DmCatD, a cathepsin D-like peptidase of the hematophagous insect Dipetalogaster maxima (Hemiptera: Reduviidae): Purification, bioinformatic analyses and the significance of its interaction with lipophorin in the internalization by developing oocytes

DmCatD, a cathepsin D-like peptidase of the hematophagous insect Dipetalogaster maxima, is synthesized by the fat body and the ovary and functions as yolk protein precursor. Functionally, DmCatD is involved in vitellin proteolysis. In this work, we purified and sequenced DmCatD, performed bioinformatic analyses and investigated the events involved in its targeting and storage in developing oocytes. By ion exchange and gel filtration chromatography, DmCatD was purified from egg homogenates and its identity was confirmed by mass spectrometry. Approximately 73% of the full-length transcript was sequenced. The phylogeny indicated that DmCatD has features which suggest its distancing from “classical” cathepsins D. Bioinformatic analyses using a chimeric construct were employed to predict post-translational modifications. Structural modeling showed that DmCatD exhibited the expected folding for this type of enzyme, and an active site with conserved architecture. The interaction between DmCatD and lipophorin in the hemolymph was demonstrated by co-immunoprecipitation. Colocalization of both proteins in developing oocyte membranes and yolk bodies was detected by immunofluorescence. Docking assays favoring the interaction DmCatD-lipophorin were carried out after modeling lipophorin of a related triatomine species. Our results suggest that lipophorin acts as a carrier for DmCatD to facilitate its further internalization by the oocytes. The mechanisms involved in the uptake of peptidases within the oocytes of insects have not been reported. This is the first experimental work supporting the interaction between cathepsin D and lipophorin in an insect species, enabling us to propose a pathway for its targeting and storage in developing oocytes.Fil: Leyria, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Fruttero, Leonardo Luis. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Pontificia Universidade Católica do Rio Grande do Sul; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Ligabue Braun, Rodrigo. Universidade Federal do Rio Grande do Sul; BrasilFil: Defferrari, Marina S.. University of Toronto; CanadáFil: Arrese, Estela L.. Oklahoma State University; Estados UnidosFil: Soulages, José L.. Oklahoma State University; Estados UnidosFil: Settembrini, Beatriz Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”; ArgentinaFil: Carlini, Célia Regina R S. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Canavoso, Lilian Etelvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentin

Structure-Function insights of Jaburetox and Soyuretox: Novel intrinsically disordered polypeptides derived from plant ureases

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) do not have a stable 3D structure but still have important biological activities. Jaburetox is a recombinant peptide derived from the jack bean (Canavalia ensiformis) urease and presents entomotoxic and antimicrobial actions. The structure of Jaburetox was elucidated using nuclear magnetic resonance which reveals it is an IDP with small amounts of secondary structure. Different approaches have demonstrated that Jaburetox acquires certain folding upon interaction with lipid membranes, a characteristic commonly found in other IDPs and usually important for their biological functions. Soyuretox, a recombinant peptide derived from the soybean (Glycine max) ubiquitous urease and homologous to Jaburetox, was also characterized for its biological activities and structural properties. Soyuretox is also an IDP, presenting more secondary structure in comparison with Jaburetox and similar entomotoxic and fungitoxic effects. Moreover, Soyuretox was found to be nontoxic to zebra fish, while Jaburetox was innocuous to mice and rats. This profile of toxicity affecting detrimental species without damaging mammals or the environment qualified them to be used in biotechnological applications. Both peptides were employed to develop transgenic crops and these plants were active against insects and nematodes, unveiling their immense potentiality for field applications.Fil: Grahl, Matheus V. Coste. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Lopes, Fernanda Cortez. Universidade Federal do Rio Grande do Sul; BrasilFil: Martinelli, Anne H. Souza. Universidade Federal do Rio Grande do Sul; BrasilFil: Carlini, Célia Regina R. S.. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Fruttero, Leonardo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin

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.Fil: Kappaun, Karine. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Martinelli, Anne H. S.. Universidade Federal do Rio Grande do Sul; BrasilFil: Broll, Valquiria. Universidade Federal do Rio Grande do Sul; BrasilFil: Zambelli, Barbara. Universidad de Bologna; ItaliaFil: Lopes, Fernanda C.. Universidade Federal do Rio Grande do Sul; BrasilFil: Ligabue-Braun, Rodrigo. Universidade Federal do Rio Grande do Sul; BrasilFil: Fruttero, Leonardo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Moyetta, Natalia Rita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Bonan, Carla D.. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Carlini, Célia Regina R. S.. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Ciurli, Stefano. Universidad de Bologna; Itali

