Jack bean (Canavalia ensiformis) urease induces eicosanoid-modulated hemocyte aggregation in the Chagas' disease vector Rhodnius prolixus

AbstractUreases are multifunctional proteins that display biological activities independently of their enzymatic function, such as induction of exocytosis and insecticidal effects. Rhodnius prolixus, a major vector of Chagas' disease, is a model for studies on the entomotoxicity of jack bean urease (JBU). We have previously shown that JBU induces the production of eicosanoids in isolated tissues of R. prolixus. In insects, the immune response comprises cellular and humoral reactions, and is centrally modulated by eicosanoids. Cyclooxygenase products signal immunity in insects, mainly cellular reactions, such as hemocyte aggregation. In searching for a link between JBU's toxic effects and immune reactions in insects, we have studied the effects of this toxin on R. prolixus hemocytes. JBU triggers aggregation of hemocytes after injection into the hemocoel and when applied to isolated cells. On in vitro assays, the eicosanoid synthesis inhibitors dexamethasone (phospholipase A2 indirect inhibitor) and indomethacin (cyclooxygenase inhibitor) counteracted JBU's effect, indicating that eicosanoids, more specifically cyclooxygenase products, are likely to mediate the aggregation response. Contrarily, the inhibitors esculetin and baicalein were inactive, suggesting that lipoxygenase products are not involved in JBU's effect. Extracellular calcium was also necessary for JBU's effect, in agreement to other cell models responsive to ureases. A progressive darkening of the medium of JBU-treated hemocytes was observed, suggestive of a humoral response. JBU was immunolocalized in the cultured cells upon treatment along with cytoskeleton damage. The highest concentration of JBU tested on cultured cells also led to nuclei aggregation of adherent hemocytes. This is the first time urease has been shown to affect insect hemocytes, contributing to our understanding of the entomotoxic mechanisms of action of this protein

Soyuretox, an intrinsically disordered polypeptide derived from soybean (Glycine max) ubiquitous urease with potential use as a biopesticide

Ureases from different biological sources display non-ureolytic properties that contribute to plant defense, in addition to their classical enzymatic urea hydrolysis. Antifungal and entomotoxic effects were demonstrated for Jaburetox, an intrinsically disordered polypeptide derived from jack bean (Canavalia ensiformis) urease. Here we describe the properties of Soyuretox, a polypeptide derived from soybean (Glycine max) ubiquitous urease. Soyuretox was fungitoxic to Candida albicans, leading to the production of reactive oxygen species. Soyuretox further induced aggregation of Rhodnius prolixus hemocytes, indicating an interference on the insect immune response. No relevant toxicity of Soyuretox to zebrafish larvae was observed. These data suggest the presence of antifungal and entomotoxic portions of the amino acid sequences encompassing both Soyuretox and Jaburetox, despite their small sequence identity. Nuclear Magnetic Resonance (NMR) and circular dichroism (CD) spectroscopic data revealed that Soyuretox, in analogy with Jaburetox, possesses an intrinsic and largely disordered nature. Some folding is observed upon interaction of Soyuretox with sodium dodecyl sulfate (SDS) micelles, taken here as models for membranes. This observation suggests the possibility for this protein to modify its secondary structure upon interaction with the cells of the affected organisms, leading to alterations of membrane integrity. Altogether, Soyuretox can be considered a promising biopesticide for use in plant protection

Ureases display biological effects independent of enzymatic activity: Is there a connection to diseases caused by urease-producing bacteria?

