RESUMO
l-asparaginases play a crucial role in the treatment of acute lymphoblastic leukemia (ALL), a type of cancer that mostly affects children and teenagers. However, it is common for these molecules to cause adverse reactions during treatment. These downsides ignite the search for novel asparaginases to mitigate these problems. Thus, this work aimed to produce and characterize a recombinant asparaginase from Phaseolus vulgaris (Asp-P). In this study, Asp-P was expressed in Escherichia coli with high yields and optimum activity at 40 °C, pH 9.0. The enzyme Km and Vmax values were 7.05 mM and 1027 U/mg, respectively. Asp-P is specific for l-asparagine, showing no activity against l-glutamine and other amino acids. The enzyme showed a higher cytotoxic effect against Raji than K562 cell lines, but only at high concentrations. In silico analysis indicated that Asp-P has lower immunogenicity than a commercial enzyme. Asp-P induced biofilm formation by Candida sp. due to sublethal dose, showing an underexplored potential of asparaginases. The absence of glutaminase activity, lower immunogenicity and optimal activity similar to physiological temperature conditions are characteristics that indicate Asp-P as a potential new commercial enzyme in the treatment of ALL and its underexplored application in the treatment of other diseases.
Assuntos
Asparaginase , Phaseolus , Proteínas Recombinantes , Asparaginase/química , Asparaginase/farmacologia , Asparaginase/genética , Asparaginase/imunologia , Phaseolus/química , Humanos , Cinética , Proteínas Recombinantes/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Leucemia/tratamento farmacológico , Células K562 , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Biofilmes/efeitos dos fármacos , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
Galectins, which correspond to a group of proteins capable of recognizing and reversibly binding to ß-galactoside carbohydrates, have been the subject of innovation and development of technological products. Galectins play biological roles, such as cell proliferation and apoptosis, and some studies showed differences in the concentrations of galectins dispersed in serum of patients with cancer. For this reason, different studies have evaluated the biotechnological potential of these proteins as biomarkers for the prognosis and/or diagnosis of physiological disorders. Thus, this review discusses recent technological advancements in targeting galectins for the treatment of cancer and using galectins for cancer prognosis and diagnosis. Data mining was performed using the search descriptors "Galectin 9* and cancer*" and the ESPACENET and Cortellis Drug Discovery Intelligence (CDDI) databases. PRISMA guidelines were followed as a basis for literature review which aimed to conduct a systematic study of galectin-9 patents related to cancer prognosis, diagnosis and treatment. Results showed the importance of galectin-9 protein patents in furthering biomedical advancements in the global fight against cancer.
Assuntos
Galectinas , Neoplasias , Humanos , Galectinas/metabolismo , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , CarboidratosRESUMO
The SfL-1 isoform from the marine red algae Solieria filiformis was produced in recombinant form (rSfL-1) and showed hemagglutinating activity and inhibition similar to native SfL. The analysis of circular dichroism revealed the predominance of ß-strands structures with spectra of ßI-proteins for both lectins, which had Melting Temperature (Tm) between 41 °C and 53 °C. The three-dimensional structure of the rSfL-1 was determined by X-ray crystallography, revealing that it is composed of two ß-barrel domains formed by five antiparallel ß chains linked by a short peptide between the ß-barrels. SfL and rSfL-1 were able to agglutinate strains of Escherichia coli and Staphylococcus aureus and did not show antibacterial activity. However, SfL induced a reduction in E. coli biomass at concentrations from 250 to 125 µg mL-1, whereas rSfL-1 induced reduction in all concentrations tested. Additionally, rSfL-1 at concentrations from 250 to 62.5 µg mL-1, showed a statistically significant reduction in the number of colony-forming units, which was not noticed for SfL. Wound healing assay showed that the treatments with SfL and rSfL-1 act in reducing the inflammatory response and in the activation and proliferation of fibroblasts by a larger and fast deposition of collagen.
