International journal of biological macromolecules, Jan 10, 2016
A lectin from Canavalia virosa, Diocleinae subtribe, was purified by affinity chromatography with... more 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, indicat...
A glycosylated lectin (CTL) with specificity for mannose and glucose has been detected and purifi... more 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.
International journal of biological macromolecules, Jan 21, 2015
Lectins are proteins that show a variety of biological activities. However, they share in common ... more Lectins are proteins that show a variety of biological activities. However, they share in common at least one domain capable of recognizing specific carbohydrates reversibly without changing its structure. The legume lectins family is the most studied family of plant lectins, in particular the Diocleinae subtribe, which possesses high degree of structural similarity, but variable biological activities. This variability lies in small differences that can be analyzed in studies based on structures. In particular, Dioclea sclerocarpa seed lectin (DSL) presents low ability to relax endothelialized rat aorta in comparison with other Dioclea lectins such as D. violacea (DVL), D. virgata (DvirL) and D. rostrata (DRL). The DSL relaxation mechanism relies on nitric oxide production and carbohydrate recognition domain (CRD). This behavior can be explained by structural differences, since DSL has a carbohydrate recognition domain design less favorable. In addition, the presence of a glutamate ...
Este trabalho mostra o efeito da vibração térmica na densidade eletrônica de cristais bidimension... more Este trabalho mostra o efeito da vibração térmica na densidade eletrônica de cristais bidimensionais, usando o programa Mathematica. É apresentado um modelo de cristal simples para ajudar no ensino de conceitos básicos relacionados à cristalografia.
The present work reports our succesfull experience concerning crystallization of four fish hemogl... more The present work reports our succesfull experience concerning crystallization of four fish hemoglobins from three Brazilian species of Teleosts: Liposarcus anisitsi, Brycon cephalus and Piaractus mesopotamicus. The data shown here is part of a systematic functional and structural study of fish hemoglobins with the aim of better understanding the outstanding range of functional and structural properties exhibited by these proteins. We also present a reduced sparse-matrix method for crystallization of fish hemoglobins, which can reduce the amount of hemoglobin initially used in the crystallization experiments.
The Abelmoschus esculentus (Malvaceae) plant originated in Africa and has spread across a number ... more The Abelmoschus esculentus (Malvaceae) plant originated in Africa and has spread across a number of tropic countries, including northeastern Brazil. The plant has been used to treat various disorders, such as cancer, microbial infections, hypoglycemia, constipation, urine retention and inflammation. The lectin of A. esculentus (AEL) was isolated by precipitation with ammonium sulfate at a saturation level of 30/60 and purified by ion exchange chromatography (Sephacel-DEAE). The electrophoresis (SDS-PAGE) profile of the AEL showed two protein bands of apparent molecular mass of approximately 15.0 and 21.0 kDa. The homogenity of the protein was confirmed by electrospray mass spectrometry (ESI-MS), which revealed the presence of a 10.29-kDa monomer and a 20.58-kDa dimer. The AEL exhibits agglutinating activity against rabbit (74.41 UH/mP) and human type ABO erythrocytes (21.00 UH/mP). This activity does not require the presence of divalent cations and is specifically inhibited by lactose, fructose and mannose. The intravenous treatment with 0.01, 0.1 and 1 mg/kg of AEL inhibited the paw edema elicited by carrageenan by approximately 15, 22 and 44 %, respectively, but not that induced by dextran. In addition, treatment with 0.1, 1 and 10 mg/kg of AEL also inhibited the abdominal writhing induced by acetic acid by approximately 52, 57 and 69 %, respectively. In conclusion, AEL is a new lectin with a molecular mass of 20.0 kDa, which is -composed of a 10.291-Da monomer and a 20.582-kDa dimer, that exhibits anti-inflammatory, antinociceptive and hemagglutinating activities. In addition, the lectin hemagglutinating property is both metallo-independent and associated with the lectin domain.
Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) see... more Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP‐HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407 ± 15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N‐acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed β(1–4) glycosidic bonds linking 2‐acetoamido‐2‐deoxy‐β‐d‐glucopyranose units in chitin. The full‐length amino acid sequence of Parkia platycephala lectin 2, determined by N‐terminal sequencing and cDNA cloning, and its three‐dimensional structure, established by X‐ray crystallography at 1.75 Å resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which...
The simulations presented here are based on the programMathematicaas a tool to present electron d... more The simulations presented here are based on the programMathematicaas a tool to present electron density maps of two-dimensional crystal structures. The models give further insights into the relationship between the thermal displacement parameters and the quality of the electron density maps. Furthermore, users can readily test the effects of several crystallographic parameters on the electron density maps, such as, the number of reflections, the thermal displacement parameters and the unit-cell dimensions.
