A MANNOSE-SPECIFIC LECTIN FROM VICIA-VILLOSA SEEDS

A lectin specific to mannose has been purified from Vicia villosa seed by (NH4)(2)SO4 fractionation, GalNAc-Sepharose and Man-Sepharose affi nity chromatography. It was defined as VVL(M), which showed a single b and on an acidic-PAGE stained with Coomassie brilliant blue. The molec ular weight of VVL(M) was 50 kDa as determined by gel filtration on Bi ogel P-100 column. The VVL(M) molecule consists of 2 distinct subunits with apparent molecular weight of 30 kDa and 22 kDa determined by SDS -PAGE. VVL(M) has at least four isolectins with similar haemagglutinat ing activity. Its extinction coefficient is calculated as A(1cm)(1) = 16.4 at 280 nm. Sugars could not be detected by phenol-sulfuric acid m ethod. The circular dichroism analysis at far UV indicated that VVL(M) was a beta-sheet-rich protein, and gave no alpha-helix, 69% beta-shee t, 14% beta-turn by Provencher and Glockner method. The lectin was inh ibited by alpha-methyl-D-mannose at 12.5 mM and glucose or GlcNAc at 5 0 mM. The carbohydrate binding specificity of VVL(M) was investigated by using affinity chromatography on a VVL(M)-Sepharose column. Among v arious Asn-linked oligosaccharides, core structure Man alpha 1-> 3(Man alpha 1 -> 6)Man beta 1 -> 4GlcNAc beta 1 -> 4GlcNAc(OT) were found t o have high affinity for VVL(M)-Sepharose. The antisera of VVL(M) did not produce precipitin fine with VVL(G) in agar double diffusion plate indicating no serological relationship between VVL, and VVL(G). Howev er VVL(M) showed similar immunodeterminants of some other lectins of m annose specificity such as Con A, PSL, LCA and VFL.