Assuntos
Lectinas , Rodófitas , Lectinas/farmacologia , Lectinas/química , Escherichia coli , Antibacterianos/farmacologia , Antibacterianos/química , Rodófitas/química , CicatrizaçãoRESUMO
The Leucaena leucocephala trypsin inhibitor (LTI) + Bacillus thuringiensis (Bt) protoxins mix has been proposed as a novel larvicide agent in order to control the vector mosquito of dengue virus, Aedes aegypti, in their aquatic breeding sites. However, use of this insecticide formulation has raised concerns about its impacts on aquatic biota. In this context, this work aimed to assess the effects of LTI and Bt protoxins, separately or in combination, in zebrafish, in regard to the evaluation of toxicity at early life stages and to the presence of LTI inhibitory effects on intestinal proteases of this fish. Results showed that LTI and Bt concentrations (250 mg/L, and 0.13 mg/L, respectively), and LTI + Bt mix (250 mg/L + 0.13 mg/L) - 10 times superior to those with insecticidal action - did not cause death nor did it induce morphological changes during embryonic and larval development (3 to 144 h post-fertilization) of zebrafish. Molecular docking analyses highlighted a possible interaction between LTI and zebrafish trypsin, especially through hydrophobic interactions. In concentrations near to those with larvicidal action, LTI (0.1 mg/mL) was able to inhibit in vitro intestinal extracts of trypsin in female and male fish by 83 % and 85 %, respectively, while LTI + Bt mix promoted trypsin inhibition of 69 % in female and 65 % in male ones. These data show that the larvicidal mix can potentially promote deleterious effects to nutrition and survival in non-target aquatic organisms, especially those with trypsin-like dependent protein digestion.
Assuntos
Inseticidas , Animais , Inseticidas/toxicidade , Peixe-Zebra , Inibidores de Proteases/farmacologia , Tripsina , Larva , Simulação de Acoplamento Molecular , Mosquitos Vetores , Inibidores da Tripsina/farmacologia , Antivirais/farmacologia , Proteínas de Bactérias/toxicidadeRESUMO
Studies on the globin family are continuously revealing insights into the mechanisms of gene and protein evolution. The rise of a new globin gene type in Pelobatoidea and Neobatrachia (Amphibia:Anura) from an α-globin precursor provides the opportunity to investigate the genetic and physical mechanisms underlying the origin of new protein structural and functional properties. This amphibian-specific globin (globin A/GbA) discovered in the heart of Rana catesbeiana is a monomer. As the ancestral oligomeric state of α-globins is a homodimer, we inferred that the ancestral state was lost somewhere in the GbA lineage. Here, we combined computational molecular evolution with structural bioinformatics to determine the extent to which the loss of the homodimeric state is pervasive in the GbA clade. We also characterized the loci of GbA genes in Bufo bufo. We found two GbA clades in Neobatrachia. One was deleted in Ranidae, but retained and expanded to yield a new globin cluster in Bufonidae species. Loss of the ancestral oligomeric state seems to be pervasive in the GbA clade. However, a taxonomic sampling that includes more Pelobatoidea, as well as early Neobatrachia, lineages would be necessary to determine the oligomeric state of the last common ancestor of all GbA. The evidence presented here points out a possible loss of oligomerization in Pelobatoidea GbA as a result of amino acid substitutions that weaken the homodimeric state. In contrast, the loss of oligomerization in both Neobatrachia GbA clades was linked to independent deletions that disrupted many packing contacts at the homodimer interface.
Assuntos
Evolução Molecular , Globinas , Animais , Globinas/genética , Filogenia , Anfíbios/genéticaRESUMO
Inflammation and oxidative stress are processes associated with different human diseases. They are treated using drugs that have several side effects. Seaweed are sources of potentially relevant natural compounds for use as treatment of these disorders. Lectins are able to reversibly interact with complex carbohydrates and modulate cell membrane glycosylated receptors through this interaction. This study aimed to determine the antinociceptive and anti-inflammatory potential of CiL-1 in adult zebrafish by modulation of TRPA1 through lectin-glycan binding. Possible neuromodulation by TRPA1 channel was also evaluated by camphor pretreatment. CiL-1 was efficacious at all tested doses, revealing anti-nociceptive and anti-inflammatory effects in adult zebrafish. This galactose-binding lectin was also able to reduce the content of ROS in brain and liver. In silico analyses showed CiL-1 interactions with both ligands tested. LacNac2 presents the most favorable binding energy with the protein. The interaction occurs at 4 subsites as an extended conformation at the site. LacNac2-Sia had a less favorable curved-shape interaction energy. Based on the predictions made for the oligosaccharides, a tetra-antenate putative glycan was schematically constructed, illustrating an interaction between TRPA1 N-glycan and CiL-1. This binding seems to be related to CiL-1 anti-inflammatory activity as result of receptor modulation.
