RESUMO
KM+, a lectin purified from Artocarpusintegrifolia seeds, is an attractant for neutrophils, and has properties similar to fMLP, IL-8 and MNCF. The endogenous lectin MNCF, inhibits carrageenan-induced neutrophil migration when intravenously administered in rats. In an attempt to mimic the activity of MNCF with KM+, we determined the effect of intravenous (iv) injection of KM+ (5 microg) on neutrophil migration to the peritoneal cavity of Wistar rats induced by KM+ (50 microg, intraperitoneal, ip), fMLP (5 ng, ip) and carrageenan (300 microg, ip). Initially we evaluated the effect of the time interval between intravenous and intraperitoneal administration of KM+. The intervals ranged from 20 to 120 min and progressively stronger inhibition was observed with increasing time intervals up to a maximum of 60 min, with effect decreasing thereafter. With injections at the optimum interval of 60 min, we observed that KM+ inhibited KM+- and carrageenan-induced neutrophil migration by 72%, and fMLP-induced migration by 56%. White cell counts for Wistar rats that only received KM+iv, performed at 0 to 120 min intervals after injection, revealed early neutropenia lasting 60 min, followed by a marked increase in circulating neutrophils that reached a maximum of twice the initial levels within 90 min and after 120 min returned to levels near to that observed before intravenous administration of KM+. These results indicate that when KM+ is present in the intravascular space, it produces an inhibitory effect on neutrophil migration similar to that caused by the intravenous administration of other chemoattractants, regardless of whether they act through a mechanism independent of carbohydrate recognition, as does IL-8, or are dependent on carbohydrate recognition, like MNCF.
Assuntos
Quimiotaxia de Leucócito/efeitos dos fármacos , Lectinas/farmacologia , Neutrófilos/efeitos dos fármacos , Animais , Injeções Intravenosas , Lectinas/administração & dosagem , Contagem de Leucócitos , Masculino , Neutrófilos/citologia , Cavidade Peritoneal/citologia , Ratos , Ratos WistarRESUMO
A lectin was purified from seeds of Erythrina velutina forma aurantiaca by affinity chromatography on cross-linked guar gum. The lectin is a potent agglutinin for human (minimal concentration of protein able to cause visible agglutination of a 2% erythrocyte suspension varying from 1 to 4 micrograms/ml), rabbit (4 micrograms/ml) and chicken erythrocytes (8 micrograms/ml) but presented low activity against cow (250 micrograms/ml) or sheep (333 micrograms/ml) blood cells. Hemagglutination of human O+ erythrocytes was inhibited by D-lactose (0.2 mM) > D-galactose (0.8 mM) > D-raffinose (2.1 mM). At pH 7.5, chromatography on a Superose 12 HR 10/30 column showed that the lectin was primarily a dimer (56.0 kDa) composed of two identical subunits (31.6 kDa each). A small amount of a tetrameric form was also apparently present. The lectin is a glycoprotein (7.3% carbohydrate), has a pI of 4.5, contains high levels of acidic (Asp and Glu, 64.2 and 51.6 residues/mol, respectively) and hydroxy amino acids (Ser and Thr, 42.9 and 38.5 residues/mol, respectively) but relatively low amounts of sulfur amino acids (Cys and Met, 1.0 and 5.0 residues/mol, respectively) and has an N-terminal sequence of Val-Glu-Thr-Ile/Leu-Pro-Phe-Ser. Its hemagglutinating activity was abolished by heating at 70 degrees C for 10 min. The activation energy (delta G') required for denaturation measured by loss of hemagglutination activity was 24.87 kcal/mol. In rats, the purified lectin (100 micrograms) induced neutrophil migration into the peritoneal cavity (3.7 +/- 0.6 x 10(6) neutrophils/ml) or into the air pouch (2.75 +/- 0.25 x 10(6) neutrophils/ml), 8 and 10 times greater than the negative control, respectively.
