Heparin and Heparan Sulfate Bind to Snake Cardiotoxin

Patel, Himatkumar V.; Vyas, Alka; Vyas, K. A.; Liu, Yi-Shiuan; Chiang, Chien-Min; Lang, Ming; Wu, Wenwen

doi:10.1074/jbc.272.3.1484

Cited by 66 publications

(36 citation statements)

References 38 publications

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“…GAGs have been shown to be a potential targets for cobra cardiotoxin with high affinity and specificity through a cationic belt at the concave surface of the polypeptide (14,15). We demonstrated that the binding site of VVA2 with GAGs was located at ␤-strands 7, 8, and ␣-helix D by affinity column chromatography, and preincubation of VVA2 with heparin prevented VVA2 oligomerization by liposomes.…”

Section: Discussionmentioning

confidence: 80%

“…2C). Because cardiac myocytes contain a higher amount of GAGs on the cell membrane, it suggests that the HBS located at ␤-strands 7, 8, and ␣-helix D of VVA2 plays an important role in its cardiotoxicity (15).…”

Section: Fig 5 Vva2 Binds To Cell Membrane By Its Ctfmentioning

confidence: 99%

“…166 -194 residues), which is conserved among cardiotoxins isolated from snake venoms (14,15). The amphipathic ␣-helix B (78 -86 residues) of the N-terminal fragment (NTF, 1-127 residues), either as an isolated fragment, NTF, or in the intact VVA2 molecule, was shown to be crucial for VVA2 oligomerization.…”

mentioning

confidence: 99%

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Functional Domains of a Pore-forming Cardiotoxic Protein, Volvatoxin A2

Weng

Lin

Hsu

et al. 2004

Journal of Biological Chemistry

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Volvatoxin A2 (VVA2), a novel pore-forming cardiotoxic protein was isolated from the mushroom Volvariella volvacea. We identified an N-terminal fragment (NTF) (1-127 residues) of VVA2 as a domain for oligomerization by limited tryptic digestion. On preincubation of NTF with VVA2, NTF was found to inhibit VVA2 hemolytic activity by inducing VVA2 oligomerization in the solution in the same manner as liposomes. By site-directed mutagenesis, the amphipathic ␣-helix B of NTF or VVA2 was shown to be indispensable for its biological functions. Interestingly, at a molar ratio of recombinant NTF (reNTF)/VVA2 as low as 0.01, reNTF was able to inhibit VVA2 hemolytic activity and induce VVA2 oligomerization. This indicates that reNTF can trigger VVA2 oligomerization by a seeding effect. Furthermore, the recombinant C-terminal fragment (128 -199 residues) was found to be a functional domain that mediates the membrane binding of VVA2. We found a fragment localized at the C-terminal half of VVA2 containing ␤6, -7, and -8, which is protected from protease digestion because of its insertion of a membrane. We also identified a putative heparin binding site (HBS) located in the VVA2 C terminus (166 -194 residues), which was conserved among 10 kinds of snake venom cardiotoxins. VVA2 or the reHBS fragment was shown to interact with sulfated glycoaminoglycans by affinity column chromatography. The finding of a higher number of glycoaminoglycans in the membrane of cardiac myocytes suggested that they could be the specific membrane target for VVA2. Taken together, these findings indicate that VVA2 contains two functional domains, NTF and CTF. The NTF domain is responsible for VVA2 oligomerization and the CTF domain for membrane binding and insertion. Our results support a model whereby the formation of VVA2 oligomeric pre-pore complexes precedes their membrane insertion.Volvatoxin A (VVA), 1 first isolated from the edible mushroom Volvariella volvacea (1, 2), is a novel pore-forming cardiotoxic protein that causes cardiac arrest by activation of Ca 2ϩ -dependent ATPase activity and inhibition of Ca 2ϩ accumulation in the microsomal fraction of the sarcoplasmic recticulum of the ventricular muscle (3). VVA is composed of VVA1 and VVA2, of which only VVA2 is endowed with hemolytic and cytotoxic activity (1). Our previous studies showed that VVA2 consists of 199 amino acid residues without cysteine residues, and that it forms pores in response to its oligomer formation in human erythrocyte membranes and liposomes. This toxic protein also permeabilized the cell membrane and allowed the passage of 70-kDa dextran rhodamine B into cultured Xenopus myocytes. Furthermore, conformational changes occurred when VVA2 interacted with liposomes (2).Naturally occurring hemolytic toxic proteins can be classified into three groups based on their mechanisms of lysis of red blood cells: (a) pore forming, (b) enzymatic degradation of membrane lipids, and (c) solubilizing cell membrane by a detergentlike action (4). Several hemolytic toxic proteins isolated...

