Purification, Characterization, and Preliminary X-Ray Diffraction Analysis of a Lactose-Specific Lectin from Cymbosema roseum Seeds

Abstract

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.

Purification and characterization of a highly thermostable GlcNAc-binding lectin from Collaea speciosa seeds

“…However, not all lectins from the Diocleinae subtribe can be purified using the same protocols. For example, purification of the mannose/glucose-specific lectins from Cratylia mollis (Iso1, Iso 2 and Iso 3) [35,36], Cymbosema roseum (CRL I and CRL II) [37,38] and lectins from Camptosema pedicellatum [39], as well as the lactose-specific lectin from Camptosema ellipticum [40], all required multiple chromatographic steps, including ion exchange chromatography and gel filtration, in addition to the affinity chromatography.…”

Purification and molecular characterization of a novel mannose‐specific lectin from Dioclea reflexa hook seeds with inflammatory activity

“…However, lectins isolated from other plant components, such as bulb and rhizome, have also been reported (Loris et al ., ; Prasanna and Venkatesh, ; Xiao et al ., ; Albuquerque et al ., ). Leguminous seeds are a rich source of lectins, and several genera of the subtribe Diocleinae (tribe Phaseoleae ) have been the main source for isolation and characterization of novel lectins (Cavada et al ., ; Peumans and Van Damme, Correia et al ., ; Van Damme et al ., ; Rocha et al ., ). Typically, Diocleinae lectins share the following patterns: (1) composed of two or four monomers linked by noncovalent interactions; (2) high homology; (3) molecular mass of 25–30 kDa, in which each monomer presents a unique, highly conserved carbohydrate‐binding site with specificity for mannose/glucose; and (4) common physicochemical properties (Brinda et al ., ).…”

Affinity Chromatography of Lectins

“…Recently, isolation of lectins from various sources has been described using numerous sugars, such as lactose (Almanza et al, 2004;Braga et al, 2006;Fujii et al, 2011;Hamako et al, 2007;Kawsar et al, 2009;Mendonça-Franqueiro et al, 2011;Naeem et al, 2007b;Pohleven et al, 2009;Rocha et al, 2009;Silva et al, 2007), sucrose and glucose (Pohleven et al, 2011), galactose (Konami et al, 1991;Lavanya Latha et al, 2006;Mo et al, 2000;Moreira et al, 1998;Naeem et al, 2007b;Nagata, 2005), mannose (Andon et al, 2003;Ooi et al, 2010;Suseelan et al, 2002), fucose (Cammarata et al, 2007;Mansour & Abdul-Salam, 2009), L-rhamnose (Jimbo et al, 2007;Watanabe et al, 2008), and sugars with 2'-acetamido groups N-acetylgalactosamine (Gerlach et al, 2005;Perçin et al, 2009;Qureshi et al, 2006), N-acetylglucosamine (Kaur et al, 2005;Kim et al, 2006;Maheswari et al, 2002;Wang & Ng, 2003), and N,N'-diacetylchitobiose (Konami et al, 1991;Koyama et al, 2002).…”