Purification and Characterization of an N-Acetyl-D-Glucosamine Binding Lectin from Datura Stramonium Seeds.

Abstract

This study reports the purification and properties of an N-acetyl-D-glucosamine binding lectin from the seeds of Datura stramonium inermis. Purification was accomplished by dialysis of a saline extract of the seeds against 0.01 M acetic acid, followed by affinity chromatography on an N,N'-diacetyl-(beta)-chitobioside - Sepharose column. A fraction of the affinity-purified lectin (40%) required further purification by gel filtration on Sephadex G-200 to remove trace amounts of a contaminating glycoprotein. The purified Datura lectin appeared homogeneous by sedimentation analysis, gel filtration, electrophoresis under both native and denaturing conditions, and immunoelectrophoresis. No trace of the protein of molecular weight 32,000 which has contaminated previously reported lectin preparations was detected. The physical properties of Datura lectin, including its amino acid and carbohydrate composition, molecular weight, and spectral properties in the presence and absence of haptenic sugar, were determined. The lectin was shown to be a dimeric glycoprotein composed of 2 nonidentical subunits (M(,r) 40,000 and 46,000) joined by disulfide bonds. There are 2 sugar-binding sites per mole lectin. The specificity of the lectin binding site was probed by sugar inhibition of the lectin - asialofetuin precipitation system. On the basis of these studies, the binding site was proposed to consist of 3 subsites, designated A, B, and C. Each subsite can accommodate a single (beta)-(1-4) linked N-acetyl-D-glucosamine residue. The (beta)-(1-4) linkage, and the presence of N-acetamido groups (in the gluco-configuration) at subsites A and C, but not B, are obligatory for binding to occur. Preliminary evidence was obtained by UV difference spectroscopy and chemical modification experiments for the involvement of tyrosinyl and tryptophanyl, and the non-participation of amino, carboxyl, histidyl, methionyl, and cysteinyl residues in the binding activity of Datura lectin.