluent was separated by SDS ⁄ PAGE and transferred to the polyvinylidene difluoride membrane, and western blots were prepared using anti-VVA2 as a standard protocol [6] Acknowledgements We would like to thank Professor Ta-Hsiu Liao for his valuable suggestions, and Professor Zee-Fen Chang, Laura Heraty and Dr Brett Hosking for their critical reading of this 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triggers cell death by inactivating a thiol-specific antioxidant protein J Biol Chem 276, 21870–21877 43 Wilchek M, Miron T & Kohn J (1984) Affinity chromatography Methods Enzymol 104, 3–55 Supplementary material The following supplementary material is available online: Table S1 Sequences of the primers used for cloning volvatoxin A1 (VVA1) cDNA and peptide competitors Fig S1 Nucleotide and deduced protein sequence of volvatoxin A1 (VVA1) The nucleotide sequence and VVA1 is a novel toxin regulator of VVA2 deduced amino acid sequence of VVA1 cDNA (GenBank accession number AY952461) The ORF consists of 393 amino acid residues The first nucleotide and amino acid of VVA1 are boxed Nucleotide and amino acid residues are numbered on the left and right, respectively Arrows mark the primers used in RACEPCR The asterisk denotes the stop codon at nucleotide position 1180 The amino acid sequence deduced from VVA1 cDNA is shown by a one-letter code Fig S2 Schematic representation of a structural model of volvatoxin A1 (VVA1) The figure shows a ribbon plot representation of the proposed models for VVA1 b-Sheets are in yellow, a-helices are in pink, and the loops are in white–blue Each VVA1 domain was modeled by swissmodel (http://swissmodel.expasy.org) using the crystal structure of volvatoxin A2 (VVA2) (PDBID:1PP0) (J Mol Biol 343, 477–491, 2004) as a template Two domains were linked manually by program O Fig S3 Effects of detergents and liposomes on the oligomerization of volvatoxin A2 (VVA2) (A) Triton X-100 induced the oligomerization of VVA2 VVA2 (45 nm) was incubated with various detergents in 50 mm Tris, pH 8.0, at 37 °C for 30 The formation of VVA2 oligomer was determined by 10% SDS ⁄ PAGE analysis (B) Oligomerization of VVA2 was induced by liposomes After treatment of VVA2 with or without mm liposomes in NaCl ⁄ Pi, the reaction products were analyzed by 10% SDS ⁄ PAGE Fig S4 Anti-volvatoxin A2 (VVA2) IgG VVA2 and volvatoxin A1 (VVA1) were analyzed by 10% SDS ⁄ PAGE, followed by western blots and anti-VVA2 IgG This material is available as part of the online article from http://www.blackwell-synergy.com FEBS Journal 273 (2006) 3160–3171 ª 2006 The Authors Journal compilation ª 2006 FEBS 3171 ... membrane Interactions between VVA1 and VVA2 B Fig Effects of volvatoxin A1 (VVA1) on the hemolytic and cytotoxic activity of volvatoxin A2 (VVA2) (A) The hemolytic activity of VVA2 regulated by VVA1... VVA2 can use VVA1 as a basis for the formation of VVA2 oligomers Interaction of VVA1 and VVA2 by amphipathic a-helix To identify the binding sites in VVA1 responsible for direct interaction with. .. competition assay (A) Schematic representation of peptide competitors (B) Binding of volvatoxin A2 (VVA2) to volvatoxin A1 (VVA1) was inhibited by the amphipathic a-helices of VVA1 The VVA2 and VVA1