Syed Abid Ali

Sea snake Hydrophis cyanocinctus venom. I. Purification, characterization and N-terminal sequence of two phospholipases A2

Two phospholipases A2 (PLA2, H1 and H2) from sea snake Hydrophis cyanocinctus venom were purified to homogeneity in a single step using reversed-phase high performance liquid chromatography on a Nucleosil 7C18 column. The molecular weights of H1 and H2, as estimated by MALDI MS, were 13588.1 and 13247.2 Da, respectively. The N-terminal 60 amino acid residues were determined by direct automated Edman degradation analysis. Since both PLA2s show close sequence homologies to those of PLA2s from other Elapid snakes (60-84%) they have been tentatively classified as belonging to group-IA and Asp-49 phospholipases A2. Despite the sequence variation (18%) between H1 and H2, their general structural organization is very similar as shown by their clearly related CD spectra. Furthermore, both enzymes are quite thermostable (60-65 degrees C) as determined by temperature variable CD spectra, indicating that the enzymes contain compact folded structure, mainly based on the core structure of disulfide bridges. However, the major PLA2 (H1) shows higher toxicity to albino rats (LD50 i.p. 0.04 mg/kg) and purification resulted in 18-fold increase in toxicity over the crude or whole venom (LD50 i.p. 0.80 mg/kg). H1 also shows edema-inducing and indirect haemolytic but no haemorrhagic activity. Unlike the toxic PLA2-H1, enzyme H2 was not toxic to albino rats but showed edema-inducing and indirect haemolytic activities.

Purification and primary structure of low molecular mass peptides from scorpion (Buthus sindicus) venom.

The primary structures of four low molecular mass peptides (Bs 6, 8, 10 and 14) from scorpion Buthus sindicus were elucidated via combination of Edman degradation and matrix-assisted laser desorption ionization mass spectrometry. Bs 8 and 14 are cysteine-rich, thermostable peptides composed of 35-36 residues with molecular weights of 3.7 and 3.4 kDa, respectively. These peptides show close sequence homologies (55-78%) with other scorpion chlorotoxin-like short-chain neurotoxins (SCNs) containing four intramolecular disulfide bridges. Despite the sequence variation between these two peptides (37% heterogeneity) their general structural organization is very similar as shown by their clearly related circular dichroism spectra. Furthermore, Bs6 is a minor component, composed of 38 residues (4.1 kDa) containing six half-cystine residues and having close sequence identities (40-80%) with charybdotoxin-like SCNs containing three disulfide bridges. The non-cysteinic, bacic and thermolabile Bs10 is composed of 34 amino acid residues (3.7 kDa), and belongs to a new class of peptides, with no sequence resemblance to any other so far reported sequence isolated from scorpions. Surprisingly, Bs10 shows some limited sequence analogy with oocyte zinc finger proteins. Results of these studies are discussed with respect to their structural similarities within the scorpion LCNs, SCNs and other biologically active peptides.

Sea snake Hydrophis cyanocinctus venom. II. Histopathological changes, induced by a myotoxic phospholipase A2 (PLA2-H1)

A toxic phospholipase A2 (PLA2-H1), isolated from the venom of the sea snake Hydrophis cyanocinctus, was tested for its ability to induce myonecrosis and histopathological changes in albino rats and mice. Induction of myonecrosis was demonstrated by their ability to release creatine kinase (CK) from damaged muscle fibers and direct histopathological examination of the injected muscles (i.m.). PLA2-H1 exhibits intense myonecrosis characterized by the changes including, necrosis and edematous appearance with cellular infiltrate, vacuolation and degenerated muscle cells with delta lesions and heavy edema in between the cells. No myoglobinuria was noted in any group of animals. The purified PLA2-H1 was also administered intraperitoneally into the experimental animals and tissue samples were taken at several time intervals. Light microscopic examination of the kidney sections revealed severe damage, evident by focal tubular necrosis, complete disquamation of epithelial lining and epithelial degeneration of tubules in all test animals. Light micrographs of liver sections after 24 h of injection shows fatty infiltration in parenchyma and squashed hepatocytes, while after 48 h, fatty vacuolation of parenchyma in a generalized pattern was observed. Furthermore, sections of the lungs of the same group of animals (48 h) show dilated bronchia and marked infiltration of inflammatory cells within alveoli. Our results suggest that the purified PLA2-H1 induced moderate myotoxicity in muscles and mild histopathological changes in other vital organs without myoglobinuria.