β-chain of ATP synthase as a lipophorin binding protein and its role in lipid transfer in the midgut of Panstrongylus megistus (Hemiptera: Reduviidae)

Lipophorin, the main lipoprotein in the circulation of the insects, cycles among peripheral tissues to exchange its lipid cargo at the plasma membrane of target cells, without synthesis or degradation of its apolipoprotein matrix. Currently, there are few characterized candidates supporting the functioning of the docking mechanism of lipophorin-mediated lipid transfer. In this work we combined ligand blotting assays and tandem mass spectrometry to characterize proteins with the property to bind lipophorin at the midgut membrane of Panstrongylus megistus, a vector of Chagas' disease. We further evaluated the role of lipophorin binding proteins in the transfer of lipids between the midgut and lipophorin. The β subunit of the ATP synthase complex (β-ATPase) was identified as a lipophorin binding protein. β-ATPase was detected in enriched midgut membrane preparations free of mitochondria. It was shown that β-ATPase partially co-localizes with lipophorin at the plasma membrane of isolated enterocytes and in the sub-epithelial region of the midgut tissue. The interaction of endogenous lipophorin and β-ATPase was also demonstrated by co-immunoprecipitation assays. Blocking of β-ATPase significantly diminished the binding of lipophorin to the isolated enterocytes and to the midgut tissue. In vivo assays injecting the β-ATPase antibody significantly reduced the transfer of [3H]-diacylglycerol from the midgut to the hemolymph in insects fed with [9,10-3H]-oleic acid, supporting the involvement of lipophorin-β-ATPase association in the transfer of lipids. In addition, the β-ATPase antibody partially impaired the transfer of fatty acids from lipophorin to the midgut, a less important route of lipid delivery to this tissue. Taken together, the findings strongly suggest that β-ATPase plays a role as a docking lipophorin receptor at the midgut of P. megistus.Fil: Fruttero, Leonardo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: De Martini, Diogo R.. Universidade Federal do Rio Grande do Sul; BrasilFil: Rubiolo, Edilberto Rene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Carlini, Célia Regina R S. Universidade Federal do Rio Grande do Sul; BrasilFil: Canavoso, Lilian Etelvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin

The entomotoxin Jack Bean Urease changes cathepsin D activity in nymphs of the hematophagous insect Dipetalogaster maxima (Hemiptera: Reduviidae)

In insects, cathepsin D is a lysosomal aspartic endopeptidase involved in several functions such as digestion, defense and reproduction. Jack Bean Urease (JBU) is the most abundant urease isoform obtained from the seeds of the plant Canavalia ensiformis. JBU is a multifunctional protein with entomotoxic effects unrelated to its catalytic activity, by mechanisms not yet fully understood. In this work, we employed nymphs of the hematophagous insect Dipetalogaster maxima as an experimental model in order to study the effects of JBU on D. maxima CatD (DmCatD). In insects without treatment, immunofluorescence assays revealed a conspicuous distribution pattern of DmCatD in the anterior and posterior midgut as well as in the fat body and hemocytes. Western blot assays showed that the active form of DmCatD was present in the fat body, the anterior and posterior midgut; whereas the proenzyme was visualized in hemocytes and hemolymph. The transcript of DmCatD and its enzymatic activity was detected in the anterior and posterior midgut as well as in fat body and hemocytes. JBU injections induced a significant increase of DmCatD activity in the posterior midgut (at 3 h post-injection) whereas in the hemolymph, such an effect was observed after 18 h. These changes were not correlated with modifications in DmCatD mRNA and protein levels or changes in the immunofluorescence pattern. In vitro experiments might suggest a direct effect of the toxin in DmCatD activity. Our findings indicated that the tissue-specific increment of cathepsin D activity is a novel effect of JBU in insects.Fil: Moyetta, Natalia Rita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Fruttero, Leonardo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Leyria, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Ramos, Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Carlini, Célia Regina R S. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Canavoso, Lilian Etelvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin

Distribution and characterization of corazonin in the central nervous system of Triatoma infestans (Insecta: Heteroptera)