Assuntos
Anti-Inflamatórios , Polissacarídeos , Peixe-Zebra , Animais , Analgésicos/farmacologia , Anti-Inflamatórios/farmacologia , Lectinas/química , Polissacarídeos/química , Polissacarídeos/farmacologiaRESUMO
The globin gene repertoire of gnathostome vertebrates is dictated by differential retention and loss of nine paralogous genes: androglobin, neuroglobin, globin X, cytoglobin, globin Y, myoglobin, globin E, and the α- and ß-globins. We report the globin gene repertoire of three orders of modern amphibians: Anura, Caudata, and Gymnophiona. Combining phylogenetic and conserved synteny analysis, we show that myoglobin and globin E were lost only in the Batrachia clade, but retained in Gymnophiona. The major amphibian groups also retained different paralogous copies of globin X. None of the amphibian presented αD-globin gene. Nevertheless, two clades of ß-globins are present in all amphibians, indicating that the amphibian ancestor possessed two paralogous proto ß-globins. We also show that orthologs of the gene coding for the monomeric hemoglobin found in the heart of Rana catesbeiana are present in Neobatrachia and Pelobatoidea species we analyzed. We suggest that these genes might perform myoglobin- and globin E-related functions. We conclude that the repertoire of globin genes in amphibians is dictated by both retention and loss of the paralogous genes cited above and the rise of a new globin gene through co-option of an α-globin, possibly facilitated by a prior event of transposition.
Assuntos
Anfíbios/genética , Globinas/genética , Animais , Evolução Molecular , Filogenia , SinteniaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Since drugs currently used to manage pain and inflammatory conditions present several side effects, the investigation of new anti-inflammatory and antinociceptive agents from folk-medicine plants is an important approach. Costus spiralis (Costaceae) has been used in Brazilian medicinal teas to treat urinary infection, cough, inflammation, arthritis, among others. AIM OF THE STUDY: The current study focused on investigating anti-inflammatory and antinociceptive effects of fractions from C. spiralis leaves using animal models. MATERIALS AND METHODS: Adults Swiss mice were used in the following experimental models: acetic acid-induced abdominal writhing, formalin-induced nociception, hot plate, zymosan-induced peritonitis, and arthritis induced by complete Freund's adjuvant. RESULTS: The presence of steroids was confirmed in all fractions. Flavonoids, condensed tannins and saponins were observed in EFL. In methanolic fraction leaves (MFL), the presence of flavonoids and pentacyclic triterpenoids was confirmed. Orally administered leaf fractions significantly reduced abdominal writhing. Fractions were ineffective in the neurogenic stage of the formalin test, but in the inflammatory stage, ethyl acetate fraction levaes (AcFL), ethanolic fraction leaves (EFL), and MFL significantly reduced paw licking time by 69.6 ± 11.9%, 58.2 ± 9.4%, and 79.6 ± 8.3%, respectively. In the hot plate test, the reaction latency was similar for treated animals and controls. However, in the peritonitis test, cell migration was significantly reduced in animals treated with chloroform fractions leaves ClFL (61.8 ± 11.4%), AcFL (58.7 ± 8.3%), EFL (39.2 ± 5.0%), and MFL (64.8 ± 4.4%). This was similar to the result observed in the chronic inflammation model, this time only the chloroform fraction was able to reduce paw edema. CONCLUSION: Our results show that leaf fractions of Costus spiralis are capable of modulating peripheral nociceptive and inflammatory responses without effects on central nervous system being potential substrates for phytochemical purification, structural and mechanistic studies.
