Chikungunya virus entry is strongly inhibited by phospholipase A2 isolated from the venom of Crotalus durissus terrificus

Chikungunya virus (CHIKV) is the etiologic agent of Chikungunya fever, a globally spreading mosquito-borne disease. There is no approved antiviral or vaccine against CHIKV, highlighting an urgent need for novel therapies. In this context, snake venom proteins have demonstrated antiviral activity against several viruses, including arboviruses which are relevant to public health. In particular, the phospholipase A2CB (PLA2CB), a protein isolated from the venom of Crotalus durissus terrificus was previously shown to possess anti-inflammatory, antiparasitic, antibacterial and antiviral activities. In this study, we investigated the multiple effects of PLA2CB on the CHIKV replicative cycle in BHK-21 cells using CHIKV-nanoluc, a marker virus carrying nanoluciferase reporter. The results demonstrated that PLA2CB possess a strong anti-CHIKV activity with a selectivity index of 128. We identified that PLA2CB treatment protected cells against CHIKV infection, strongly impairing virus entry by reducing adsorption and post-attachment stages. Moreover, PLA2CB presented a modest yet significant activity towards post-entry stages of CHIKV replicative cycle. Molecular docking calculations indicated that PLA2CB may interact with CHIKV glycoproteins, mainly with E1 through hydrophobic interactions. In addition, infrared spectroscopy measurements indicated interactions of PLA2CB and CHIKV glycoproteins, corroborating with data from in silico analyses. Collectively, this data demonstrated the multiple antiviral effects of PLA2CB on the CHIKV replicative cycle, and suggest that PLA2CB interacts with CHIKV glycoproteins and that this interaction blocks binding of CHIKV virions to the host cells

Refolding by High Pressure of a Toxin Containing Seven Disulfide Bonds: Bothropstoxin-1 from Bothrops jararacussu

Aggregation is a serious obstacle for recovery of biologically active heterologous proteins from inclusion bodies (IBs) produced by recombinant bacteria. E. coli transformed with a vector containing the cDNA for Bothropstoxin-1 (BthTx-1) expressed the recombinant product as IBs. In order to obtain the native toxin, insoluble and aggregated protein was refolded using high hydrostatic pressure (HHP). IBs were dissolved and refolded (2 kbar, 16 h), and the effects of protein concentration, as well as changes in ratio and concentration of oxido-shuffling reagents, guanidine hydrochloride (GdnHCl), and pH in the refolding buffer, were assayed. A 32% yield (7.6 mg per liter of bacterial culture) in refolding of the native BthTx-1 was obtained using optimal conditions of the refolding buffer (Tris–HCl buffer, pH 7.5, containing 3 mM of a 2:3 ratio of GSH/GSSG, and 1 M GdnHCl). Scanning electron microscopy (SEM) showed that that disaggregation of part of IBs particles occurred upon compression and that the morphology of the remaining IBs, spherical particles, was not substantially altered. Dose-dependent cytotoxic activity of high-pressure refolded BthTx-1 was shown in C2C12 muscle cells

BOTHROPSTOXIN-I - AMINO-ACID-SEQUENCE AND FUNCTION

The complete amino acid sequence of bothropstoxin-I (BthTX-I), a myotoxin isolated from Bothrops jararacussu snake venom, is reported. The results show that BthTX-I is a Lys49 phospholipase A2 (PLA2)-like protein composed of a single polypeptide chain of 121 amino acid residues (M(r) = 13,720), containing one methionine and 14 half-cystines. Although deprived of any detectable PLA2 activity, BthTX-1 reveals a high degree of sequence homology with Asp49-PLA2s and with other Lys49-myotoxins. Critical mutations-such as Leu5 for Phe5; Gln11 for X11; Asn28 for Tyr28; Leu32 for Gly32; Lys4, for Asp49; and Asp71 for Asn71-which are apparently involved with the decreasing or elimination of PLA2 activity, have been detected. The same mutations occurred in myotoxin II from Bothrops asper venom, but five extra changes-namely, Pro90 for Ser90; Gly111 for Asn111; His120 for Tyr120; Phe124 for Leu124; and Pro132 for Ala132-have been found relative to myotoxin II.121576

CRYSTALLIZATION AND PRELIMINARY DIFFRACTION DATA OF BOTHROPSTOXIN-I ISOLATED FROM THE VENOM OF BOTHROPS-JARARACUSSU

The myotoxic Lys-49 phospholipase bothropstoxin I was crystallized, and X-ray diffraction data were collected to 3.5 Angstrom resolution. Preliminary analysis reveals the presence of four molecules in the asymmetric unit

Landbrugstoldkommissionens Flertalsbetænkning.

