RESUMO
The unique carbohydrate-binding property of lectins makes them invaluable tools in biomedical research. Here, we report the purification, partial primary structure, carbohydrate affinity characterization, crystallization, and preliminary X-ray diffraction analysis of a lactose-specific lectin from Cymbosema roseum seeds (CRLII). Isolation and purification of CRLII was performed by a single step using a Sepharose-4B-lactose affinity chromatography column. The carbohydrate affinity characterization was carried using assays for hemagglutination activity and inhibition. CRLII showed hemagglutinating activity toward rabbit erythrocytes. O-glycoproteins from mucine mucopolysaccharides showed the most potent inhibition capacity at a minimum concentration of 1.2 microg mL(-1). Protein sequencing by mass spectrometry was obtained by the digestion of CRLII with trypsin, Glu-C, and AspN. CRLII partial protein sequence exhibits 46% similarity with the ConA-like alpha chain precursor. Suitable protein crystals were obtained with the hanging-drop vapor-diffusion method with 8% ethylene glycol, 0.1 M Tris-HCl pH 8.5, and 11% PEG 8,000. The monoclinic crystals belong to space group P2(1) with unit cell parameters a = 49.4, b = 89.6, and c = 100.8 A.
Assuntos
Fabaceae/química , Lactose/metabolismo , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Sementes/química , Sequência de Aminoácidos , Animais , Cromatografia de Afinidade , Cristalização , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Hemaglutinação , Humanos , Dados de Sequência Molecular , Peptídeos/química , Filogenia , Lectinas de Plantas/isolamento & purificação , Coelhos , Alinhamento de Sequência , Análise de Sequência de Proteína , Espectrometria de Massas em TandemRESUMO
A lectin from Canavalia maritima seeds (ConM) was purified and submitted to crystallization experiments. The best crystals were obtained using the vapour-diffusion method at a constant temperature of 293 K and grew in 7 d. A complete structural data set was collected to 2.1 A resolution using a synchrotron-radiation source. The ConM crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 67.15, b = 70.90, c = 97.37 A. A molecular-replacement search found a solution with a correlation coefficient of 69.2% and an R factor of 42.5%. Crystallographic refinement is under way.