Correlating biological activity to thermo-structural analysis of the interaction of CTX with synthetic models of macrophage membranes.

The important pharmacological actions of Crotoxin (CTX) on macrophages, the main toxin in the venom of Crotalus durissus terrificus, and its important participation in the control of different pathophysiological processes, have been demonstrated. The biological activities performed by macrophages are related to signaling mediated by receptors expressed on the membrane surface of these cells or opening and closing of ion channels, generation of membrane curvature and pore formation. In the present work, the interaction of the CTX complex with the cell membrane of macrophages is studied, both using biological cells and synthetic lipid membranes to monitor structural alterations induced by the protein. Here we show that CTX can penetrate THP-1 cells and induce pores only in anionic lipid model membranes, suggesting that a possible access pathway for CTX to the cell is via lipids with anionic polar heads. Considering that the selectivity of the lipid composition varies in different tissues and organs of the human body, the thermostructural studies presented here are extremely important to open new investigations on the biological activities of CTX in different biological systems.

Proteomic, toxicological and immunogenic characterization of Mexican west-coast rattlesnake (Crotalus basiliscus) venom and its immunological relatedness with the venom of Central American rattlesnake (Crotalus simus).

The venom of the Mexican west-coast rattlesnake (Crotalus basiliscus) was characterized for its protein composition, toxicological profile and immunogenic properties. This venom is composed of 68% Zn2+-dependent metalloproteinases (SVMPs), 14% phospholipases A2 (PLA2s), 11% serine proteinases, 4% SVMPs-inhibitor tripeptides (SVMP-ITs), 2% bradykinin-potentiating peptides (BPPs), 0.6% cysteine-rich secretory proteins (CRISPs), and 0.2% l-amino acid oxidases (LAAOs). SVMPs present in the venom are responsible for azocasein hydrolysis and hemorrhagic activity, but their contribution to the lethal activity of the venom in mice is masked by the neurotoxic activity of PLA2s, which in addition are also responsible for myotoxic activity. Despite its relatively high content of serine proteinases, the venom of C. basiliscus did not exert in vitro coagulant or in vivo defibrinogenating activities. The ability of antivenoms raised against the venoms of C. basiliscus and C. simus (from Costa Rica) to neutralize homologous and heterologous venoms revealed antigenic similarities between toxins of both venoms. Preclinical evaluation of an antivenom produced by using the venom of C. basiliscus as immunogen demonstrated that it is able to neutralize not only the most relevant toxic activities of C. basiliscus venom, but also those exerted by Costa Rican C. simus venom, including coagulant and defibrinogenating activities. BIOLOGICAL SIGNIFICANCE: The Central American rattlesnake (Crotalus simus) is widely distributed from Mexico to west central Costa Rica, and induces an important number of envenomations in this region. On the other hand, the immunogenic mixture used by Laboratorios de Biológicos y Reactivos de Mexico S.A. (Birmex) to produce the snake antivenom more frequently used in Mexico does not include the venom of C. simus. This immunogenic mixture is composed by the venoms of the Fer-de-lance (Bothrops asper) and the Mexican west-coast rattlesnake (Crotalus basiliscus). We studied the protein composition, toxicological profile and immunogenic properties of the venom of C. basiliscus, and evaluated the ability of the Birmex antivenom to neutralize the venom of C. basiliscus and whether it cross-neutralizes the venom of C. simus from Costa Rica. Using proteomics analysis, in combination with in vitro and mouse tests, we determined that the venom of C. basiliscus is mainly composed by SVMPs, which confer proteolytic and hemorrhagic activities to the venom. Other major components of the venom of C. basiliscus are PLA2s, which are responsible for the myotoxic activity and are the main contributors to the lethal activity. Non-clotting SVSPs correspond to 11% of the venom. Minor components include SVMP-ITs, BPPs, CRISPs and LAAOs, which have not been associated with toxicity. The antibodies induced in horses by the venom of C. basiliscus are able to neutralize not only the most relevant toxic activities of the homologous venom, but also those exerted by Costa Rican C. simus venom, including coagulant and defibrinogenating activities. Our preclinical evaluation suggests that Birmex antivenom can be used to treat envenomations by Costa Rican adult C. simus snakebites, despite this venom not being included in the immunizing mixture.

