Habu Snake Venom Research Articles

Roles of lysine-69 in dimerization and activity of Trimeresurus flavoviridis venom aspartate-49-phospholipase A2.

Trimeresurus flavoviridis (Habu snake) venom aspartate-49-phospholipase A2 (Asp-49-PLA2) was reacted at pH 9.0 with a 2-fold molar excess of 2,4,6-trinitrobenzenesulfonate in the absence of Ca2+ and two trinitrophenylated derivatives were isolated by HPLC. One was a derivative modified at Lys-11 and its activity was mostly retained. The other was a derivative modified at both Lys-11 and Lys-72 and its activity was 40% that of unmodified enzyme. Trinitrophenylation of Lys-72 appeared to bring about a conformational disorder at the lipid-water interface recognition site and thus a reduction of activity. When the enzyme was modified in the presence of Ca2+, activity decreased at a rate much faster than that in the absence of Ca2+ and Lys-69 came to be modified. These results suggested that conformational displacement of Asp-49-PLA2 of a local to global type occurs upon the binding of Ca2+. The derivative modified at Lys-69 had 28% activity and existed as a monomer. This supports a previous assumption that Lys-69 participates in dimerization of group II Asp-49-PLA2s [Brunie et al. (1985) J. Biol. Chem. 260, 9742-9749] and shows that dimerization is not necessarily essential for activity manifestation.

Purification and primary structure of a myotoxic Lysine-49 phospholipase A 2 with low lipolytic activity from Trimeresurus gramineus venom

Four acidic phospholipase A 2 (PLA 2) isozymes named PLA 2-I, II, III and IV have previously been isolated from Trimeresurus gramineus (green habu snake) venom and sequenced [Oda et al. (1991) Toxicon 29, 157; Fukagawa et al. (1992) Toxicon 30, 1131; Fukagawa et al. (1993) Toxicon 31, 957]. They contain aspartate-49 which is known to bind Ca 2+, essential for catalysis. In the present study, a basic PLA 2 named PLA 2-V containing lysine-49 was newly isolated from the same snake venom. Its isoelectric point was 9.4 and considerably higher than those ( c. 4.5) of PLA 2-I-IV. PLA 2-V was 1.1% as active as PLA 2-I toward egg-yolk emulsion but exhibited strong myotoxicity. The amino acid sequence of PLA 2-V was determined by sequencing the S-carboxamidomethylated derivative and its peptide fragments produced by enzymatic (clostripain, chymotrypsin, Achromobacter protease I and Staphylococcus aureus V8 protease) cleavages. PLA 2-V consists of 122 amino acid residues and is highly homologous (72–78%) to Lys-49 PLA 2s so far isolated from Viperidae snake venoms but less homologous (52%) to PLA 2-I. The presence of Asn-28, which is characteristic of Lys-49 PLA 2s, was confirmed.

Endothelial regeneration during the repair process following Habu-snake venom induced glomerular injury.

In this study, the kinetics of glomerular endothelial cells during the repair process following glomerular injury was investigated in a model of mesangial proliferative glomerulonephritis induced by Habu-snake venom (HSV) in rats. Intravenous injection of HSV led to a cystic ballooning type lesion at day 1. Subsequently a marked segmental proliferative lesion was observed in the cystic areas at day 5. Thereafter cellularity decreased and reconstruction of the glomerular tuft was gradually observed with time. The histological structure of the glomeruli had almost returned to normal 21 days following HSV injection. After prominent depletion at day 1, the number of endothelial cells increased rapidly and reached a plateau at day 7, not significantly different from that of the control group. Morphologically endothelial cell elongation from the vascular pole into the cystic lesion was seen together with premature capillary formation in the proliferative lesion. Accompanying the reduction of mesangial expansion, the endothelial cells gradually formed definite capillary lumens. We conclude that the mesangial proliferative glomerulonephritis induced by HSV recovers to its original structural state and that the migration and proliferation of endothelial cells with accompanying capillary formation are essential for the repair process, in addition to mesangial cell proliferation.