The distribution of corazonin in the central nervous system of the heteropteran insect Triatoma infestans was studied by immunohistochemistry. The presence of corazonin isoforms was investigated using MALDI-TOF mass spectrometry in samples containing the brain, the subesophageal ganglion, the corpora cardiaca-corpus allatum complex and the anterior part of the aorta. Several groups of immunopositive perikarya were detected in the brain, the subesophageal ganglion and the thoracic ganglia. Regarding the brain, three clusters were observed in the protocerebrum. One of these clusters was formed by somata located near the entrance of the ocellar nerves whose fibers supplied the aorta and the corpora cardiaca. The remaining groups of the protocerebrum were located in the lateral soma cortex and at the boundary of the protocerebrum with the optic lobe. The optic lobe housed immunoreactive somata in the medial soma layer of the lobula and at the level of the first optic chiasma. The neuropils of the deutocerebrum and the tritocerebrum were immunostained, but no immunoreactive perikarya were detected. In the subesophageal ganglion, immunostained somata were found in the soma layers of the mandibular and labial neuromeres, whereas in the mesothoracic ganglionic mass, they were observed in the mesothoracic, metathoracic and abdominal neuromeres. Immunostained neurites were also found in the esophageal wall. The distribution pattern of corazonin like immunoreactivity in the central nervous system of this species suggests that corazonin may act as a neurohormone. Mass spectrometric analysis revealed that [Arg 7]-corazonin was the only isoform of the neuropeptide present in T. infestans tissue samples.Fil: Settembrini, Beatriz Patricia. Universidad Austral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”; ArgentinaFil: De Pasquale, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”; ArgentinaFil: Postal, Melissa. Universidade Federal do Rio Grande do Sul; BrasilFil: Pinto, Paulo M.. Universidade de Caxias do Sul; BrasilFil: Carlini, Célia Regina R S. Universidade Federal do Rio Grande do Sul; BrasilFil: Villar, Marcelo Jose. Universidad Austral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Structural Analysis of the Interaction between Jaburetox, an Intrinsically Disordered Protein, and Membrane Models

Jack bean urease is entomotoxic to insects with cathepsin-like digestive enzymes, and its toxicity is mainly caused by a polypeptide called Jaburetox (Jbtx), released by cathepsin-dependent hydrolysis of the enzyme. Jbtx is intrinsically disordered in aqueous solution, as shown by CD and NMR. Jbtx is able to alter the permeability of membranes, hinting to a role of Jbtx-membrane interaction as the basis for its toxicity. The present study addresses the structural aspects of this interaction by investigating the behaviour of Jbtx when in contact with membrane models, using nuclear magnetic resonance and circular dichroism spectroscopies in the absence or presence of micelles, large unilamellar vesicles, and bicelles. Fluorescence microscopy was also used to detect protein-insect membrane interaction. Significant differences were observed depending on the type of membrane model used. The interaction with negatively charged SDS micelles increases the secondary and tertiary structure content of the polypeptide, while, in the case of large unilamellar vesicles and bicelles, conformational changes were observed at the terminal regions, with no significant acquisition of secondary structure motifs. These results were interpreted as suggesting that the Jbtx-lipids interaction anchors the polypeptide to the cellular membrane through the terminal portions of the polypeptide and that, following this interaction, Jbtx undergoes conformational changes to achieve a more ordered structure that could facilitate its interaction with membrane-bound proteins. Consistently with this hypothesis, the presence of these membrane models decreases the ability of Jbtx to bind cellular membranes of insect nerve cord. The collected evidence from these studies implies that the biological activity of Jbtx is due to protein-phospholipid interactions.Fil: Broll, Valquiria. Universidade Federal do Rio Grande do Sul; BrasilFil: Martinelli, Anne Helene S.. Universidade Federal do Rio Grande do Sul; BrasilFil: Lopes, Fernanda C.. Universidade Federal do Rio Grande do Sul; BrasilFil: Fruttero, Leonardo Luis. Pontificia Universidade Católica do Rio Grande do Sul; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Zambelli, Barbara. Universidad de Bologna; ItaliaFil: Salladini, Edoardo. Universidad de Bologna; ItaliaFil: Dobrovolska, Olena. Universidad de Bologna; ItaliaFil: Ciurli, Stefano. Universidad de Bologna; ItaliaFil: Carlini, Célia Regina R S. Pontificia Universidade Católica do Rio Grande do Sul; Brasil. Universidade Federal do Rio Grande do Sul; Brasi