Assuntos
Analgésicos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Artrite Experimental/tratamento farmacológico , Costus , Medição da Dor/efeitos dos fármacos , Extratos Vegetais/uso terapêutico , Analgésicos/isolamento & purificação , Analgésicos/farmacologia , Animais , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/farmacologia , Artrite Experimental/induzido quimicamente , Artrite Experimental/metabolismo , Feminino , Masculino , Camundongos , Medição da Dor/métodos , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Folhas de PlantaRESUMO
The Pisum arvense lectin (PAL), a legume protein belonging to the Vicieae tribe, is capable of specific recognition of mannose, glucose and its derivatives without altering its structure. In this work, the three-dimensional structure of PAL was determined by X-ray crystallography and studied in detail by a combination of molecular docking and molecular dynamics (MD). Crystals belonging to monoclinic space group P21 were grown by the vapor diffusion method at 293 K. The structure was solved at 2.16 Å and was similar to that of other Vicieae lectins. The structure presented Rfactor and Rfree of 17.04% and 22.08%, respectively, with all acceptable geometric parameters. Molecular docking was performed to analyze interactions of the lectin with monosaccharides, disaccharides and high-mannose N-glycans. PAL demonstrated different affinities on carbohydrates, depending on bond orientation and glycosidic linkage present in ligands. Furthermore, the lectin interacted with representative N-glycans in a manner consistent with the biological effects described for Vicieae lectins. Carbohydrate-recognition domain (CRD) in-depth analysis was performed by MD, describing the behavior of CRD residues in complex with ligand, stability, flexibility of the protein over time, CRD volume and topology. This is a first report of its kind for a lectin of the Vicieae tribe.
Assuntos
Fabaceae/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Lectinas de Plantas/química , Polissacarídeos/química , Cristalografia por Raios XRESUMO
The three-dimensional structure of Dioclea reflexa seed lectin (DrfL) was studied in detail by a combination of X-ray crystallography, molecular docking and molecular dynamics. DrfL was purified by affinity chromatography using Sephadex G-50 matrix. Its primary structure was obtained by mass spectrometry, and crystals belonging to orthorhombic space group P212121 were grown by the vapor diffusion method at 293K. The crystal structure was solved at 1.765Å and was very similar to that of other lectins from the same subtribe. The structure presented Rfactor and Rfree of 21.69% and 24.89%, respectively, with no residues in nonallowed regions of Ramachandran plot. Similar to other Diocleinae lectins, DrfL was capable of relaxing aortic rings via NO induction, with CRD participation, albeit with low intensity (32%). In silico analysis results demonstrated that DrfL could strongly interact with complex N-glycans, components of blood vessel glycoconjugates. Despite the high similarity among Diocleinae lectins, it was also reported that each lectin has unique CRD properties that influence carbohydrate binding, resulting in different biological effects presented by these molecules.
Assuntos
Dioclea/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Sementes/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Manosídeos/química , Manosídeos/metabolismo , Lectinas de Plantas/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Domínios Proteicos , Ratos , Vasodilatadores/química , Vasodilatadores/metabolismo , Vasodilatadores/farmacologiaRESUMO
A lectin from Canavalia virosa, Diocleinae subtribe, was purified by affinity chromatography with Sephadex G-50 matrix and named ConV. The primary structure of ConV was obtained by mass spectrometry and crystals were obtained by the vapor diffusion method at 293K and belonged to orthorhombic space group P21221 with two molecules in its asymmetric unit. The structure obtained presented Rfactor and Rfree of 18.91% and 24.92% respectively, with no residues in nonallowed regions of Ramachandran plot. The crystal structure was solved at 2.53Å and was demonstrated to be very similar to other lectins from the same subtribe. In inflammatory tests, ConV elicited paw edema, but incubation of lectin with glucose beforehand was able to reduce the edematogenic effect, indicating the involvement of the carbohydrate recognition domain in this process. The lectin also showed toxicity to rat C6 glioma cells, disrupting the mitochondrial membrane potential (ΔYm) and decreasing cell viability, indicating an anticancer potential for ConV. In silico studies confirmed that ConV interacts strongly with carbohydrates that comprise the N-glycans of glycoproteins. This finding corroborates the hypothesis which holds that the lectin domain interacts with glycans in molecular targets and that this contributes to the effects observed in biological activities.