Assuntos
Erythrina/química , Lectinas/química , Plantas Medicinais , Animais , Brasil , Humanos , Lectinas/isolamento & purificação , Lectinas de Plantas , Ratos , Ratos Wistar , Sementes/químicaRESUMO
A lectin was purified from seeds of Erythrina velutina forma aurantiaca by affinity chromatography on cross-linked guargum. The lectin is a potent agglutinin for human (minimal concentration of protein able to cause visible agglutination of a 2 per cent erythrocyte suspension varying from 1 to 4 mug/ml), rabbit(4 mug/ml) and chicken erythrocytes (8 mug/ml) but presented low activity against cow (250 mug/ml) or sheep (333 mug/ml) blood cells. Hemagglutination of human O+ erythrocytes was inhibited by D-lactose (0.2 mM) > D-galactose(0.8 mM) > D-raffinose (2.1 mM). At pH 7.5, chromatography on a Superose 12 HR 10/30 column showed that the lectin was primarily a dimer (56.0 kDa) composed of two identical subunits (31.6 kDa each). A small amount of a tetrameric form was also apparently present. The lectin is a glycoprotein (7.3 per cent carbohydrate), has a pI of 4.5, contains high levels of acidic (Asp and Glu, 64.2 and 51.6 residues/mol, respectively) and hydroxy amino acids (Ser and Thr, 42.9 and 38.5 residues/mol, respectively) but relatively low amounts of sulfur amino acids (Cys and Met, 1.0 and 5.0 residues/mol, respectively) and has an N-terminal sequence of Val-Glu-Thr-Ile/Leu-Pro-Phe-Ser. Its hemagglutinating activity was abolished by heating at 70 degrees Celsius for 10 min. The activation energy (delta G') required for denaturation measured by loss of hemagglutination activity was 24.87 kcal/mol. In rats, the purified lectin (100 mug) induced neutrophil migration into the peritoneal cavity (3.7 ñ 0.6 x 10(6) neutrophils/ml) or into the air pouch (2.75 ñ 0.25 x 10(6) neutrophils/ml), 8 and 10 times greater than the negative control, respectively.
Assuntos
Humanos , Animais , Ratos , Erythrina/química , Lectinas/química , Brasil , Lectinas/isolamento & purificação , Ratos Wistar , Sementes/químicaRESUMO
A neutrophil migration-inducing protein has been isolated from the saline extract of Artocarpus integrifolia seeds by successive sugar affinity chromatography steps during which the protein was not absorbed by D-galactose resin, and then was absorbed to and eluted from D-mannose resin by 0.1 M D-mannose. Gel filtration on Superdex 75 HR indicated a molecular mass of 52 kDa when 0.1 M D-mannose was present in the elution buffer. A single band of apparent molecular mass of 13 kDa was demonstrable by SDS-PAGE only after heating, both in the presence and absence of reducing agent, suggesting that the molecule is a tetramer formed by the noncovalent association of 13 kDa chains. Isoelectric forms corresponding to isoelectric points of 4.0, 4.2, 5.0, and 5.2 were demonstrable by isoelectric focusing-PAGE, and four active forms having the same isoelectric points were separated by chromatofocusing. The minimal m.w. calculated from amino acid analysis data was 13,193. The protein, denoted KM+, stimulated neutrophil migration in the rat peritoneal cavity assay in a dose-related manner in the range of 1 to 300 micrograms per rat. The dose-response curve of the in vitro chemotactic activity of KM+ was bell shaped and its ascending limb was dose dependent in the range of 1 ng to 10 micrograms/well. D-Mannose (0.1 M) inhibited the in vitro (80%) and in vivo (60%) neutrophil migration-inducing activities of KM+ and also its hemmaglutinating activity. The chemotactic activity was shown to be caused by haptotaxis rather than chemokinesis. The physical and biologic properties of KM+ suggest that this lectin may attract neutrophils by a mechanism involving a haptotactic gradient as has been proposed for IL-8. KM+ might be used as tool to study protein-carbohydrate interactions during neutrophil migration through the extracellular matrix.