Protein complexes in snake venom.

Snake venom contains mixture of bioactive proteins and polypeptides. Most of these proteins and polypeptides exist as monomers, but some of them form complexes in the venom. These complexes exhibit much higher levels of pharmacological activity compared to individual components and play an important role in pathophysiological effects during envenomation. They are formed through covalent and/or non-covalent interactions. The subunits of the complexes are either identical (homodimers) or dissimilar (heterodimers; in some cases subunits belong to different families of proteins). The formation of complexes, at times, eliminates the non-specific binding and enhances the binding to the target molecule. On several occasions, it also leads to recognition of new targets as protein-protein interaction in complexes exposes the critical amino acid residues buried in the monomers. Here, we describe the structure and function of various protein complexes of snake venoms and their role in snake venom toxicity.

Primary culture of venom gland cells from the South American rattlesnake (Crotalus durissus terrificus).

Primary cultures of venom gland cells from the South American rattlesnake (Crotalus durissus terrificus) were attempted. At first, six different cell types were obtained including potentially secreting epithelial-like cells. Nonepithelial cell cultures were later invaded by fibroblast-like cells. Cultures of epithelial-like gland cells were successfully maintained, after testing different culture conditions by varying the media, incubation temperature, use of dissociating agents and adhesion substrates. The best results were achieved using plates precoated with rattlesnake skin collagen and incubation in CMRL 1415 modified for snake gland cells plus 10% fetal calf serum at 30 degrees C. The presence of venom could be demonstrated in the supernatant of five out of six epithelial-like gland cell cultures tested by ELISA, in the very first passages. After the third passage, however, venom amounts dropped to undetectable values. A total of 23 venom gland cell lines were obtained and are kept frozen in the laboratory; among them, five epithelial-like gland cell lines with up to 12 passages, that were continuously cultured for more than 30 weeks. The methodology described here was successfully applied to C. d. terrificus kidney cells culturing, developed to be used as negative control.

Crocalbin: a new calcium-binding protein that is also a binding protein for crotoxin, a neurotoxic phospholipase A2.

Utilizing Marathon-ready cDNA library and a gene-specific primer corresponding to a partial amino acid sequence determined previously, the complete nucleotide sequence for the cDNA of crocalbin, which binds crotoxin (a phospholipase A2) and Ca2+, was obtained by polymerase chain reaction. The open reading frame of the cDNA encodes a novel polypeptide of 315 amino acid residues, including a signal sequence of 19 residues. This protein contains six potential Ca(2+)-binding domains, one N-glycosylation site, and a large amount of acidic amino acid residues. The ability to bind Ca2+ has been ascertained by calcium overlay experiment. Evidenced by sequence similarity in addition, it is concluded that crocalbin is a new member of the reticulocalbin family of calcium-binding proteins.

Purification and partial amino acid sequence of a novel protein of the reticulocalbin family.

Binding proteins in neuronal membranes for a phospholipase A2 with presynaptic neurotoxicity have been purified. Three polypeptides of 87, 65, and 50 K Da were obtained from the synaptic membrane fraction of guinea pig brain utilizing an immobilized crotoxin (a phospholipase A2) column. For large scale purification, porcine brain was used instead, and two polypeptides of 50 and 18 K Da were found. The 65 and 18 K polypeptides may represent hitherto unidentified components of the crotoxin-binding proteins. Partial N-terminal amino acid sequence and a partial sequence for an internal peptide fragment have been determined for the 50 K polypeptide. Search of protein data bank reveals that this polypeptide or protein is a novel member of the reticulocalbin family of calcium-binding proteins.

Regulation of epidermal growth factor receptor activity by crotoxin, a snake venom phospholipase A2 toxin. A novel growth inhibitory mechanism.