Expression of plasminogen activator-inhibitor-1 (PAI-1) during cellular remodeling in proliferative glomerulonephritis in the rat.

Pericellular proteolysis involves the plasminogen activator/plasmin system and plays an important role in cell remodeling involving cell migration and extracellular matrix turnover. Studies in this laboratory have previously characterized a model of proliferative glomerulonephritis induced by Habu snake venom (HSV) in the rat that involves cell migration, proliferation, and extracellular matrix accumulation. Because plasminogen activator-inhibitor-1 (PAI-1) has been used as a marker for cell migration as well as matrix accumulation, we were interested in examining the temporal and spatial expression and cellular sources of PAI-1 mRNA and translated protein over the course of HSV-induced proliferative glomerulonephritis. The results showed a highly localized and progressive expression of PAI-1 mRNA and translated protein by in situ hybridization and immunohistochemistry at the margins and periphery of glomerular lesions 8 and 24 hr after HSV. The expression of PAI-1 in glomerular lesions localized to the same sites as mesangial cell marker proteins, desmin and Thy-1.1, indicating that mesangial cells synthesize this important regulator proteolysis. Few cells expressed PAI-1 in the central aspects of glomerular lesions at later time intervals (48 and 72 hr) when cell proliferation and expression of extracellular matrix (fibronectin protein and mRNA) were maximal. Therefore, the expression of PAI-1 in this model was associated more with early events related to cell migration than with proliferation or extracellular matrix synthesis. These observations support the hypothesis that the plasminogen activator/plasmin system is involved in cell migration in early remodeling during glomerular disease.

Darwinian evolution of Trimeresurus flavoviridis venom gland phospholipase A2 isozymes

Abstract

Purification, sequencing and characterization of single amino acid-substituted phospholipase A2 isozymes from Trimeresurus Gramineus (green habu snake) venom

T. Fukagawa, T. Nose, Y. Shimohigashi, T. Ogawa, N. Oda, K. Nakashima, C.-C. Chang and M. Ohno. Purification, sequencing and characterization of single amino acid-substituted phospholipase A2 isozymes from Trimeresurus gramineus (green habu snake) venom. Toxicon31, 957–967, 1993.—Two phospholipases A2 named PLA2-III and IV were newly isolated from Trimeresurus gramineus (green habu snake) venom in addition to PLA2-I and II reported previously [Odaet al. (1991) Toxicon29, 157; Fukagawaet al. (1992) Toxicon30, 133]. Their isoelectric points were determined to be about 4.5. PLA2-III and IV exhibited almost unchanged lipolytic activity toward egg-yolk when compared with PLA2-I. The amino acid sequences were determined by sequencing the native proteins and the peptides produced by enzymatic (Achromobacter protease I and clostripain) and chemical (hydroxylamine) cleavages of the S-carboxamidomethylated derivative of the proteins. Both proteins consisted of 122 amino acid residues. When compared with PLA2-I, PLA2-III showed only a single amino acid substitution at the N-terminal position; namely from His to Asn. PLA2-IV also showed a single substitution from Ala to Asp at position 72. It was inferred that these amino acid substitutions between PLA2-I and PLA2-III or IV are due to the single base substitution at the corresponding codons of genes, which might be preserved independently. The unique presence of Phe at position 28, where Tyr is commonly located and assumed to be a part of the Ca2+-binding loop, was conserved in both PLA2-III and IV as in PLA2-I. There was no significant difference in the dissociation constants (4.3–5.2 × 10−4 M) for Ca2+ between these PLA2s and Tyr-28-containing PLA2s. These results suggested that the p-hydroxy group of Try-28 does not play a crucial role in binding of PLA2s to Ca2+.

Accelerated evolution of Trimeresurus flavoviridis venom gland phospholipase A2 isozymes.