Ureases are enzymes from plants, fungi and bacteria that catalyze the hydrolysis of urea to form ammonia and carbon dioxide. While fungal and plant ureases are homo-oligomers of 90-kDa subunits, bacterial ureases are multimers of two or three subunit complexes. We showed that some isoforms of jack bean urease, canatoxin and the classical urease, bind to glycoconjugates and induce platelet aggregation. Canatoxin also promotes release of histamine from mast cells, insulin from pancreatic cells and neurotransmitters from brain synaptosomes. In vivo it induces rat paw edema and neutrophil chemotaxis. These effects are independent of ureolytic activity and require activation of eicosanoid metabolism and calcium channels. Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach mucosa, causes gastric ulcers and cancer by a mechanism that is not understood. H. pylori produces factors that damage gastric epithelial cells, such as the vacuolating cytotoxin VacA, the cytotoxin-associated protein CagA, and a urease (up to 10% of bacterial protein) that neutralizes the acidic medium permitting its survival in the stomach. H. pylori whole cells or extracts of its water-soluble proteins promote inflammation, activate neutrophils and induce the release of cytokines. In this paper we review data from the literature suggesting that H. pylori urease displays many of the biological activities observed for jack bean ureases and show that bacterial ureases have a secretagogue effect modulated by eicosanoid metabolites through lipoxygenase pathways. These findings could be relevant to the elucidation of the role of urease in the pathogenesis of the gastrointestinal disease caused by H. pylori

Blood Glucose Alterations Induced In Rats By Canatoxin, A Protein Isolated From Jack Bean (canavalia Ensiformis) Seeds

Canatoxin, a lethal convulsant protein isolated from the seeds of Canatoxin ensiformis (jack bean), induced a biphasic alteration in blood glucose levels when injected i.v. into rats and mice. After administration of subconvulsant doses of canatoxin the rats showed initially hyperglycemia during the first 30 min, followed by a long-lasting hypoglycemia. Return to basal glucose levels was not seen up to 15 days. The hyperglycemic effect was dose-dependent and occurred in starved and well-fed animals. A parallelism between convulsant and hyperglycemic activity was observed throughout all canatoxin purification steps. Mice were about 20-fold less sensitive (on a weight basis) than rats to canatoxin-induced hyperglycemia. The hyperglycemic phase was not affected by pretreatment of the rats with alphaor beta-adrenergic blockers or chlorpromazine, but was potentiated by reserpine and haloperidol. Diazepam and hexamethonium were able to block the hyperglymic phase of canatoxin's effect. The results suggest that the hyperglycemic alterations induced by canatoxin in rats and mice are probably mediated via the central nervous system. © 1986.248775782Bellville, William, Gary, A method for investigating the role of calcium in the shape change, aggregation and serotonin release of rat platelets (1979) J. Physiol., Lond., 297, p. 289Bilski, Dorries, Fitzgerald, Jessup, Tucker, Wale, ICI 118.551: a potent beta 2 adrenoceptor antagonist (1980) Br. J. Pharmac., 69, p. 292. , 3PBlackard, Small, Lundman, Inhibition of insulin binding by concanavalin A (1980) Proc. Soc. exp. Biol. Med., 153, p. 213Carlini, Guimarães, Isolation and characterization of a toxic protein from Canavalia ensiformis (jack bean) seeds, distinct from concanavalin A (1981) Toxicon, 19, p. 667Carlini, Gomes, Guimarães, Markus, Sato, Trolin, Central nervous effect of the convulsant protein canatoxin (1984) Acta pharmac. tox., 54, p. 161Carlini, Guimarães, Ribeiro, Platelet release reaction and aggregation induced by canatoxin, a convulsant protein: evidence for the involvement of the lipoxygenase pathway (1985) Br. J. Pharmac., 84, p. 551Cuatrecasas, Interaction of concanavalin A and wheat germ agglutinin with insulin receptor of fat cells and liver (1973) J. biol. Chem., 248, p. 3528Cuatrecasas, Tell, Insulin-like activity of concanavalin A and wheat germ agglutinin: direct interactions with insulin receptors (1973) Proc. natn. Acad. Sci. U.S.A., 70, p. 485Felig, Wahren, Fuel homeostasis in exercise (1975) New Engl. J. Med., 293, p. 1078Furman, Tayo, Inhibitory effect of propranolol on insulin secretion (1973) Br. J. Pharmac., 49, p. 145Guimarães, Ubatuba, Carlini, Physical - chemical properties of canatoxin, a hemilectin displaying toxic and convulsant activity (1985) Anais Acad. bras. Cienc., 57, p. 388Harvey, Hypnotics and sedatives (1980) The Pharmacological Basis of Therapeutics, p. 339. , Goodman, Gilman, 6th Edn, Macmillan, New YorkHelting, Zwisler, Structure of tetanus toxin. I. Breakdown of the toxic molecule and discrimination between polypeptides fragments (1977) J. biol. Chem., 252, p. 187Leblanc, Lachelin, Abu-Fadil, Yen, The effect of dopamine infusion on insulin and glucagon secretion in man (1977) J. clin. Endocr., 44, p. 196Litchfield, Jr., Wilcoxon, A simplified method for evaluating dose - effects experiments (1949) J. Pharmac. exp. Ther., 96, p. 99Maier, Schneider, Schatz, Pfeiffer, Interactions of concanavalin A with isolated pancreatic islets (1975) Hoppe-Seyler's Z. physiol. Chem., 356, p. 887Nelson, A photometric adaptation of the Somogyi method for the determination of glucose (1944) J. biol. Chem., 153, p. 375Nirmul, Severin, Taub, In vivo effects of concanavalin A II Distribution histopathological changes and toxicity (1976) Mt Sinai J Med, 43, p. 238Nopanitaya, Hanker, Tyan, Concanavalin A toxicity: histological studies (1976) Proc. Soc. exp. Biol. Med., 153, p. 213Olsnes, Saltvedt, Pihl, Isolation and comparison of galactose-binding lectins from Abrus precatorius and Ricinus communis (1974) J. biol. Chem, 249, p. 803Poretz, Goldstein, A study of turbidity as it relates to concanavalin A-glycan interaction (1968) Immunology, 14, p. 165Smith, Pork, Jr, Robertson, Neural regulation of endocrine pancreas (1979) Proceedings of the First International Symposium on the Endocrinology of the Pancreas and Diabetes, p. 64. , J. Pierluissi, Elsevier, AmsterdamSouza, Masur, Blood glucose and body temperature alterations induced by ethanol in rats submitted to different levels of food deprivation (1981) Pharmacology Biochemistry and Behavior, 15, p. 551Souza, Masur, Does hypothermia play a relevant role in the glycemic alterations induced by ethanol (1982) Pharmac. Biochem. Behav., 16, p. 903Unger, Physiology of glucagon (1971) New Engl. J. Med., 285, p. 443Virji, Steffes, Estensen, Concanavalin A and alloxan interactions on glucose-induced insulin secretion and biosynthesis from islets of Langerhans (1984) Diabetes, 33, p. 164Woods, Porte, Jr, Neural control of the endocrine pancreas (1974) Physiol. Rev., 54, p. 59

