Jorge A. Guimarães

Structural and functional identification of GP57/51 antigen of Trypanosoma cruzi as a cysteine proteinase

Purified GP57/51, a Trypanosoma cruzi glycoprotein earlier identified as a major antigen in infected humans, was subjected to N-terminal sequence analysis. Alignment of the first 30 amino acids revealed that its N-terminal region is virtually identical to that reported for a cysteine-proteinase isolated from the Tulahuen strain, including the presence of active site cysteine at position 25. The finding of serine at position 24 of GP57/51 (Y strain) has further increased the homology between this protozoan antigen with other members of the eukaryotic family of cysteine proteases, including human cathepsin L. Functional analysis of GP57/51 indicated that the antigen is indeed an active thiol proteinase, which is active across a wide pH range (5-7.5). This was shown using either human IgG or gelatin substrates co-polymerized into polyacrylamide gels prepared for electrophoresis, and also by enzyme assays peformed with the synthetic substrate Z-phe-arg-NMec. The enzyme was activated by thiol containing reagents, and was strongly inhibited by low concentrations of E-64 (IC50 0.1 microM), cystatin (IC50 1 microM), leupeptin (IC50 0.1 microM) and antipain (IC50 0.1 microM). Monoclonal antibodies directed against distinct epitopes of GP57/51 absorbed the hydrolytic activity from purified preparations, demonstrating that the antigenic and enzymatic activities were indeed expressed by the same molecular entities. The subcellular localization of immunoreactive molecules was investigated by electron microscopy; immunogold staining was conspicuously found in vesicles belonging to the endosomal-lysosomal system, in tissue culture trypomastigotes as well as in epimastigotes. The possibility that this highly antigenic protease is actively secreted and/or leaked out of damaged parasites is under investigation; its release to tissues and to the circulation may contribute to pathology, considering that it (i) can degrade proteins across a wide pH range and (ii) stimulates immune T cells from chronic chagasic patients.

Lipoxygenase-mediated secretory effect of canatoxin the toxic protein from Canavalia ensiformis seeds

Canatoxin was shown to induce serotonin release from rabbit platelets and rat brain synaptosomes, as well as to release insulin from isolated pancreatic islets. All these effects were dose-dependent and were inhibited by lipoxygenase inhibitors, such as nordihydroguaiaretic acid and esculetin, but not by indomethacin, a cyclooxygenase inhibitor. The data suggest that canatoxin-induced secretory effect results from the activation of the lipoxygenase pathway which would elicit exocytosis. Thus, canatoxin might be a useful tool for the study of biological events that involve lipoxygenase mediation.

Plant and microbial toxic proteins as hemilectins: Emphasis on canatoxin

Ribosome-inactivating plant toxic proteins and ADP-ribosylating microbial toxins share a common structural organization. These proteins present domains displaying different biological properties: a target cell membrane-binding component (B-subunit or haptomer) and an enzymatically active component (A-subunit or effectomer). Interactions of these toxins with the target cells are mediated by the hemilectin-like haptomer, which recognizes and specifically binds to a given glycoderivative present at the cell surface. After binding the holoprotein is internalized via endocytosis. Inside the endocytic compartment the toxin is processed to release its effectomer moiety which catalytically modifies a cytoplasmic component, and this step accounts for its toxic effect. The structural relationships between toxic hemilectins and plant lectins are discussed, with emphasis on the example of canatoxin and concanavalin A, both present in the seeds of the jack bean Canavalia ensiformis. Contrary to other plant toxic proteins, which inhibit protein synthesis, canatoxin-induced toxicity includes central nervous system-mediated effects. In vivo as well as in vitro canatoxin acts as lipoxygenase-mediated secretagogue in several types of cells: blood platelets, mast cells, pancreatic islets and synaptosomes. Elucidation of structure vs biological activity relationships of canatoxin and other toxic proteins may provide data for their utilization as pharmacological tools and as therapeutic agents.