Six Trimeresurus flavoviridis (Habu snake) venom gland phospholipase A2 (PLA2) isozyme genes were found to consist of four exons and three introns and to encode proteins of 138 amino acid residues, including the signal sequence of 16 amino acid residues. Comparison of the nucleotide sequences showed that the introns are much more homologous than the protein-coding regions of exons except for the signal peptide-coding region of the first exon. The numbers of nucleotide substitutions per site (KN) for introns are approximately one-fourth of the numbers of nucleotide substitutions per synonymous site (KS) for the protein-coding regions, indicating that the introns are unusually conserved. The absence of an apparent functional role for the introns suggests that the protein-coding regions, except for the signal peptide-coding domains, have evolved at greater substitution rates than introns. The fact that the numbers of nucleotide substitutions per nonsynonymous site (KA) are close to or larger than KS values for relevant pairs of genes revealed that Darwinian-type accelerated substitutions have occurred in the protein-coding regions or exons. This is compatible with the presence of PLA2 species with diverse physiological activities in the venom.

Sequence determination and characterization of a phospholipase A2 isozyme from Trimeresurus gramineus (green habu snake) venom

In addition to phospholipase A2-I (PLA2-I) reported previously (Odaet al., 1991, Toxicon29, 157), a new PLA2 named PLA2-II was isolated from Trimeresurus gramineus (green habu snake) venom, and its amino acid sequence was determined by sequencing the native protein and the peptides produced by enzymatic (Achromobacter protease I and clostripain) cleavages of the carboxamidomethylated derivative of the protein. The protein consisted of 122 amino acid residues and His-47, Asp-48, and Asp-98 which have been assumed to be essential for PLA2 activity were conserved. Its sequence similarity to PLA2-I was 79%, with 26 residual differences. In contrast to the unique presence of Phe-28 in PLA2-I, PLA2-II contains Tyr-28 as seen in most of other PLA2s. There was no significant difference between the dissociation constants of PLA2-I and PLA2-II for Ca2+. Secondary structure compositions of PLA2-II were similar to those of PLA2-I and Crotalus atrox PLA2. A striking difference was found between these isozymes in contractile activity of isolated smooth muscle preparation of guinea-pig ileum. PLA2-II was over ten times more potent than PLA2-I, although its lipolytic activity toward egg-yolk was even slightly weaker (73%) than that of PLA2-I. The difference in contractile activities of PLA2-I and PLA2-II could be assumed to be due to discriminative lipid recognition brought about by different amino acid residues at the 58th position (Asp for PLA2-I and Asn for PLA2-II).

Refolding of Trimeresurus flavoviridis Phospholipases A2

Abstract Trimeresurus flavoviridis (Habu snake) venom contains phospholipase A2 (PLA2) isozymes which are rich in disulfide bond and show diverse activities in spite of their highly homologous structures. When reduced form of Tyr(NO2)-67-PLA2, which is a mimic of Asp-49-PLA2 mutant, was oxidized at pH 8.0 in the presence of 5 mM L-cysteine and 5 mM Ca2+, native form of Tyr(NO2)-67-PLA2 was produced as being judged from both activity regeneration and adequate HPLC profile. Reduced forms of two Lys-49-PLA2 isozymes with extremely low lipolytic activity, which are called basic proteins I and II, also generated native forms of proteins when oxidized under the same conditions as above. Protein disulfide isomerase accelerated proper folding of reduced Tyr(NO2)-67-PLA2 at an initial phase of oxidation and was effective for rearrangement of incorrectly paired disulfide bonds of Asp-49-PLA2 to native construction. However, this isomerase failed to convert reduced form of partially active L-fragment, Asp-49-PLA2 lacking N-terminal octapeptide, to the native form, indicating that the entire sequence of protein in a nascent state is primarily required for proper folding. The present data together with the fact that reduced Asp-49-PLA2 folded properly (S. Tanaka et al. J. Biochem., 96, 1443 (1984)) afforded a strong basis for the structure and function study of T. flavoviridis PLA2s by means of in vitro mutagenesis technique.