International journal of biological macromolecules, Jan 10, 2016
A lectin from Canavalia virosa, Diocleinae subtribe, was purified by affinity chromatography with... more 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, indicat...
A glycosylated lectin (CTL) with specificity for mannose and glucose has been detected and purifi... more 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.
International journal of biological macromolecules, Jan 21, 2015
Lectins are proteins that show a variety of biological activities. However, they share in common ... more Lectins are proteins that show a variety of biological activities. However, they share in common at least one domain capable of recognizing specific carbohydrates reversibly without changing its structure. The legume lectins family is the most studied family of plant lectins, in particular the Diocleinae subtribe, which possesses high degree of structural similarity, but variable biological activities. This variability lies in small differences that can be analyzed in studies based on structures. In particular, Dioclea sclerocarpa seed lectin (DSL) presents low ability to relax endothelialized rat aorta in comparison with other Dioclea lectins such as D. violacea (DVL), D. virgata (DvirL) and D. rostrata (DRL). The DSL relaxation mechanism relies on nitric oxide production and carbohydrate recognition domain (CRD). This behavior can be explained by structural differences, since DSL has a carbohydrate recognition domain design less favorable. In addition, the presence of a glutamate ...
Este trabalho mostra o efeito da vibração térmica na densidade eletrônica de cristais bidimension... more Este trabalho mostra o efeito da vibração térmica na densidade eletrônica de cristais bidimensionais, usando o programa Mathematica. É apresentado um modelo de cristal simples para ajudar no ensino de conceitos básicos relacionados à cristalografia.
The present work reports our succesfull experience concerning crystallization of four fish hemogl... more The present work reports our succesfull experience concerning crystallization of four fish hemoglobins from three Brazilian species of Teleosts: Liposarcus anisitsi, Brycon cephalus and Piaractus mesopotamicus. The data shown here is part of a systematic functional and structural study of fish hemoglobins with the aim of better understanding the outstanding range of functional and structural properties exhibited by these proteins. We also present a reduced sparse-matrix method for crystallization of fish hemoglobins, which can reduce the amount of hemoglobin initially used in the crystallization experiments.
The Abelmoschus esculentus (Malvaceae) plant originated in Africa and has spread across a number ... more The Abelmoschus esculentus (Malvaceae) plant originated in Africa and has spread across a number of tropic countries, including northeastern Brazil. The plant has been used to treat various disorders, such as cancer, microbial infections, hypoglycemia, constipation, urine retention and inflammation. The lectin of A. esculentus (AEL) was isolated by precipitation with ammonium sulfate at a saturation level of 30/60 and purified by ion exchange chromatography (Sephacel-DEAE). The electrophoresis (SDS-PAGE) profile of the AEL showed two protein bands of apparent molecular mass of approximately 15.0 and 21.0 kDa. The homogenity of the protein was confirmed by electrospray mass spectrometry (ESI-MS), which revealed the presence of a 10.29-kDa monomer and a 20.58-kDa dimer. The AEL exhibits agglutinating activity against rabbit (74.41 UH/mP) and human type ABO erythrocytes (21.00 UH/mP). This activity does not require the presence of divalent cations and is specifically inhibited by lactose, fructose and mannose. The intravenous treatment with 0.01, 0.1 and 1 mg/kg of AEL inhibited the paw edema elicited by carrageenan by approximately 15, 22 and 44 %, respectively, but not that induced by dextran. In addition, treatment with 0.1, 1 and 10 mg/kg of AEL also inhibited the abdominal writhing induced by acetic acid by approximately 52, 57 and 69 %, respectively. In conclusion, AEL is a new lectin with a molecular mass of 20.0 kDa, which is -composed of a 10.291-Da monomer and a 20.582-kDa dimer, that exhibits anti-inflammatory, antinociceptive and hemagglutinating activities. In addition, the lectin hemagglutinating property is both metallo-independent and associated with the lectin domain.
Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) see... more Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP‐HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407 ± 15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N‐acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed β(1–4) glycosidic bonds linking 2‐acetoamido‐2‐deoxy‐β‐d‐glucopyranose units in chitin. The full‐length amino acid sequence of Parkia platycephala lectin 2, determined by N‐terminal sequencing and cDNA cloning, and its three‐dimensional structure, established by X‐ray crystallography at 1.75 Å resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which...
The simulations presented here are based on the programMathematicaas a tool to present electron d... more The simulations presented here are based on the programMathematicaas a tool to present electron density maps of two-dimensional crystal structures. The models give further insights into the relationship between the thermal displacement parameters and the quality of the electron density maps. Furthermore, users can readily test the effects of several crystallographic parameters on the electron density maps, such as, the number of reflections, the thermal displacement parameters and the unit-cell dimensions.
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