Crotoxin (CT), a phospholipase A2 (PLA2) derived from the venom of Crotalus durissus terrificus, is a heterodimeric protein composed of subunit B with enzymatic activity and a binding regulatory subunit (A) without enzyme activity. Although the PLA2 activity of CT may be important in its anti-proliferative activity, its cytostatic mechanism is unknown. In this study, we examined the cytostatic effect of PLA2-associated CT activity on squamous carcinoma cells expressing distinct levels of epidermal growth factor receptor (EGFr). CT was most effective in suppressing growth on cells expressing high intrinsic levels of EGFr. Cardiotoxin, another membrane active toxin with no intrinsic PLA2 activity, had no differential anti-proliferative activity on cells expressing high EGFr levels, suggesting a correlation between EGFr expression and CT-directed anti-proliferative activity. Both chemically modified CT (MCT) devoid of PLA2 activity and covalently cross-linked CT (CCT), which is functionally unable to utilize cellular membranes as PLA2 substrate, were also without growth inhibitory activity. No evidence for direct binding of CT to EGFr was found, although pretreatment with EGF was able to partially suppress the anti-proliferative activity of CT. Tyrosine phosphorylation of EGFr, however, was stimulated by CT in intact A431 cells. Tyrosine phosphorylation of EGFr was concentration-dependently stimulated (3- to 8-fold) in cellular membranes of A431 cells treated in vitro with CT but not with anti-proliferatively inactive MCT or CCT. The data provide evidence for transmembrane receptors involved in growth signaling (namely EGFr) as cellular targets and potential effectors of PLA2-mediated anti-proliferative activity of snake venom.

Binding proteins on synaptic membranes for crotoxin and taipoxin, two phospholipases A2 with neurotoxicity.

Crotoxin and taipoxin are both neurotoxic phospholipases A2 capable of affecting the presynaptic activity to bring about ultimate blockade of synaptic transmission. The enzymatic activity has generally been considered to be necessary but not sufficient for the blockade. Since many phospholipases A2 with comparable or even higher enzymatic activity are not toxic, it has been postulated that the difference lies in the affinity of binding to the presynaptic membrane. In confirmation of this proposition, we and others have previously shown that iodinated crotoxin and taipoxin bind specifically with high affinity to the isolated synaptic membrane fraction from guinea-pig brain, whereas specific binding is not detected with the nontoxic pancreatic phospholipase A2. Experiments based on photoaffinity labeling and simple chemical cross-linking techniques have led to the identification of three polypeptides preferentially present in neuronal membranes as (subunits of) the binding protein(s) for crotoxin and/or taipoxin. Some, but not all, other toxic phospholipases A2 also appear to be ligands for the three polypeptides. We now report studies on partial purification of these polypeptides using affinity chromatography and other techniques. In order to learn the normal physiological roles played by the toxin-binding proteins, the phospholipase-independent effects of the toxins on the synaptosomes have been sought. We have found that under Ca(2+)-free condition, taipoxin or crotoxin inhibits with IC50 of 20-1000 nM the Na(+)-dependent uptake of norepinephrine, dopamine and serotonin by the synaptosomes. In contrast, choline uptake is not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Conversion of bovine pancreatic phospholipase A2 at a single site into a competitor of neurotoxic phospholipases A2 by site-directed mutagenesis.

A 45-kDa polypeptide preferentially present in neuronal membranes was previously identified as a subunit of a binding (or receptor) protein for several phospholipase A2 variants with neurotoxicity, including crotoxin, by chemical cross-linking experiments (Yen, C.-H., and Tzeng, M.-C. (1991) Biochemistry 30, 11473-11477). The binding of crotoxin to this receptor protein was completely suppressed by sufficient F22Y, a mutated bovine pancreatic phospholipase A2 generated by site-directed mutagenesis of Phe22 of the wild-type enzyme to Tyr. The IC50 of this inhibition was estimated to be 1 microM. In sharp contrast, the wild-type enzyme gave no effect even at 50 microM. This mutation resulted in only minor and localized structural perturbations with little effect on enzymatic activity. Other phospholipase A2 molecules capable of competing with crotoxin for this binding invariably have Tyr at this position. It was concluded that this Tyr residue is an important determinant for the binding of a number of phospholipase A2 variants to the 45-kDa receptor.