Crystallization and preliminary X-ray study of blood coagulation factor IX/factor X-binding protein with anticoagulant activity from Habu snake venom

Crystals of a blood anticoagulant from the venom of the Habu snake, Trimeretsurus flavoviridis, have been obtained using ammonium sulfate by the vapor diffusion method. The crystals belong to the orthorhombic space group P2 1 2 1 2 with cell dimensions a = 172 A ̊ , b= 86 A ̊ , c = 65 A ̊ , and diffract to at least 4.0 Å resolution.

Glomerular localization of platelet secretory proteins in mesangial proliferative lesions induced by habu snake venom.

Platelets have been implicated in mesangial cell proliferation in experimental and clinical glomerular disease. In this study, the temporal relationship between release of platelet secretory cationic proteins (PSCP) and progression of mesangial hyperplasia was examined in a model of mesangial proliferative glomerulonephritis induced by Habu snake venom (HSV). Intravenous injection of HSV (2 mg/kg body wt) led to capillary dilatation and ballooning into cysts filled with prominent platelet aggregates at 8 hr and 24 hr. At 48 hr, lesions were heterogeneous, some exclusively cystic, others exclusively nodular (comprised of confluent proliferative mesangial cells). Most lesions were mixed, showing features of cystic lesions containing clusters of proliferating cells. At 72 hr, all lesions were exclusively nodular. These lesions were associated with persistent localization of PSCP, as demonstrated by immunofluorescence microscopy. At 8 hr, PSCP were restricted primarily to platelets, became more intensified and diffuse at later time intervals, and by 72 hr was demonstrated in a homogeneous pattern interspaced throughout the nodular lesions. Studies utilizing antiserum to a specific platelet secretory protein, platelet factor 4 (PF4), showed an identical pattern of glomerular localization. Thus, before and during the proliferative phase of nodular formation, mesangial cells are exposed to a milieu replete with PSCP, some of which are presumably biologically active, suggesting a potential role for platelet-secreted proteins in mesangial hyperplasia in this model.

メサンギウムの増生性変化とｍｅｓａｎｇｉｏｌｙｓｉｓ ハブ毒による腎糸球体の障害

Proliferative changes of the glomerular mesangium of the kidney are generally considered to be a progressive inflammatory process of glomerulonephritis. There are several reports on experimental glomerulonephritis induced by snake venom which is known to cause demonstrable mesangiolytic changes in the glomerulus. The author investigated the sequential morphological changes of the renal glomerulus by light and electron microscopy after administration of 1.0 mg/kg of Habu (Trimeresurus flavoviridis) snake venom to 56 guinea-pigs via tail vein injection. In addition, an analysis of proliferated cells in the glomerulus was attempted by applying the immunostaining method for the antibody to BrdU as the marker of the dividing cells. Various degrees of mesangiolytic changes of the glomerulus were induced after Habu venom injection. In this model, however, the majority of mesangiolytic changes were mild. Subsequent segmental nodular proliferation of the mesangial cells was observed in mesangiolytic areas. Mitotic figures appeared from 24 hours to 7 days after the venom injection, as shown by BrdU staining. New capillary lumens were formed among the proliferated cells of the postmesangiolytic segment 4 to 5 days after the venom injection. Formation of capillary lumens and reconstruction of the glomerular loops had been gradually established through the time course. The histological structure of the glomeruli returned almost to normal 15 weeks after the venom injection, with occasional features of remolded-healing, although a small number of glomeruli still showed persisted mild segmental mesangial proliferation as well as mild increase of PAM-positive substance in the mesangial area. These results suggest that mesangial proliferation is related to the reconstruction of the glomerulus rather than the progression of the glomerular lesions, at least in this model. The author postulates that the mesangial cell has the functional ability to repair the injured glomerular structure. The application of the BrdU immunostaining method turns out to be a useful tool for analyzing the relationship between cell mitosis and cell proliferation. In the guinea-pig, the Habu-snake venom induced mesangiolytic lesions are milder than those in the rabbit and the rat and the degree of subsequent mesangial proliferative changes is less prominent.

