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Ureases catalyze the hydrolysis of urea into carbamate and ammonia. Well-conserved proteins, most plant ureases are hexamers of a single chain subunit, like the most abundant isoform of the jack bean ( Canavalia ensiformis ) urease (JBU). Canatoxin (CNTX) was originally isolated from these seeds as a neurotoxic protein, and later characterized as an isoform of JBU with lower molecular mass and enzyme activity. Inactive CNTX oligomers form upon storage and stabilization of CNTX was achieved by treatment with low concentration of formaldehyde, avoiding its oligomerization. Here, nano-LC-MS/MS-based peptide analysis of CNTX revealed 804 amino acids identical to those of JBU's sequence (840 amino acids). De novo sequencing of CNTX revealed 15 different peptides containing substitution of amino acid residues, denoting CNTX as a product of a paralog gene of JBU. The MS/MS analysis of formaldehyde-treated CNTX showed that amino acid residues located at the trimer-trimer interface of JBU's hexamer were modified. The data confirmed that CNTX is an isoform of JBU and elucidated that stabilization by formaldehyde treatment occurs by modification of amino acids at the protein's surface that prevents the formation of the hexamer and of higher molecular mass inactive aggregates. HIGHLIGHTSCanatoxin (CNTX) is an isoform of jack bean urease (JBU, hexamer of 90 kDa chains)MS/MS sequencing of CNTX showed 804 amino acids identical in JBU (840 residues)Formaldehyde treatment of CNTX stabilizes its toxicity and avoids oligomerizationModified amino acid residues in CNTX are at the trimer-trimer interface of JBUCommunicated by Ramaswamy H. Sarma.

Ureases are microbial virulence factors either because of the enzymatic release of ammonia or due to many other non-enzymatic effects. Here we studied two neurotoxic urease isoforms, Canatoxin (CNTX) and Jack Bean Urease (JBU), produced by the plant Canavalia ensiformis, whose mechanisms of action remain elusive. The neurotoxins provoke convulsions in rodents (LD 50 ∼2 mg/kg) and stimulate exocytosis in cell models, affecting intracellular calcium levels. Here, electrophysiological and brain imaging techniques were applied to elucidate their mode of action. While systemic administration of the toxins causes tonic-clonic seizures in rodents, JBU injected into rat hippocampus induced spike-wave discharges similar to absence-like seizures. JBU reduced the amplitude of compound action potential from mouse sciatic nerve in a tetrodotoxin-insensitive manner. Hippocampal slices from CNTX-injected animals or slices treated in vitro with JBU failed to induce long term potentiation upon tetanic stimulation. Rat cortical synaptosomes treated with JBU released L-glutamate. JBU increased the intracellular calcium levels and spontaneous firing rate in rat hippocampus neurons. MicroPET scans of CNTX-injected rats revealed increased [18] Fluoro-deoxyglucose uptake in epileptogenesis-related areas like hippocampus and thalamus. Curiously, CNTX did not affect voltage-gated sodium, calcium or potassium channels currents, neither did it interfere on cholinergic receptors, suggesting an indirect mode of action that could be related to the ureases' membrane-disturbing properties. Understanding the neurotoxic mode of action of C. ensiformis ureases could help to unveil the so far underappreciated relevance of these toxins in diseases caused by urease-producing microorganisms, in which the human central nervous system is affected., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Ureases are metalloenzymes that catalyze the hydrolysis of urea to ammonia and carbon dioxide. Jack bean (Canavalia ensiformis) produces three isoforms of urease (Canatoxin, JBU and JBURE-II). Canatoxin and JBU display several biological properties independent of their ureolytic activity, such as neurotoxicity, exocytosis-inducing and pro-inflammatory effects, blood platelets activation, insecticidal and antifungal activities. The Canatoxin entomotoxic activity is mostly due to an internal peptide, named pepcanatox, released upon the hydrolysis of the protein by insect cathepsin-like digestive enzymes. Based on pepcanatox sequence, Jaburetox-2Ec was produced in Escherichia coli. JBU and its peptides were shown to permeabilize membranes through an ion channel-based mechanism. Here we studied the JBU and Jaburetox-2Ec interaction with platelet-like multilamellar liposomes (PML) using Dynamic Light Scattering and Small Angle X-ray Scattering techniques. We also analyzed the interaction of JBU with giant unilamellar vesicles (GUVs) using Fluorescence Microscopy. The interaction of vesicles with JBU led to a slight reduction of hydrodynamic radius, and caused an increase in the lamellar repeat distance of PML, suggesting a membrane disordering effect. In contrast, Jaburetox-2Ec decreased the lamellar repeat distance of PML membranes, while also diminishing their hydrodynamic radius. Fluorescence microscopy showed that the interaction of GUVs with JBU caused membrane perturbation with formation of tethers. In conclusion, JBU can interact with PML, probably by inserting its Jaburetox "domain" into the PML external membrane. Additionally, the interaction of Jaburetox-2Ec affects the vesicle's internal bilayers and hence causes more drastic changes in the PML membrane organization in comparison with JBU., (Copyright © 2016 Elsevier B.V. All rights reserved.)