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Section: Discussionmentioning

confidence: 80%

Section: Fig 5 Vva2 Binds To Cell Membrane By Its Ctfmentioning

confidence: 99%

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Functional Domains of a Pore-forming Cardiotoxic Protein, Volvatoxin A2

Weng

Lin

Hsu

et al. 2004

Journal of Biological Chemistry

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“…Hep-HMW accentuates the penetration of CTX into phospholipid membranes, as analyzed by Langmuir monolayer measurement under physiological salt condition (6). A similar heparin-enhanced binding of CTX A3 toward LPC micelles can also be monitored by Tyr intrinsic fluorescence intensity change of CTX A3 under 150 mM NaCl and 10 mM phosphate buffer at pH 7.4 ( Fig.…”

Section: Heparin-enhanced Ctx Binding To Lpc Micelles As Reflected Bymentioning

confidence: 85%

“…Heparin not only binds to homologous CTXs, with different specificity, but also promotes its penetration into phospholipid monolayer under physiological ionic conditions (6). Analysis of binding of 10 CTX homologues to heparin reveals a new heparin-binding structural motif by involving several positive residues at the concave surface of the three-finger toxin (7).…”

Section: From the Department Of Life Sciences National Tsing Hua Unimentioning

confidence: 99%

Heparin Binding Stabilizes the Membrane-bound Form of Cobra Cardiotoxin

Sue

Chien

Huang

et al. 2002

Journal of Biological Chemistry

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It has been shown previously that the long chain fragments of heparin bind to the ␤-strand cationic belt of the three-finger cobra cardiotoxin (or cytotoxin, CTX) and hence enhance its penetration into phospholipid monolayer under physiological ionic conditions. By taking lysophosphatidylcholine (LPC) micelles as a membrane model, we have shown by 1 H NMR study that the binding of heparin-derived hexasaccharide (Hep-6) to CTX at the ␤-strand region can induce conformational changes of CTX near its membrane binding loops and promote the binding activity of CTX toward LPC. The Fourier-transform infrared spectra and NMR nuclear Overhauser effect of Hep-6⅐CTX and CTX⅐LPC complex in aqueous buffer also supplemented the aforementioned observation. Thus, the detected conformational change may presumably be the result of structural coupling between the connecting loops and its ␤-strands. This is the first documentation of results showing how the association of hydrophilic carbohydrate molecules with amphiphilic proteins can promote hydrophobic protein-lipid interaction via the stabilization of its membrane-bound form. A similar mechanism involving tripartite interactions of heparin, protein, and lipid molecules may be operative near the extracellular matrix of cell membranes.Cobra cardiotoxins (or cytotoxins, CTXs) 1 are highly basic, three-fingered polypeptides with the capability of inducing direct lytic effect on many cell types, including cardiac myocytes (1, 2). The available x-ray and NMR structures of CTXs are similar with a short antiparallel double-stranded ␤-sheet connected by loop I and with a long antiparallel triple-stranded ␤-sheet connected by loop II and III. Two distinct types of CTXs, i.e. P-and S-type, can be distinguished by the presence of Pro 30 and Ser 28 , respectively, near the tip of second finger (loop II) based on its binding activity toward neutral phospholipid bilayers and micelles (3). P-type CTXs, including the major CTX A3 from Taiwan cobra (Naja atra) venom, have been shown to induce a reversible formation of an extra outwardly rectifying conductance in bullfrog atrial myocytes (4) and also redistribute themselves between penetrating and peripheral binding states in phosphatidylcholine (PC) dispersion under favorable condition (5). Despite of the apparent perturbing effect of CTX toward phospholipid membranes, it is not clear how CTXs might act specifically toward different cell types.Recently, it was suggested that the sulfated polysaccharides such as glycosaminoglycans (GAGs), which are abundant in the extracellular matrix of most animal cells, are potential targets for CTX action (6 -8). Heparin not only binds to homologous CTXs, with different specificity, but also promotes its penetration into phospholipid monolayer under physiological ionic conditions (6). Analysis of binding of 10 CTX homologues to heparin reveals a new heparin-binding structural motif by involving several positive residues at the concave surface of the three-finger toxin (7). The positive residues flanking the...

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