Assuntos
Anti-Inflamatórios/química , Antineoplásicos Fitogênicos/química , Extratos Vegetais/química , Sequência de Aminoácidos , Animais , Anti-Inflamatórios/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Sítios de Ligação , Canavalia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sequência Conservada , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Ligação de Hidrogênio , Masculino , Manosídeos/química , Camundongos , Simulação de Acoplamento Molecular , Extratos Vegetais/farmacologia , Lectinas de Plantas/química , Ligação Proteica , Conformação Proteica em Folha beta , Estrutura Quaternária de Proteína , Ratos , Sementes/químicaRESUMO
The relation structure-activity of the Mimosoideae lectins of Parkia platycephala (PPL) and Parkia biglobosa (PBL) was analyzed in this study. PBL was solved by X-ray crystallography at a resolution of 2.1Å, and the crystal structure belonged to the C2221 space group. Structural organization and binding sites were also characterized. Specifically, PBL monomer consists of three ß-prism domains tandemly arranged with each one presenting a different carbohydrate recognition domain (CRD). PPL showed antinociceptive activity in the mouse model of acetic acid-induced writhes with maximal inhibitory effect by 74% at 1mg/mL. PPL also demonstrated anti-inflammatory effect causing inhibition of leukocyte migration induced by both direct and indirect chemoattractants. These PPL activities were compared to that of PBL described previously. Molecular docking of both PBL and PPL demonstrated some differences in carbohydrate-lectin interaction energy. Comparing structure and biological effects of the two lectins provided new data about their structure and the relation with its biological activities.
Assuntos
Fabaceae/química , Lectinas/química , Lectinas/farmacologia , Sequência de Aminoácidos , Analgésicos/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Sítios de Ligação , Movimento Celular/efeitos dos fármacos , Leucócitos/citologia , Camundongos , Simulação de Acoplamento Molecular , Domínios Proteicos , Estrutura Secundária de Proteína , Alinhamento de Sequência , Eletricidade EstáticaRESUMO
A glycosylated lectin (CTL) with specificity for mannose and glucose has been detected and purified from seeds of Centrolobium tomentosum, a legume plant from Dalbergieae tribe. It was isolated by mannose-sepharose affinity chromatography. The primary structure was determined by tandem mass spectrometry and consists of 245 amino acids, similar to other Dalbergieae lectins. CTL structures were solved from two crystal forms, a monoclinic and a tetragonal, diffracted at 2.25 and 1.9 Å, respectively. The carbohydrate recognition domain (CRD), metal-binding site and glycosylation site were characterized, and the structural basis for mannose/glucose-binding was elucidated. The lectin adopts the canonical dimeric organization of legume lectins. CTL showed acute inflammatory effect in paw edema model. The protein was subjected to ligand screening (dimannosides and trimannoside) by molecular docking, and interactions were compared with similar lectins possessing the same ligand specificity. This is the first crystal structure of mannose/glucose native seed lectin with proinflammatory activity isolated from the Centrolobium genus.
Assuntos
Edema/induzido quimicamente , Fabaceae/química , Lectina de Ligação a Manose , Simulação de Acoplamento Molecular , Lectinas de Plantas , Sementes/química , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Edema/patologia , Feminino , Glicosilação , Inflamação/induzido quimicamente , Inflamação/patologia , Lectina de Ligação a Manose/química , Lectina de Ligação a Manose/toxicidade , Espectrometria de Massas , Lectinas de Plantas/química , Lectinas de Plantas/toxicidade , Pegadas de Proteínas , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
An L-rhamnose-binding lectin named ELEL was isolated from eggs of the rock boring sea urchin Echinometra lucunter by affinity chromatography on lactosyl-agarose. ELEL is a homodimer linked by a disulfide bond with subunits of 11 kDa each. The new lectin was inhibited by saccharides possessing the same configuration of hydroxyl groups at C-2 and C-4, such as L-rhamnose, melibiose, galactose and lactose. The amino acid sequence of ELEL was determined by tandem mass spectrometry. The ELEL subunit has 103 amino acids, including nine cysteine residues involved in four conserved intrachain disulfide bonds and one interchain disulfide bond. The full sequence of ELEL presents conserved motifs commonly found in rhamnose-binding lectins, including YGR, DPC and KYL. A three-dimensional model of ELEL was created, and molecular docking revealed favorable binding energies for interactions between ELEL and rhamnose, melibiose and Gb3 (Galα1-4Galß1-4Glcß1-Cer). Furthermore, ELEL was able to agglutinate Gram-positive bacterial cells, suggesting its ability to recognize pathogens.
