Isolation from lima bean lectin of a peptide containing a cysteine residue essential for carbohydrate binding activity.
The location and amino acid sequence surrounding a cysteine residue required for carbohydrate binding in the lima bean lectin (LBL) was determined. Following selective conversion of the sulfhydryl group to its S-cyano derivative, LBL was cleaved at the essential cysteine residue to give two fragments, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in two buffer systems to have molecular masses of 16.5-19 kDa and 10.5-11 kDa. The larger fragment, which contained the glycosyl moiety of the lectin, was shown by sequence analysis to contain the NH2-terminal sequence of LBL. The smaller COOH-terminal fragment was found to contain the cysteine residue involved in the intersubunit disulfide bond of LBL. Digestion of LBL with pepsin and trypsin yielded four peptides containing the essential cysteine. Sequencing of the three major peptides gave a single consensus sequence, Val-Glu-Phe-Asp-Thr-Cys-His-Asn-Leu-Asp-, for the primary sequence surrounding the cysteine. The peptide sequence and site of cyanylation cleavage were used to predict alignment of the LBL peptide with the primary sequence of concanavalin A. Maximum homology was found with a sequence in concanavalin A beginning at valine 7. Implications of this alignment to the function of the cysteine in carbohydrate and metal ion binding of LBL, and for conservation of carbohydrate binding site residues in legume lectins are discussed.
D000595:Amino Acid Sequence; D050260:Carbohydrate Metabolism; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D037102:Lectins / Q000032:analysis*; D010446:Peptide Fragments / Q000302:isolation & purification* / Q000378:metabolism; D037121:Plant Lectins
D037102:Lectins; D010446:Peptide Fragments; D037121:Plant Lectins; C031887:lima bean lectin; D003545:Cysteine