Convulsions Induced By Canatoxin In Rats Are Probably A Consequence Of Hypoxia

[No abstract available]22787788

Biochemical and structural studies on native and recombinant Glycine max UreG: a detailed characterization of a plant urease accessory protein

Urea is the nitrogen fertilizer most utilized in crop production worldwide. Understanding all factors involved in urea metabolism in plants is an essential step towards assessing and possibly improving the use of urea by plants. Urease, the enzyme responsible for urea hydrolysis, and its accessory proteins, necessary for nickel incorporation into the enzyme active site and concomitant activation, have been extensively characterized in bacteria. In contrast, little is known about their plant counterparts. This work reports a detailed characterization of Glycine max UreG (GmUreG), a urease accessory protein. Two forms of native GmUreG, purified from seeds, were separated by metal affinity chromatography, and their properties (GTPase activity in absence and presence of Ni(2+) or Zn(2+), secondary structure and metal content) were compared with the recombinant protein produced in Escherichia coli. The binding affinity of recombinant GmUreG (rGmUreG) for Ni(2+) and Zn(2+) was determined by isothermal titration calorimetry. rGmUreG binds Zn(2+) or Ni(2+) differently, presenting a very tight binding site for Zn(2+) (K (d) = 0.02 \ub1 0.01 μM) but not for Ni(2+), thus suggesting that Zn(2+) may play a role on the plant urease assembly process, as suggested for bacteria. Size exclusion chromatography showed that Zn(2+) stabilizes a dimeric form of the rGmUreG, while NMR measurements indicate that rGmUreG belongs to the class of intrinsically disordered proteins. A homology model for the fully folded GmUreG was built and compared to bacterial UreG models, and the possible sites of interaction with other accessory proteins were investigated