Determination of the carbohydrate-binding site of Bauhinia purpurea lectin by affinity chromatography.
To determine the carbohydrate-binding site of Bauhinia purpurea lectin (BPA), a D-galactose- and lactose-binding lectin, a peptide which interacts with lactose was purified from endoproteinase Asp-N digests of BPA by chromatography on a lactose-Sepharose column. It consists of nine amino acids and its amino acid sequence is Asp-Thr-Trp-Pro-Asn-Thr-Glu-Trp-Ser. A tryptic fragment with the ability to interact with lactose was also purified and found to contain this sequence, consisting of nine amino acids. This nonapeptide was aligned in a part of the metal-binding region conserved in all legume lectins. The chemical synthesis of the nonapeptide was carried out by a solid-phase method and the synthetic peptide showed a lactose-specific binding activity in the presence of calcium. A chimeric lectin gene was constructed using a cDNA coding BPA in which the nonapeptide sequence was replaced by the corresponding region of the alpha-D-mannose binding Lens culinaris lectins. Although BPA is specific for beta-D-galactose, the chimeric lectin expressed in Escherichia coli was found to bind alpha-D-mannosyl-bovine serum albumin and this binding was inhibited by D-mannose.
Yamamoto
K
K
Division of Chemical Toxicology and Immunochemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Konami
Y
Y
Osawa
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002241:Carbohydrates / Q000737:chemistry*; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D037102:Lectins / Q000737:chemistry*; D008358:Mannose / Q000494:pharmacology; D008969:Molecular Sequence Data; D037121:Plant Lectins; D010944:Plants / Q000737:chemistry*; D010957:Plasmids; D011485:Protein Binding; D012710:Serum Albumin, Bovine; D014357:Trypsin
D002241:Carbohydrates; D037102:Lectins; D037121:Plant Lectins; D012710:Serum Albumin, Bovine; D014357:Trypsin; D008358:Mannose