Assuntos
Lectinas/química , Óvulo/química , Ouriços-do-Mar/química , Sequência de Aminoácidos , Animais , Cátions Bivalentes , Concentração de Íons de Hidrogênio , Lectinas/isolamento & purificação , Lectinas/metabolismo , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica , Ramnose/química , Ramnose/metabolismo , Alinhamento de Sequência , TemperaturaRESUMO
Plant lectins have been studied as histological markers and promising antineoplastic molecules for a long time, and structural characterization of different lectins bound to specific cancer epitopes has been carried out successfully. The crystal structures of Vatairea macrocarpa (VML) seed lectin in complex with GalNAc-α-O-Ser (Tn antigen) and GalNAc have been determined at the resolution of 1.4Å and 1.7Å, respectively. Molecular docking analysis of this new structure and other Tn-binding legume lectins to O-mucin fragments differently decorated with this antigen provides a comparative binding profile among these proteins, stressing that subtle alterations that may not influence monosaccharide binding can, nonetheless, directly impact the ability of these lectins to recognize naturally occurring antigens. In addition to the specific biological effects of VML, the structural and binding similarities between it and other lectins commonly used as histological markers (e.g., VVLB4 and SBA) strongly suggest VML as a candidate tool for cancer research.
Assuntos
Antígenos Glicosídicos Associados a Tumores/química , Antígenos Glicosídicos Associados a Tumores/metabolismo , Fabaceae/química , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Acetilgalactosamina/química , Sítios de Ligação , Cristalografia por Raios X , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Mucina-2/química , Homologia Estrutural de Proteína , TermodinâmicaRESUMO
Plant lectins, especially those purified from species of the Leguminosae family, represent the best-studied group of carbohydrate-binding proteins. Lectins purified from seeds of the Diocleinae subtribe exhibit a high degree of sequence identity notwithstanding that they show very distinct biological activities. Two main factors have been related to this feature: variance in key residues influencing the carbohydrate-binding site geometry and differences in the pH-dependent oligomeric state profile. In this work, we have isolated a lectin from Canavalia boliviana (Cbol) and solved its x-ray crystal structure in the unbound form and in complex with the carbohydrates Man(α1-3)Man(α1-O)Me, Man(α1-4)Man(α1-O)Me and 5-bromo-4-chloro-3-indolyl-α-D-mannose. We evaluated its oligomerization profile at different pH values using Small Angle X-ray Scattering and compared it to that of Concanavalin A. Based on predicted pKa-shifts of amino acids in the subunit interfaces we devised a model for the dimer-tetramer equilibrium phenomena of these proteins. Additionally, we demonstrated Cbol anti-inflammatory properties and further characterized them using in vivo and in vitro models.
Assuntos
Anti-Inflamatórios/farmacologia , Canavalia/química , Edema/tratamento farmacológico , Manosídeos/química , Peritonite/tratamento farmacológico , Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Sementes/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Movimento Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Cristalografia por Raios X , Edema/induzido quimicamente , Manosídeos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Peritonite/induzido quimicamente , Conformação Proteica , Ratos , Ratos Wistar , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Lectins are comprised of a large family of proteins capable of the specific and reversible recognition of carbohydrates. Legume lectins, the most studied plant lectins, show high structural similarity, but with modifications that imply a variation in the intensity of some biological activities. In this work, the primary and tertiary structures of Canavalia grandiflora (ConGF) were determined. ConGF, a lectin isolated from C. grandiflora seeds, is able to induce relaxant activity in rat aortic rings. The complete sequence of ConGF comprises 237 amino acids. This particular protein has primary sequence variations commonly found in lectins from Dioclea and Canavalia genera. The protein structure was solved at 2.3 Å resolution by X-ray crystallography. An X-Man molecule was modeled into the carbohydrate recognition domain. Still, ConGF (30 and 100 µg mL(-1)) elicited 25% of vasorelaxation (IC50=34.48 ± 5.07 µg mL(-1)) in endothelialized aortic rings. A nonselective inhibitor of nitric oxide blocked ConGF relaxant effect, showing mediation by nitric oxide. Key distances between ConGF carbohydrate recognition domain residues were determined in order to explain this effect, in turn revealing some structural aspects that could differentiate lectins from the Canavalia genera with respect to different efficacy in vasorelaxant effect.