The role of low levels of phospholipase A(2) (PLA(2)) activity and intracellular Ca2+ stores in the pharmacological action of bothropstoxin (BthTX), a myotoxic Lys49 PLA(2) homologue isolated from the venom of Bothrops jararacussu, was investigated. We examined the muscular effects of BthTX in the mouse diaphragm and its PLA, activity in radiolabeled human and rat primary cultures of skeletal muscle. Although it is a Lys49 PLA(2) homologue, BthTX had a low, but easily detectable, level of enzymatic activity relative to two Asp49 PLA(2) enzymes from Naja naja kaouthia and Naja naja atra venoms, and this activity was reduced by about 85% in the presence of Sr2+ (4.0 mM). However, the replacement of 1.8 mM Ca2+ by 4 mM Sr2+ did not alter the BthTX-induced contracture and blockade of the muscle twitch tension. In addition, Sr2+ decreased by 50% the time required to cause 50% paralysis, and evoked approximately a four-fold increase in the number of spontaneous spikes. In isolated sarcoplasmic reticulum preparations, BthTX opened the intracellular Ca2+ release channel (ryanodine receptor) and lowered the threshold of Ca2+-induced Ca2+ release by a second, as yet unidentified, mechanism. However, in intact muscle, dantrolene, an antagonist of some forms of intracellular Ca2+ release, had no effect on the actions of BthTX. These findings do not support any role for the low levels of PLA(2) activity, or dantrolene-sensitive intracellular Ca2+ stores, in the action of BthTX. The mechanism whereby Sr2+ stimulates the pharmacological activity of BthTX remains to be clarified.33111479148

Crytallization and high-resolution X-ray diffraction data collection of an Asp49 PLA(2) from Bothrops jararacussu venom both in the presence and absence of Ca2+ ions

Snake venom PLA(2)s have been extensively studied due to their role in mediating and disrupting physiological processes such as coagulation, platelet aggregation and myotoxicity. The Ca2+ ion bound to the putative calcium-binding loop is essential for hydrolytic activity. We report the crystallization in the presence and absence of Ca2+ and X-ray diffraction data collection at 1.60 Angstrom (with Ca2+) and 1.36 Angstrom (without Ca2+) of an Asp49 PLA(2) from Bothrops jararacussu venom. The crystals belong to orthorhombic space group C222(1). Initial refinement and electron density analysis indicate significant conformational. changes upon Ca2+ binding. (C) 2004 Elsevier B.V. All fights reserved

Signalling pathways regulating human neutrophil migration induced by secretory phospholipases A(2)

This study was designed to elucidate the signalling pathways by which secretory phospholipases A(2) (sPLA(2)s) induce in vitro neutrophil migration. The cell migration assays were performed with Naja mocambique venom PLA(2) (sPLA(2) with high catalytic activity), bothropstoxin-I (sPLA2 devoid of catalytic activity) and platelet-activating factor (PAF), using a 48-well microchemotaxis chamber. Both the non-selective protein kinase inhibitor staurosporine (30-300 nM) and the selective protein kinase C (PKC) inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpyperazine (H7; 50-200 muM) as well as the Gi inactivator pertussis toxin (30-300 nM) caused a concentration-dependent inhibition of the neutrophil migration induced by either N. mocambique venom PLA(2) (100 mug/ml) or bothropstoxin-I (100 mug/ml). Pertussis toxin nearly abolished PAF-induced migration, while staurosporine and H7 partly (but significantly) inhibited the chemotactic responses to PAR The dual inhibitor of cytosolic PLA(2) and Ca2+-independent PLA(2) (iPLA(2)), arachidonil-trifluoromethyl-ketone (ATK; 0.2-20 muM), or the specific iPLA(2) inhibitor bromoenol lactone (1-30 muM) caused a concentration-dependent inhibition of the migration induced by either sPLA(2)s. At the maximal concentration used for each compound, the migration was almost suppressed. In contrast, both of these compounds caused only slight inhibitions of PAF-induced migration. No rise in intracellular Ca2+ Was observed in neutrophil-stimulated sPLA(2), as determined in cells preloaded with fura 2-AM. In the experimental condition used, pertussis toxin, staurosporine, H7, ATK or bromoenol lactone did not induce cytotoxic effects, according to MTT assay. Our results suggest that activation of an endogenous PLA(2) through activation of GTP-binding protein and PKC is the main mechanism by which exogenous sPLA(2)s cause neutrophil migration. (C) 2004 Elsevier Ltd. All rights reserved.44547348

Crystallization of bothropstoxin II isolated from the venom of Bothrops jararacussu

A myotoxic phospholipase A(2), bothropstoxin II, which exhibits low hydrolytic activity, was crystallized and X-ray diffraction data were collected to a resolution of 2.2 Angstrom. Preliminary analysis reveals the presence of three molecules in the asymmetric unit. Copyright (C) 1996 Elsevier B.V. Ltd

The amino acid sequence of bothropstoxin-II, an Asp-49 myotoxin from Bothrops jararacussu (Jararacucu) venom with low phospholipase A(2) activity

The complete amino acid sequence of bothropstoxin-II (BthTX-II), a myotoxin isolated from Bothrops jararacussu snake venom, is reported. The results show that BthTX-II is an Asp-49 phospholipase A(2) (PLA(2))-like protein composed of a single polypeptide chain of 120 amino acid residues (M-r = 13,976), containing one methionine and 14 half-cystines. Despite a high degree of homology with other PLA(2)'s and the presence of the strategic residues known to compose the Ca2+-binding loop, namely Tyr-28, Gly-30, Gly-32, and especially Asp-49, besides His-48, Tyr-52, and Asp-99, all of them directly or indirectly involved in catalysis, BthTX-II revealed a very low PLA(2) activity when assayed on egg yolk phosphatidylcholine. We attribute this low catalytic activity to the existence of extra mutations, e.g., Trp-5 for Phe-5, which points to the need of considering other strategic positions, since only Lys-49 PLA(2)'s have been considered to be devoid of this enzymatic activity.17438138