Phosphatidylinositol 3'-kinase and tyrosine-phosphatase activation positively modulate Convulxin-induced platelet activation. Comparison with collagen.

In this report we have studied the role of phosphatidylinositol 3′‐kinase (PI3‐K) and tyrosine phosphatase activation on platelet activation by Convulxin (Cvx). Wortmannin, a specific PI3‐K inhibitor, and phenylarsine oxide (PAO), a sulfhydryl reagent that inhibits tyrosine phosphatase (PTPase), block Cvx‐induced platelet aggregation, granule secretion, inositol phosphate production, and increase in [Ca2+]i. However, PAO does not inhibit Cvx‐induced tyrosine phosphorylation of platelet proteins, including Syk and PLCγ2, but blocked collagen‐induced platelet aggregation as well as tyrosine phosphorylation of PLCγ2. In contrast, Cvx‐induced PLCγ2 tyrosyl phosphorylation was partially inhibited by wortmannin. We conclude that (i) although Cvx and collagen activate platelets by a similar mechanism, different regulatory processes are specific to each agonist; (ii) mechanisms other than tyrosine phosphorylation regulate PLCγ2 activity; and (iii) besides protein tyrosine kinases, PI3‐K (and PTPase) positively modulate platelet activation by both Cvx and collagen, and this enzyme is required for effective transmission of GPVI‐Fc receptor γ chain signal to result in full activation and tyrosine phosphorylation of PLCγ2 in Cvx‐stimulated platelets.

BOTHROPS SP. SNAKE VENOMS : COMPARISON OF SOME BIOCHEMICAL AND PHYSICOCHEMICAL PROPERTIES AND INTERFERENCE IN PLATELET FUNCTIONS

Procoagulant, proteolytic, phospholipase and platelet pro-aggregating and inhibiting activities were screened for pooled venoms of seven Bothrops species as well as Crotalus durissus terrificus and Lachesis muta snakes typical of the Brazilian territory. As reported by other authors, we also found that examination of the electrophoretic and gel filtration patterns of Bothrops snakes venoms could not be used for identification of the species of a given venom because of the lack of marked interspecific differences within the same genus. Our data indicated that B. cotiara, B. alternatus and B. atrox possess no platelet pro-aggregating activity, low inhibitory effect on platelet aggregation and very low or intermediate levels for the other activities. B. moojeni, B. neuwiedi and B. jararacussu whose venoms possess high procoagulant, platelet pro-aggregating and phospholipase activities are low in both proteolytic and platelet inhibitory activities. B. jararaca venom showed the highest inhibitory effect on platelet aggregation and very low platelet pro-aggregating activity. Compared with the Bothrops venoms studied, L. muta venom showed that highest proteolytic activity while C. d. terrificus venom presented remarkable high platelet pro-aggregating and phospholipase activities. In all venoms, proteolytic activity could be completely inhibited by EDTA (2 mM) alone. In contrast, the presence of phenylmethylsulfonyl fluoride (5 mM) inhibited partially the caseinolytic activity of all venoms, except that L. muta venom, which was almost completely blocked by this reagent. Altogether, these data confirm the presence of high levels of metalloproteinases in the venoms of Crotalinae snakes. Most of these enzymes are dependent of the availability of Ca2+, being much less the same concerning the presence of serine residues in their active sites. The data indicated that the presence and levels of procoagulant, azocaseinolytic and phospholipase A2 activities alone could not differentiated the species of the Bothrops venoms studied, particularly in the cases of B. jararaca, B. moojeni and B. atrox. However, the platelet inhibiting property of low doses of B. jararaca venom can be useful to differentiate it from B. moojeni venom. In the same way, the platelet pro-aggregating activity of high doses of B. jararaca venom may be used to distinguish it from B. atrox crude venom, otherwise very similar but incapable to activate platelets. In conclusion, our comparative screening of biological properties has indicated that platelet studies may serve as a tool to distinguish among venoms that otherwise behave biochemically in a very similar way. Although promising, the general applicability of platelet activation studies by snake venoms for classification or taxanomical purposes has yet to be extended to other family of snakes to be proven useful.