Comparison of amino terminal region of three isoenzymes of phospholipases A 2 (TFV PL-Ia, TFV PL-Ib, TFV PL-X) from Trimeresurus flavoviridis (habu snake) venom and the complete amino acid sequence of the basic phospholipase, TFV PL-X

R. M. Kini, S.-I. Kawabata and S. Iwanaga. Comparison of amino terminal region of three isoenzymes of phospholipases A 2 (TFV OL-Ia, TFV PL-Ib, TFV PL-X) from Trimeresurus flavoviridis (habu snake) venom and the complete amino acid sequence of the basic phospholipase, TFV PL-X. Toxicon 24, 1117 – 1129, 1986.—Three phospholipases (PLA 2s) isolated from Japanese habu snake ( Trimeresurus flavoviridis) venom differ from each other in the physiological symptoms induced in animals. The amino acid compositions and amino terminal sequences of these isoenzymes have been compared. TFV PL-Ia and TFV PL-Ib resemble each other very closely, but TFV PL-X has a distinctly different amino acid composition and amino terminal region. The complete amino acid sequence of the basic PLA 2, TFV PL-X, was determined by sequencing the four peptides obtained by the cleavage of Asp - Pro bonds. The overlapping peptide was obtained by cleaving tryptophanyl bond and the carboxy terminal region was determined by sequencing the chymotryptic peptide. TFV PL-X consisted of a single chain of 122 amino acid residues, including 14 half-cystine residues. It is a group II A PLA 2 and is homologous with the phospholipase super family. However, it has a distinctly different sequence compared with the other PLA 2s from the venom of Trimeresurus species.

Effects of pH and lysine during detoxification of a hemorrhagic principle of Habu snake (Trimeresurus flavoviridis) venom with formalin on the immunogenicity of the toxoid.

A hemorrhagic principle (HR1B) isolated from the venom of Habu snake (Trimeresurus flavoviridis) was detoxified with formalin at pH 5, 7 and 9. Higher pH shortened the period needed for detoxification and lowered the final formalin concentration required for complete detoxification. The extent of polymerization and the immunogenicity of the toxoid formed were dependent upon pH during detoxification; the lower the pH, the higher the extent of polymerization and the immunogenicity of the toxoid. The higher immunogenicity possessed by the toxoid with higher extent of polymerization was confirmed by fractionation of the toxoid prepared at pH 7 on Sephadex G-200 and by comparing the immunogenicity of the toxoid fractions. The presence of lysine during detoxification brought about a partially polymerized toxoid having new immunogenic characteristics and a higher immunogenicity.

Rapid method for separation and purification of four isoenzymes of phosphodiesterase from trimeresurus flavoviridis (habu snake) venom

Four isoenzymes of phosphodiesterase were identified in Trimeresurus flavoviridis venom. A rapid and highly reproducible column chromatographic procedure on CM-Sephadex C-25, QAE-Sephadex A-25 and Sephadex G-100 was developed for the purification of these isoenzymes with a yield of 83%. All four isoenzymes are metalloglycoproteins having negligible amounts of phosphomonoesterase activity.

Enhancement of hepatic microsomal aniline hydroxylation by Habu snake (Trimeresurus flavoviridis) venom fractions.

Enhancement of aniline hydroxylation by Habu snake venom was studied in vitro. The fraction which was isolated from Habu venom through column chromatography caused the enhancement of aniline hydroxylation of hepatic microsomes obtained from untreated rats. This fraction was heat stable and free from phospholipase activity. Enhancement of aniline hydroxylation was not clear in crude Habu venom or the unheated fraction, but appeared after heating of the fraction. This fraction enhanced aniline hydroxylation at low concentrations, followed by inhibition at high concentrations, but inhibited aminopyrine N-demethylation dose-dependently. Aniline hydroxylation was enhanced by the heated fraction at more than 1 mM aniline, and the enhancement was rather decreased at more than 10 mM aniline. The degree of the enhancement by the heated fraction increased with increasing pH.