Jaburetox affects gene expression and enzyme activities in Rhodnius prolixus, a Chagas’ disease vector

Jaburetox, a recombinant peptide of ∼11 kDa derived from one of the Canavalia ensiformis (Jack Bean) urease isoforms, is toxic and lethal to insects belonging to different orders when administered orally or via injection. Previous findings indicated that Jaburetox acts on insects in a complex fashion, inhibiting diuresis and the transmembrane potential of Malpighian tubules, interfering with muscle contractility and affecting the immune system. In vitro, Jaburetox forms ionic channels and alters permeability of artificial lipid membranes. Moreover, recent data suggested that the central nervous system (CNS) is a target organ for ureases and Jaburetox. In this work, we employed biochemical, molecular and cellular approaches to explore the mode of action of Jaburetox using Rhodnius prolixus, one of the main Chagas’ disease vectors, as experimental model. In vitro incubations with fluorescently labeled Jaburetox indicated a high affinity of the peptide for the CNS but not for salivary glands (SG). The in vitro treatment of CNS or SG homogenates with Jaburetox partially inhibited the activity of nitric oxide synthase (NOS), thus disrupting nitrinergic signaling. This inhibitory effect was also observed in vivo (by feeding) for CNS but not for SG, implying differential modulation of NOS in these organs. The inhibition of NOS activity did not correlate to a decrease in expression of its mRNA, as assessed by qPCR. UDP-N-acetylglucosamine pyrophosphorylase (UAP), a key enzyme in chitin synthesis and glycosylation pathways and a known target of Jaburetox in insect CNS, was also affected in SG, with activation of the enzyme seen after both in vivo or in vitro treatments with the peptide. Unexpectedly, incubation of Jaburetox with a recombinant R. prolixus UAP had no effect on its activity, implying that the enzyme's modulation by the peptide requires the participation of other factor(s) present in CNS or SG homogenates. Feeding Jaburetox to R. prolixus decreased the mRNA levels of UAP and chitin synthase, indicating a complex regulation exerted by the peptide on these enzymes. No changes were observed upon Jaburetox treatment in vivo and in vitro on the activity of the enzyme acid phosphatase, a possible link between UAP and NOS. Here we have demonstrated for the first time that the Jaburetox induces changes in gene expression and that SG are another target for the toxic action of the peptide. Taken together, these findings contribute to a better understanding of the mechanism of action of Jaburetox as well as to the knowledge on basic aspects of the biochemistry and neurophysiology of insects, and might help in the development of optimized strategies for insect control.Fil: Fruttero, Leonardo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Pontificia Universidade Católica do Rio Grande do Sul; Brasil. Universidade Federal do Rio Grande do Sul; BrasilFil: Moyetta, Natalia Rita. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Krug, Monique Siebra. Universidade Federal do Rio Grande do Sul; BrasilFil: Broll, Valquiria. Universidade Federal do Rio Grande do Sul; BrasilFil: Grahl, Matheus V. Coste. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Real Guerra, Rafael. Universidade Federal do Rio Grande do Sul; BrasilFil: Stanisçuaski, Fernanda. Universidade Federal do Rio Grande do Sul; BrasilFil: Carlini, Célia Regina R S. Universidade Federal do Rio Grande do Sul; Brasil. Pontificia Universidade Católica do Rio Grande do Sul; Brasi

Jaburetox-induced toxic effects on the hemocytes of Rhodnius prolixus (Hemiptera: Reduviidae)

