Wen-guey Wu

Heparin and heparan sulfate bind to snake cardiotoxin: Sulfated oligosaccharides as a potential target for cardiotoxin action

Cardiotoxins (CTXs) from cobra venom show cytotoxicity toward several cell types. They cause systolic heart arrest and severe tissue necrosis. Their interaction with phospholipids is established but by itself fails to explain the specificity of these toxins; other component(s) of membrane must, therefore, intervene to direct them toward their target. We herein show, for the first time, that sulfated glycosaminoglycans, heparin, heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS), interact with CTX A3, a major component of Taiwan cobra venom, by use of affinity chromatography, circular dichroism, absorbance, and fluorescence intensity and anisotropy measurements. The relative strength of binding, determined by the NaCl concentration required to dissociate the CTX-glycosaminoglycan complex, varied as follows: heparin > DS > CS > HS. In physiological buffer (8 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4, 138 mM NaCl, pH 7.4), however, only heparin and HS bound to CTX, with respective dissociation constants of 1.4 and 16 μM, while CS and DS failed to exhibit well defined binding behavior, as indicated by fluorescence measurements. We estimate that CTX makes 3-4 ionic contacts with heparin based on a salt-dependent binding study and that ∼40% of binding free energy is derived from purely electrostatic interactions under physiological conditions. Sulfated pentasaccharide may be sufficient to bind to CTX. We also found that heparin accentuates the penetration of CTX into phospholipid membranes as analyzed by Langmuir monolayer measurement. In view of these results we propose that heparin-like moieties of the cell surface may modulate the action of CTX.

Cobra CRISP functions as an inflammatory modulator via a novel Zn2+- and heparan sulfate- dependent transcriptional regulation of endothelial cell adhesion molecules

Cysteine-rich secretory proteins (CRISPs) have been identified as a toxin family in most animal venoms with biological functions mainly associated with the ion channel activity of cysteine-rich domain (CRD). CRISPs also bind to Zn2+ at their N-terminal pathogenesis-related (PR-1) domain, but their function remains unknown. Interestingly, similar the Zn2+-binding site exists in all CRISP family, including those identified in a wide range of organisms. Here, we report that the CRISP from Naja atra (natrin) could induce expression of vascular endothelial cell adhesion molecules, i.e. intercellular adhesion molecule-1, vascular adhesion molecule-1, and E-selectin, to promote monocytic cell adhesion in a heparan sulfate (HS)- and Zn2+-dependent manner. Using specific inhibitors and small interfering RNAs, the activation mechanisms are shown to involve both mitogen-activated protein kinases and nuclear factor-κB. Biophysical characterization of natrin by using fluorescence, circular dichroism, and x-ray crystallographic methods further reveals the presence of two Zn2+-binding sites for natrin. The strong binding site is located near the putative Ser-His-Glu catalytic triad of the N-terminal domain. The weak binding site remains to be characterized, but it may modulate HS binding by enhancing its interaction with long chain HS. Our results strongly suggest that natrin may serve as an inflammatory modulator that could perturb the wound-healing process of the bitten victim by regulating adhesion molecule expression in endothelial cells. Our finding uncovers a new aspect of the biological role of CRISP family in immune response and is expected to facilitate future development of new therapeutic strategy for the envenomed victims.

Glycosphingolipid-facilitated Membrane Insertion and Internalization of Cobra Cardiotoxin THE SULFATIDE·CARDIOTOXIN COMPLEX STRUCTURE IN A MEMBRANE-LIKE ENVIRONMENT SUGGESTS A LIPID-DEPENDENT CELL-PENETRATING MECHANISM FOR MEMBRANE BINDING POLYPEPTIDES