Distinct bothrojaracin isoforms produced by individual jararaca (Bothrops jararaca) snakes

Bothrojaracin (apparent mol. wt 27,000) is a potent inhibitor of thrombin previously isolated from the venom of Bothrops jararaca. Several molecular variants (isoforms) have been identified in a pool of venom collected from a large number of animals. In order to determine whether an individual snake produces a single type of bothrojaracin or multiple isoforms, we analyzed the bothrojaracin content of venoms collected individually from six adult B. jararaca snakes. Bothrojaracin was found in all venoms, but its activity was especially high in three of them. After purification on an alpha-thrombin affinity column, followed by gel filtration on Superose 12 HR, proteins from these three venoms migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as single bands of 27,000 and their amino-terminal sequences (residues 1-28) revealed extensive homology with bothrojaracin. In contrast, the material purified from the three venoms with low bothrojaracin activity consisted of bothrojaracin together with inactive proteins. Differences in the sequences obtained for bothrojaracin isolated from individual venoms indicated the existence of more than one isoform in the venom of an individual snake.

Immunoreactivity for canatoxin and concanavalin A among proteins of leguminous seeds

Abstract Thejack bean (Canavalia ensiformis) is the natural source of concanavalin A and also of canatoxin, a recently described neurotoxic protein. Among some other examples, the seeds of the euphorbiaceous plant Ricinus communis contain ricin, a toxic monovalent lectin, and also an agglutinin which are closely related molecules sharing a common polypeptide subunit. Thus, seeds containing two distinct and related proteins, an atoxic lectin and a highly toxic protein which behaves as an monovalent lectin or a hemilectin, seem to be widespread. In this paper we describe the simultaneous presence of both toxic proteins and lectins in the seed extracts of 13 out of 16 different leguminous plants examined. Immunodifusion studies with anticanatoxin and anticoncanavalin A IgG antibodies indicated that proteins structurally resembling canatoxin were preserved in almost all the leguminous seeds extract except in peanut and R. communis. Proteins immunogically related to coneanavalin A weredetected only for the Canavalia genus despite the ubiquitous presence of lectins in seeds. The data suggest that canatoxin-like proteins were conservatively preserved, indicating that this toxic protein as well as other toxic hemilectins may play an important physiological role in plants.

Conversion of T-kinin to bradykinin by the rat kidney

Isolated rat kidneys were perfused with T-kinin (TK, Ile-Ser-BK) and bradykinin (BK). HPLC analysis of perfusate samples taken at 2-10 min during the TK perfusion (0.5 nmol/mL initial concentration) showed two peptide peaks, the first one eluting at 14.42 min, the same retention time for standard BK, and the second at 16.20 min, corresponding to that of TK. When BK (0.5 nmol/mL) was perfused, only its corresponding peak was obtained although total BK recovery was reduced quickly, as expected. Using both HPLC analysis and a kinin bioassay on the isolated guinea pig ileum, it was found that 12% of the added TK was converted to BK during the first perfusion cycle (2 min). While the BK recovered (12-14% from the initial TK concentration) was maintained at a similar proportion between the 2nd and the 10th min of perfusion, the rate of TK disappearance, as well as its full recovery from the perfusate, indicated further fragmentation of peptides during kinin perfusion. In the presence of 5 microM DL-mercaptomethyl-3-guanidino-ethylthiopropanoic acid (Mergetpa), an inhibitor of plasma carboxypeptidase N (EC 3.4.17.3), the rate of conversion of TK to BK was not affected. On the other hand, the kinase II inhibitor bradykinin potentiating peptide 9a (BPP9a) increased both the proportion of TK converted to BK and the disappearance rate of TK from the perfusate. In the presence of BPP9a, the rate of BK production increased from 1.5 +/- 0.2 to 7.6 +/- 0.9 nmol/min. Furthermore, the recovery of BK was reduced during the first 2 min of perfusion to 7.6% and the conversion rate to 0.9 nmol/min when TK was perfused into the kidney in the presence of 10 microM bestatin, a known inhibitor of aminopeptidases. These data indicate that in the kidney TK is converted to BK, probably by aminopeptidase M, thus suggesting that BK is, in fact, an additional and functional kinin, inducing physiological and/or pathophysiological effects in the rat kidney in which TK is the main kinin released.