Jaburetox is a recombinant peptide derived from a Canavalia ensiformis urease that presents toxic effects uponseveral species of insects, phytopathogenic fungi and yeasts of medical importance. So far, no toxicity of Jaburetoxto mammals has been shown. Previous reports have identified biochemical targets of this toxic peptide ininsect models, although its mechanism of action is not completely understood. In this work, we aimed to characterizethe effects of Jaburetox in hemolymphatic insect cells. For this purpose, the model insect and Chagas´disease vector Rhodnius prolixus was used. In vivo and in vitro experiments indicated that Jaburetox interacts witha subset of hemocytes and it can be found in various subcellular compartments. In insects injected with Jaburetoxthere was an increase in the gene expression of the enzymes UDP-N-acetylglucosamine pyrophosphorylase(UAP), chitin synthase and nitric oxide synthase (NOS). Nevertheless, the expression of NOS protein, the enzymeactivities of UAP and acid phosphatase (a possible link between UAP and NOS) as well as the phosphorylationstate of proteins remained unchanged upon the in vivo Jaburetox treatment. Nitric oxide (NO) imaging using fluorescentprobes showed that Jaburetox augmented NO production in the hemocyte aggregates when comparedto controls. Even though Jaburetox activated the hemocytes, as demonstrated by wheat germ agglutinin bindingassays, the peptide did not lead to an increase of their phagocytic behavior. Taken together, these findingscontribute to our understanding of toxic effects of Jaburetox, a peptide with biotechnological applications and aprospective tool for rational insect control.Fil: Moyetta, Natalia Rita. Pontificia Universidade Católica do Rio Grande do Sul; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Broll, Valquiria. Universidade Federal do Rio Grande do Sul; BrasilFil: Perin, Ana Paula A.. Universidade Federal do Rio Grande do Sul; BrasilFil: Uberti, Augusto F.. Universidade Federal do Rio Grande do Sul; BrasilFil: Coste Grahl, Matheus V.. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Staniscuaski, Fernanda. Universidade Federal do Rio Grande do Sul; BrasilFil: Carlini, Célia Regina R S. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Fruttero, Leonardo Luis. Pontificia Universidade Católica do Rio Grande do Sul; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin

Soybean ubiquitous urease with purification facilitator: An addition to the moonlighting studies toolbox

Ureases are nickel-dependent enzymes that catalyze the hydrolysis of urea to ammonia and carbon dioxide. In soybean (Glycine max), the embryo-specific urease (eSBU), the ubiquitous urease (uSBU), and a third isoform (SBU-III) are synthesized. Our group has previously demonstrated that eSBU, purified from seeds, has antifungal properties against phytopathogenic fungi, entomotoxicity against Dysdercus peruvianus, the ability to induce blood platelet aggregation, and these properties are independent of its enzymatic activity. Here we describe the biological properties of apo-uSBU fused to glutathione S-transferase (GST) produced in Escherichia coli. Removal of GST affected apo-uSBU stability. We performed a Response Surface Methodology to optimize GST-uSBU production to 5 mg per liter and then bioassays were carried out. The recombinant protein exhibited inhibitory effects on filamentous fungi and affected fungal secondary metabolism. Candida albicans and C. tropicalis were also susceptible to GST-uSBU and formed pseudo-hyphae. The fusion protein was toxic against Rhodnius prolixus, with the toxicity being accompanied by in vivo and in vitro hemocyte aggregation. Rabbit platelet also aggregated in the presence of GST-uSBU. Thus, uSBU displayed similar biological properties as previously described for eSBU even when fused to GST, reinforcing the proposed role of ureases in plant defense.Fil: Martinelli, Anne H.S.. Universidade Federal do Rio Grande do Sul; BrasilFil: Lopes, Fernanda C.. Universidade Federal do Rio Grande do Sul; BrasilFil: Broll, Valquiria. Universidade Federal do Rio Grande do Sul; BrasilFil: Defferrari, Marina S.. University of Toronto; CanadáFil: Ligabue-Braun, Rodrigo. Universidade Federal do Rio Grande do Sul; BrasilFil: Kappaun, Karine. Pontificia Universidade Católica do Rio Grande do Sul; BrasilFil: Tichota, Deise M.. Universidade Federal do Rio Grande do Sul; BrasilFil: Fruttero, Leonardo Luis. Pontificia Universidade Católica do Rio Grande do Sul; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Moyetta, Natalia Rita. Pontificia Universidade Católica do Rio Grande do Sul; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Demartini, Diogo R.. Universidade Federal do Rio Grande do Sul; BrasilFil: Postal, Melissa. Universidade Federal do Rio Grande do Sul; BrasilFil: Medeiros-Silva, Monica. Universidade Federal do Rio Grande do Sul; BrasilFil: Becker-Ritt, Arlete Beatriz. Universidade Luterana Do Brasil; BrasilFil: Pasquali, Giancarlo. Universidade Federal do Rio Grande do Sul; BrasilFil: Carlini, Célia Regina R S. Pontificia Universidade Católica do Rio Grande do Sul; Brasi