Purification, characterization, and preliminary X-ray diffraction analysis of a lactose-specific lectin from Cymbosema roseum seeds.
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.
Rocha
Bruno A M
BA
BioMol-Lab, Departmento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, P. O. Box 6043, 60.455-970, Fortaleza, Ceará, Brazil.
Moreno
Frederico B M B
FB
Delatorre
Plínio
P
Souza
Emmanuel P
EP
Marinho
Emmanuel S
ES
Benevides
Raquel G
RG
Rustiguel
Joane Kathelen Rodrigues
JK
Souza
Luis A G
LA
Nagano
Celso S
CS
Debray
Henri
H
Sampaio
Alexandre H
AH
de Azevedo
Walter F
WF
Cavada
Benildo S
BS
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000737:chemistry*; D006384:Hemagglutination; D006801:Humans; D007785:Lactose / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010802:Phylogeny; D037121:Plant Lectins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D012639:Seeds / Q000737:chemistry*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D053719:Tandem Mass Spectrometry
D010455:Peptides; D037121:Plant Lectins; D007785:Lactose