Variability of bothrojaracin isoforms and other venom principles in individual jararaca (Bothrops jararaca) snakes maintained under seasonally invariant conditions

Bothrojaracin (BJC) is a potent thrombin inhibitor isolated from the venom of Bothrops jararaca. Venoms from individual snakes have been shown to vary in BJC content, and more than one molecular variant (isoform) has been identified in the same venom. In order to determine whether the production of this protein and its isoforms varies under seasonally invariant conditions, an analysis was made of BJC isolated from venoms collected individually once a month for 10 months from two female B. jararaca snakes kept under conditions of constant temperature and photoperiod. The crude venom from each individual snake exhibited a characteristic pattern of protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with no noticeable variation throughout the collecting period. BJC from individual venoms was purified by gel filtration on Sephacryl S-200 followed by an affinity column (PPACK-thrombin Sepharose). BJC content and other activities such as phospholipase A2, azocaseinolytic activity and inhibition of thrombin-induced platelet aggregation varied considerably among the samples. Purified BJC from both snakes inhibited fibrin coagulation and migrated as a single band of 27,000 mol. wt on SDS-PAGE. However, the BJC pattern on non-denaturing PAGE differed between the two snakes, with four to six bands per sample each month, which were all recognized by polyclonal anti-BJC antibodies. The isoelectric focusing pattern of BJC was also characteristic for each snake, with only minor differences throughout the collecting period. These results indicate that under seasonally invariant conditions: (1) there was a considerable variation over the 10-month period in the production of BJC and other important venom activities such as phospholipase A2 and proteinases; (2) individual B. jararaca snakes produced a distinctive array of BJC isoforms; and (3) despite quantitative differences, there were essentially no qualitative differences in the production of BJC isoforms by individual snakes during the 10-month period.

Bothrops jararaca snakes produce several bothrojaracin isoforms following an individual pattern.

More than one isoform of bothrojaracin (BJC), a potent and specific thrombin inhibitor isolated from Bothrops jararaca venom, has been found in individual venoms collected from adult snakes. Variations in snake venom composition have previously been associated with factors such as age, sex, geographic origin, season of the year and diet. In order to obtain further information concerning individual patterns of expression of BJC isoforms, we have analyzed five individual Bothrops jararaca snake venoms collected at the same time from adult female snakes from the same geographic region. As expected, crude venoms showed a similar migration pattern on SDS-PAGE. BJC was purified using a procedure which includes an affinity chromatography step (PPACK-thrombin Sepharose). A slight variation in the amount of BJC obtained from individual venom samples was noticed. Inhibition of thrombin-induced platelet aggregation as well as migration pattern on SDS-PAGE (under reducing and non-reducing conditions) and isoelectric focusing varied considerably among BJC samples from the five snakes. The amino-terminal sequences (residues 1-34) of individual BJC samples were compared with the sequence deduced from isolated cDNAs encoding alpha and beta chains of BJC. A high degree of homology was detected, although some residues differed from one sample to other. Altogether, data confirmed the heterogeneity found for BJC purified from individual snakes. Thus, the results indicate that: (1) individual specimens of Bothrops jararaca have different patterns of BJC isoform expression; and (2) it seems that genetic factors, at least in part, determine the variability found in BJC production.