Cobra cardiotoxins, a family of basic polypeptides having lipid- and heparin-binding capacities similar to the cell-penetrating peptides, induce severe tissue necrosis and systolic heart arrest in snakebite victims. Whereas cardiotoxins are specifically retained on the cell surface via heparan sulfate-mediated processes, their lipid binding ability appears to be responsible, at least in part, for cardiotoxin-induced membrane leakage and cell death. Although the exact role of lipids involved in toxin-mediated cytotoxicity remains largely unknown, monoclonal anti-sulfatide antibody O4 has recently been shown to inhibit the action of CTX A3, the major cardiotoxin from Taiwan cobra venom, on cardiomyocytes by preventing cardiotoxin-induced membrane leakage and CTX A3 internalization into mitochondria. Here, we show that anti-sulfatide acts by blocking the binding of CTX A3 to the sulfatides in the plasma membrane to prevent sulfatide-dependent CTX A3 membrane pore formation and internalization. We also describe the crystal structure of a CTX A3-sulfatide complex in a membrane-like environment at 2.3 Å resolution. The unexpected orientation of the sulfatide fatty chains in the structure allows prediction of the mode of toxin insertion into the plasma membrane. CTX A3 recognizes both the headgroup and the ceramide interfacial region of sulfatide to induce a lipid conformational change that may play a key role in CTX A3 oligomerization and cellular internalization. This proposed lipid-mediated toxin translocation mechanism may also shed light on the cellular uptake mechanism of the amphiphilic cell-penetrating peptides known to involve multiple internalization pathways.

Membrane packing geometry of diphytanoylphosphatidylcholine is highly sensitive to hydration: Phospholipid polymorphism induced by molecular rearrangement in the headgroup region

Diphytanoylphosphatidylcholine (DPhPC) has often been used in the study of protein-lipid interaction and membrane channel activity, because of the general belief that it has high bilayer stability, low ion leakage, and fatty acyl packing comparable to that of phospholipid bilayers in the liquid-crystalline state. In this solid-state 31P and 2H NMR study, we find that the membrane packing geometry and headgroup orientation of DPhPC are highly sensitive to the temperature studied and its water content. The phosphocholine headgroup of DPhPC starts to change its orientation at a water content as high as approximately 16 water molecules per lipid, as evidenced by hydration-dependent 2H NMR study at room temperature. In addition, a temperature-induced structural transition in the headgroup orientation is detected in the temperature range of approximately 20-60 degrees C for lipids with approximately 8-11 water molecules per DPhPC. Dehydration of the lipid by one more water molecule leads to a nonlamellar, presumably cubic, phase formation. The lipid packing becomes a hexagonal phase at approximately 6 water molecules per lipid. A phase diagram of DPhPC in the temperature range of -40 degrees C to 80 degrees C is thus constructed on the basis of NMR results. The newly observed hydration-dependent DPhPC lipid polymorphism emphasizes the importance of molecular packing in the headgroup region in modulating membrane structure and protein-induced pore formation of the DPhPC bilayer.

Non-cytotoxic cobra cardiotoxin A5 binds to αvβ3 integrin and inhibits bone resorption: Identification of cardiotoxins as non-rgd integrin-binding proteins of the Ly-6 family

Severe tissue necrosis with a retarded wound healing process is a major symptom of a cobra snakebite. Cardiotoxins (CTXs) are major components of cobra venoms that belong to the Ly-6 protein family and are implicated in tissue damage. The interaction of the major CTX from Taiwan cobra, i.e. CTX A3, with sulfatides in the cell membrane has recently been shown to induce pore formation and cell internalization and to be responsible for cytotoxicity in cardiomyocytes (Wang, C.-H., Liu, J.-H., Lee, S.-C., Hsiao, C.-D., and Wu, W.-g. (2006) J. Biol. Chem. 281, 656-667). We show here that one of the non-cytotoxic CTXs, i.e. CTX A5 or cardiotoxin-like basic polypeptide, from Taiwan cobra specifically bound to αvβ3 integrin and inhibited bone resorption activity. We found that both membrane-bound and recombinant soluble αvβ3 integrins bound specifically to CTX A5 in a dose-dependent manner. Surface plasmon resonance analysis showed that human soluble αvβ3 bound to CTX A5 with an apparent affinity of ∼0.3 μm. Calf pulmonary artery endothelial cells, which constitutively express αvβ3, showed a CTX A5 binding profile similar to that of membrane-bound and soluble αvβ3 integrins, suggesting that endothelial cells are a potential target for CTX action. We tested whether CTX A5 inhibits osteoclast differentiation and bone resorption, a process known to be involved in αvβ3 binding and inhibited by RGD-containing peptides. We demonstrate that CTX A5 inhibited both activities at a micromolar range by binding to murine αvβ3 integrin in osteoclasts and that CTX A5 co-localized with β3 integrin. Finally, after comparing the integrin binding affinity among CTX homologs, we propose that the amino acid residues near the two loops of CTX A5 are involved in integrin binding. These results identify CTX A5 as a non-RGD integrin-binding protein with therapeutic potential as an integrin antagonist.