Assuntos
Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Vasodilatadores/química , Vasodilatadores/farmacologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Técnicas In Vitro , Masculino , Manose/química , Manose/metabolismo , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Lectinas de Plantas/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Análise de Sequência , Relação Estrutura-Atividade , Vasodilatadores/metabolismoRESUMO
Indole-3-acetic acid (IAA) bound is considered a storage molecule and is inactive. However, some studies have proposed an additional possible regulatory mechanism based on the ability of lectins to form complexes with IAA. We report the first crystal structure of ConM in complex with IAA at 2.15 Å resolution. Based on a tetrameric model of the complex, we hypothesize how the lectin controls the availability of IAA during the early seedling stages, indicating a possible new physiological role for these proteins. A free indole group is also bound to the protein. The ConM interaction with different forms of IAA is a strategy to render the phytohormone unavailable to the cell. Thus, this new physiological role proposed for legume lectins might be a novel mechanism by which IAA levels are decreased in addition to the destruction and formation of new complexes in the later stages of seed germination.
Assuntos
Canavalia/fisiologia , Ácidos Indolacéticos/metabolismo , Lectinas de Plantas/metabolismo , Sementes/metabolismo , Animais , Canavalia/metabolismo , Hemaglutinação/efeitos dos fármacos , Simulação de Acoplamento Molecular , Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Ligação Proteica , Conformação Proteica , CoelhosRESUMO
Lectins from Diocleinae subtribe belong to the family of legume lectins and are characterized by high identity between their amino acids sequences. It has been shown that punctual differences in amino acid sequences, such as one single amino acid or an alternative conformation, represent changes in biological activities caused by these lectins. Therefore, a more detailed understanding of three-dimensional structures of these proteins is essential for accurate analyzing the relationship between structure and function. In this study lectins purified from the seeds of Dioclea violacea (DVL) and Dioclea rostrata (DRL) were compared with regard to crystal structure and vasorelaxant properties. Differences in structure of lectins were found to be reflected in differences in vasorelaxant effects based on their high specificity and selectivity for cell glycans. Binding activity was related to the position of specific residues in the carbohydrate recognition domain (CRD). DVL complexed structure was solved by X-ray crystallography and was compared to native DVL and DRL. Therefore, DVL was co-crystallized with X-Man, and a molecular modeling with X-Man complexed with DVL was done to compare the complexed and native forms adjusted fit. The relatively narrow and deep CRD in DVL promotes little interaction with carbohydrates; in contrast, the wider and shallower CRD in DRL favors interaction. This seems to explain differences in the level of relaxation induced by DVL (43%) and DRL (96%) in rat aortic rings.
Assuntos
Dioclea/química , Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Vasodilatadores/química , Vasodilatadores/farmacologia , Sequência de Aminoácidos , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Cristalografia por Raios X , Técnicas In Vitro , Masculino , Manose/química , Manose/metabolismo , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Lectinas de Plantas/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Especificidade da Espécie , Vasodilatadores/metabolismoRESUMO
Two new lectins named Halilectin 1 (H-1) and Halilectin 2 (H-2) were isolated from the marine sponge Haliclona caerulea using a combination of affinity chromatography on stroma fixed onto Sephadex G-25 and cation and anion exchange chromatography. H-1 is a monomeric protein with a molecular mass of 40 kDa estimated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and 15 kDa estimated using a TSK gel. Conversely, H-2 is a homodimeric protein with 15 kDa monomers linked via weak interactions. H-1 more effectively agglutinates trypsinized rabbit erythrocytes, whereas H-2 more effectively agglutinates native rabbit erythrocytes. The hemagglutinating activity of H-1 could be not inhibited by any tested sugars, but H-2 was inhibited by orosomucoid and porcine stomach mucin. Neither lectin was dependent on divalent ions. H-1 was stable at basic pH range and temperatures up to 50 °C, whereas H-2 was stable at acid pH range and temperatures up to 80 °C. The H. caerulea lectins exhibited dose-dependent toxicity against Artemia nauplii. Additionally, 76% of the primary structure of H-2 was determined using tandem mass spectrometry to contain a unique amino acid sequence with no similarity to any members of the animal lectin family.