ENHANCEMENT OF HEPATIC MICROSOMAL ANILINE HYDROXYLATION BY HABU SNAKE (Trimeresurus flavoviridis) VENOM FRACTIONS

Enhancement of aniline hydroxylation by Habu snake venom was studied in vitro. The fraction which was isolated from Habu venom through column chromatography caused the enhancement of aniline hydroxylation of hepatic microsomes obtained from untreated rats. This fraction was heat stable and free from phospholipase activity. Enhancement of aniline hydroxylation was not clear in crude Habu venom or the unheated fraction, but appeared after heating of the fraction. This fraction enhanced aniline hydroxylation at low concentrations, followed by inhibition at high concentrations, but inhibited aminopyrine N-demethylation dose-dependently. Aniline hydroxylation was enhanced by the heated fraction at more than 1 mM aniline, and the enhancement was rather decreased at more than 10 mM aniline. The degree of the enhancement by the heated fraction increased with increasing pH.

Ultrastructural changes and release reaction of platelets induced by an aggregation inducer purified from Trimeresurus mucrosquamatus (Formosan habu) snake venom

Ultrastructural changes and release reaction of washed rabbit platelets were observed after exposure to a purified aggregation inducer from the venom of Trimeresurus mucrosquamatus (Formosan habu snake). The venom inducer, at a concentration of 1 μg/ml, caused 90% aggregation and 40% release of the total platelet factor 4 activity in platelets. The venom inducer also caused, but at higher concentrations, aggregation of thrombin-treated, degranulated platelets but caused less than 2% release of platelet factor 4 from these platelets. Platelets exposed to the purified venom inducer lost their discoid shape and developed irregular projections, which were devoid of organelles. Microtubules formed a collar around organelles which tended to shift toward the platelet center. Finally, most alpha granules and dense bodies disappeared, and elements of tubular system remained in the ballooned cytoplasm in the periphery of the aggregate.

Quinacrine mustard: A fluorescent platelet label. In vitro studies and in vivo localisation in habu venom glomerulonephritis in rats

Platelets labelled with Quinacrine mustard fluoresce green with ultra-violet light. Quinacrine labelled platelets (QLP) were prepared in vitro and 0.5 ml injected i.v. into rats (conc n 7–10 × 10 5/mm 3). No adverse effects were detected. QLP comprised 10–19% of total blood platelets. Frozen sections of lung, liver, kidney and spleen up to 4 hours after i.v. QLP showed no abnormal sequestration. Platelet distribution in blood and tissues was studied in glomerulonephritis (gn) induced by Habu snake venom from 10 minutes up to 4 hours after venom. In the initial phase of thrombocytopenia (10 minutes after venom), QLP localised in lung, liver and glomeruli, glomerular distribution being identical with previous electron microscopic findings. In vitro studies of QLP showed aggregation with sodium arachidonate and collagen, but not with ADP. 5HT levels were within the normal range. Rat platelets labelled in vitro with Quinacrine appear to function well in vitro, and in vivo and can be identified in tissues. This may be a valuable method for studying the fate of platelets activated in vivo.

KIDNEY FUNCTION IN HABU VENOM NEPHROPATHY IN RATS

The injection of Habu snake venom into rats has been discussed as a useful model for studying focal mesangioproliferative glomerulonephritis(GN) (Cattell et al. Am.J.Pathol.87:511[1977]).Morphological,immune fluorescence and clearance studies were performed for further characterisation of this model.50 inbred Lewis rats received 3.3mg/kg Habu venom i.v.Three different phases of response could be observed:I.One to 12hrs after injection shock,coagulopathy,hemolysis and thrombocytopenia occurred suggesting DIC.Mortality rate was high (40%),many organs showed hemostasis and diffuse bleeding.Mesangiolyis was not observed.II.One to 3d after injection blood pressure,coagulation and thrombocytes returned to normal levels.Kidney function remained normal.Mortality was low.Mesangiolysis was rare.III.Four to 30d after injection kidney function did not differ from controls although there was focal mesangial hypercellularity.Glomerular immune fluorescence was negative for IgM, IgG and C3 during all three stages of Habu nephropathy. Habu venom induced a variety of alterations in rats including shock,DIC,vasculopathy and mesangioproliferative glomerulonephritis,however,kidney function did not show acute or chronic deterioration.