Ureases de canavalia ensiformis : estudos estruturais e da atividade pró-convulsivante

Abstract

Ureases, metalloenzymes that catalyze the hydrolysis of urea into carbamate and ammonia, are well conserved proteins although differing in their quaternary structures in different organisms. Most plant ureases are hexamers of a single chain subunit, such as the most abundant isoform of the jack bean (Canavalia ensiformis) urease (JBU). Canatoxin (CNTX), originally isolated as a neurotoxic protein from jack bean seeds, was later characterized as an isoform of JBU, with a smaller molecular mass and lower ureolytic activity. Purified CNTX is unstable upon storage, forming inactive oligomers. Treatment of CNTX with 0.02 % v/v formaldehyde stabilizes its toxicity and avoids oligomerization. Canatoxin (CNTX) and Jack Bean Urease (JBU), isoforms of Canavalia ensiformis urease, displays a potent neurotoxicity whose mechanisms of action is not yet completely understood. The neurotoxins provokes convulsions preceding death in mice and rats (Ld50 ~2 mg/kg). Besides promoting hypoxia, hypothermia and inflammation in vivo, the proteins stimulates exocytosis in several cell models, induces neurotransmitter release from synaptosomes and affects calcium transport in platelets and in vesicles of sarcoplasmatic reticulum. For all these effects, CNTX acts by triggering eicosanoid signaling. In this work, we aimed to further characterize CNTX and elucidate the mechanism of its stabilization by formaldehyde treatment and to better understand its neurotoxic convulsant activity through a series of electrophysiological and brain imaging techniques. In this work, we successfully characterized CNTX as a trimeric state of JBU. The mechanism by which the formaldehyde treatment of CNTX avoids its oligomerization and stabilizes its biologically active conformation was elucidated and attributed to modifications introduced in the side chain of amino acid residues located at the interface between the two trimers that form the hexameric JBU. There could be multiple mechanisms underlying the seizures induced by C. ensiformis ureases. The mode of action seems to be an indirect one as no effect was detected on several isolated ion channels. It is more likely that CNTX and JBU could be acting at a neuronal network level, thereby disturbing electroencephalographic rhythms and causing metabolic alterations in key areas related to epileptogenesis and to neurogenic pulmonary edema. In the context of neuroinflammation, modulation of lipoxygenase pathways in the CNS appears as an interesting target to focus aiming to unveil the action of these neurotoxins. The lack of LTP induction is more likely caused by a neuroprotection mechanism and/or release of lipoxygenase products than direct interaction of CNTX or JBU with receptors and ion channels. The L-glutamate release induced by JBU could be a result of its membrane disturbing properties or of a lipoxygenase mediated mechanism, as observed for CNTX-induced exocytosis in many cell types. FDGmicroPET data indicated an increase in whole brain metabolism, and in areas related to epileptogenesis such as hippocampus and thalamus. A neurogenic pulmonary edema could be implicated in the convulsant activity of canatoxin, as suggested by the higher [18]FDG uptake in areas responsible for breathing control. Altogether the data emphasize the complexity of the neurotoxic mechanism of action of C. ensiformis ureases. Considering that non-enzymatic properties are shared by ureases of different kingdoms, we hope that deepening the knowledge on the mechanism of neurotoxic action of C. ensiformis ureases would help to establish the relevance of these toxins in many infectious diseases caused by urease-producing microorganisms, particularly those affecting the human central nervous system.