Structures of two elapid snake venom metalloproteases with distinct activities highlight the disulfide patterns in the D domain of ADAMalysin family proteins

Abstract The structures of snake venom metalloproteases (SVMPs) are proposed to be useful models to understand the structural and functional relationship of ADAM (a disintegrin and metalloprotease) which are membrane-anchored proteins involved in multiple human diseases. We have purified, sequenced and determined the structures of two new P-III SVMPs – atragin and kaouthiagin-like (K-like) from Naja atra. Atragin exhibits a known C-shaped topology, whereas K-like adopts an I-shaped conformation because of the distinct disulfide pattern in the disintegrin-like (D) domain. K-like exhibits an enzymatic specificity toward pro-TNFα with less inhibition of cell migration, but atragin shows the opposite effect. The specificity of the enzymatic activity is indicated to be dominated mainly by the local structures of SVMP in the metalloprotease (M) domain, whereas the hyper-variable region (HVR) in the cysteine-rich (C) domain is involved in a cell-migration activity. We demonstrate also a pH-dependent enzymatic activity of atragin that we correlate with the structural dynamics of a Zn2+-binding motif and the Met-turn based on the structures determined with a pH-jump method. The structural variations between the C- and I-shapes highlight the disulfide bond patterns in the D domain of the ADAM/adamalysin/reprolysins family proteins.

Cobra venom proteome and glycome determined from individual snakes of Naja atra reveal medically important dynamic range and systematic geographic variation.

UNLABELLED Recent progress in snake venomics has shed much light on the intra-species variation among the toxins from different geographical regions and has provided important information for better snakebite management. Most previous reports on snake venomics were based on venoms pooled from different snakes. In this study, we present the proteomic and glycomic profiles of venoms from individual Naja atra snakes. The results reveal wide dynamic range of three-finger toxins. Systematic classification based on cardiotoxin (CTX-) profiles of A2/A4 and A6, respectively, allowed the identification of two putative subspecies of Taiwan cobra from the eastern and western regions. We also identified four major N-glycan moieties on cobra snake venom metalloproteinase on the bi-antennary glycan core. ELISA showed that these glycoproteins (<3%) could elicit much higher antibody response in antiserum when compared to other high-abundance cobra venom toxins such as small molecular weight CTXs (~60%). By removing these high-molecular weight glycoproteins from the immunogen, we demonstrated better protection than that achieved with conventional crude venom immunization in mice challenged by crude venom. We conclude that both intra-species and inter-individual variations of proteomic and glycomic profiles of snake venomics should be considered to provide better antivenomic approach for snakebite management. BIOLOGICAL SIGNIFICANCE Based on the proteomic and glycomic profiles of venoms obtained from individual snakes, we demonstrated a surprisingly wide dynamic range and geographical variation of three-finger toxins in cobra venomics. This provides a reasonable explanation for the variable neutralization effects of antivenom treatment on victims suffering from cobra snakebite and suggests a simple and economic method to produce potent antivenom with better efficacy. Since two major venomic profiles with distinct dynamic ranges were observed for Taiwan cobra venoms isolated from the eastern and western regions, the current venomic profile should be used as a quality control for future production of antivenom in clinical applications.