Solid phase synthesis, NMR structure determination of α-KTx3.8, its in silico docking to Kv1.x potassium channels, and electrophysiological analysis provide insights into toxin-channel selectivity

Animal venoms, such as those from scorpions, are a potent source for new pharmacological substances. In this study we have determined the structure of the α-KTx3.8 (named as Bs6) scorpion toxin by multidimensional (1)H homonuclear NMR spectroscopy and investigated its function by molecular dynamics (MD) simulations and electrophysiological measurements. Bs6 is a potent inhibitor of the Kv1.3 channel which plays an important role during the activation and proliferation of memory T-cells (TEM), which play an important role in autoimmune diseases. Therefore, it could be an interesting target for treatment of autoimmune diseases. In this study, Bs6 was synthesised by solid phase synthesis and its three-dimensional (3D) structure has been determined. To gain a deeper insight into the interaction of Bs6 with different potassium channels like hKv1.1 and hKv1.3, the protein-protein complex was modelled based on known toxin-channel structures and tested for stability in MD simulations using GROMACS. The toxin-channel interaction was further analysed by electrophysiological measurements of different potassium channels like hKv1.3 and hKv7.1. As potassium channel inhibitors could play an important role to overcome autoimmune diseases like multiple sclerosis and type-1 diabetes mellitus, our data contributes to the understanding of the molecular mechanism of action and will ultimately help to develop new potent inhibitors in future.

Oxygen transport proteins: III. Structural studies of the scorpion (Buthus sindicus) hemocyanin, partial primary structure of its subunit Bsin1

The hemocyanin (Hc) from Buthus sindicus, studied in the native state, demonstrated to be an aggregate of eight different types of subunits arranged in four cubic hexamers. Both, the top and the side views of the native molecule have been identified from the negatively stained specimens using transmission electron microscopy. Out of these, eight different polypeptide chains, the partial primary structure (68%) of a subunit Bsin1 (Mr = 72422.7 Da) was established using a combination of automated Edman degradation and mass spectrometry. A multiple sequence alignment with other closely related cheliceratan Hc subunits revealed average identities of ca. 60%. Most of the structurally important residues, i.e. copper and calcium-binding ligands, as well as the residues involved in the presumed oxygen entrance pathway, proved to be strictly conserved in Bsin1. Sequence variations have been observed around the functionally important chloride-binding site, not only for the B. sindicus subunit Bsin1, but also for the subunit Aaus-6 of the scorpion A. australis and the subunit Ecal-a from the spider Eurypelma californicum Hcs. Deviation in the primary structure related to the chloride-binding site suggest that the effect of chloride ions may vary in different hemocyanins. Furthermore, the secondary structural contents of the Hc subunit Bsin1 were determined by circular dichroism revealing ca. 33% alpha-helix, 18%, beta-sheet, 19% beta-turn, and 30% random coil composition. These values are in good agreement with the crystal structure of the closely related Hc subunit Lpol-II from horseshoe crab L. polyphemus. Electron microscopic studies of the purified Hc subunit under native conditions revealed that Bsin1 has self aggregation properties. Results of these studies are discussed.

Oxygen transport proteins: II. Chemical and spectroscopic properties of scorpion (Buthus sindicus) native hemocyanin and purified subunit Bsin1

Out of eight different polypeptide chains, present in the native hemocyanin molecule of scorpion Buthus sindicus, subunit Bsin1 was preparatively purified and characterized by amino acid composition, laser desorption mass spectrometry and spectroscopic techniques. Thermal stability of the intact respiratory protein and its subunit was studied by following the change in ellipticity at 222 nm as a function of temperature. The copper-dioxygen system at the binuclear active site has a stabilizing effect and the oxy-proteins are significantly more thermostable than the apo-forms. The thermal stability of the scorpion Hc from B. sindicus is considerably smaller than that of the tarantula (Eurypelma californicum) Hc [Sterner R et al., FEBS Lett 1995;364:9–12], although both, scorpion and spider, belong to the subphylum chelicerata. The fluorescence properties of the scorpion Hc and Bsin1 suggest that the indol groups are in rather nonpolar environment, deeply ‘buried’ in the interior of the aggregates and structural subunits. At the same time, these chromophores are located on subunit interfaces in the C. sapidus and O. vulgaris Hcs, participating in protein–protein interactions [Ricchelli F et al., Arch Biochem Biophys 1984;235:461–469; Stoeva S et al., Spectrochimica Acta A 1995;51:1965–1974].