Functional divergence between 2 chemokines is conferred by single amino acid change

CXCL4 and CXCL4L1 are 2 closely related CXC chemokines that exhibit potent antiangiogenic activity. Because interactions with glycosaminoglycans play a crucial role in chemokines activity, we determined the binding parameters of CXCL4 and CXCL4L1 for heparin, heparan sulfate, and chondroitin sulfate B. We further demonstrated that the Leu67/His67 substitution is critical for the decrease in glycan binding of CXCL4L1 but also for the increase of its angiostatic activities. Using a set of mutants, we show that glycan affinity and angiostatic properties are not completely related. These data are reinforced using a monoclonal antibody that specifically recognizes structural modifications in CXCL4L1 due to the presence of His67 and that blocks its biologic activity. In vivo, half-life and diffusibility of CXCL4L1 compared with CXCL4 is strongly increased. As opposed to CXCL4L1, CXCL4 is preferentially retained at its site of expression. These findings establish that, despite small differences in the primary structure, CXCL4L1 is highly distinct from CXCL4. These observations are not only of great significance for the antiangiogenic activity of CXCL4L1 and for its potential use in clinical development but also for other biologic processes such as inflammation, thrombosis or tissue repair.

Structure and dynamics of primary hydration shell of phosphatidylcholine bilayers at subzero temperatures

Deuterium NMR relaxation and intensity measurements of the 2H-labeled H2O/dimyristoyl phosphatidylcholine bilayer were performed to understand the molecular origin of the freezing event of phospholipid headgroup and the structure and dynamics of unfrozen water molecules in the interbilayer space at subzero temperatures. The results suggest that about one to two water molecules associated with the phosphate group freeze during the freezing event of phospholipid headgroups, whereas about five to six waters near the trimethylammonium group behave as a water cluster and remain unfrozen at temperatures as low as -70 degrees C. In addition, temperature-dependent T1 and T2 relaxation times suggest that dynamic coupling occurs not only between the phosphate group and its bound water, but also between the methyl group and the adjacent water molecules. Based on these observations, the primary hydration shell of phosphatidylcholine headgroup at subzero temperatures is suggested to consist of two distinct regions: a clathrate-like water cluster, most likely a water pentamer, near the hydrophobic methyl group, and hydration water molecules associated with the phosphate group.

Crystal structures of bacillus cereus NCTU2 chitinase complexes with chitooligomers reveal novel substrate binding for catalysis: a chitinase without chitin-binding and insertion domains

Chitinases hydrolyze chitin, an insoluble linear polymer of N-acetyl-d-glucosamine (NAG)n, into nutrient sources. Bacillus cereus NCTU2 chitinase (ChiNCTU2) predominantly produces chitobioses and belongs to glycoside hydrolase family 18. The crystal structure of wild-type ChiNCTU2 comprises only a catalytic domain, unlike other chitinases that are equipped with additional chitin binding and insertion domains to bind substrates into the active site. Lacking chitin binding and chitin insertion domains, ChiNCTU2 utilizes two dynamic loops (Gly-67—Thr-69 and Ile-106–Val-112) to interact with (NAG)n, generating novel substrate binding and distortion for catalysis. Gln-109 is crucial for direct binding with substrates, leading to conformational changes of two loops with a maximum shift of ∼4.6 Å along the binding cleft. The structures of E145Q, E145Q/Y227F, and E145G/Y227F mutants complexed with (NAG)n reveal (NAG)2, (NAG)2, and (NAG)4 in the active site, respectively, implying various stages of reaction: before hydrolysis, E145G/Y227F with (NAG)4; in an intermediate state, E145Q/Y227F with a boat-form NAG at the −1 subsite, −1-(NAG); after hydrolysis, E145Q with a chair form −1-(NAG). Several residues were confirmed to play catalytic roles: Glu-145 in cleavage of the glycosidic bond between −1-(NAG) and +1-(NAG); Tyr-227 in the conformational change of −1-(NAG); Asp-143 and Gln-225 in stabilizing the conformation of −1-(NAG). Additionally, Glu-190 acts in the process of product release, and Tyr-193 coordinates with water for catalysis. Residues Asp-143, E145Q, Glu-190, and Tyr-193 exhibit multiple conformations for functions. The inhibitors zinc ions and cyclo-(l-His-l-Pro) are located at various positions and confirm the catalytic-site topology. Together with kinetics analyses of related mutants, the structures of ChiNCTU2 and its mutant complexes with (NAG)n provide new insights into its substrate binding and the mechanistic action.