title: Purification, physicochemical characterization, saccharide specificity, and chemical modification of a Gal/GalNAc specific lectin from the seeds of Trichosanthes dioica.
abstract: A new galactose-specific lectin has been purified from the extracts of Trichosanthes dioica seeds by affinity chromatography on cross-linked guar gum. The purified lectin (T. dioica seed lectin, TDSL) moved as a single symmetrical peak on gel filtration on Superose-12 in the presence of 0.1 M lactose with an M(r) of 55 kDa. In the absence of ligand, the movement was retarded, indicating a possible interaction of the lectin with the column matrix. In SDS-PAGE, in the presence of beta-mercaptoethanol, two non-identical bands of M(r) 24 and 37 kDa were observed, whereas in the absence of beta-mercaptoethanol, the lectin yielded a single band corresponding to approximately 55,000 Da, indicating that the two subunits of TDSL are connected by one or more disulfide bridges. TDSL is a glycoprotein with about 4.9% covalently bound neutral sugar. Analysis of near-UV CD spectrum by three different methods (CDSSTR, CONTINLL, and SELCON3) shows that TDSL contains 13.3% alpha-helix, 36.7% beta-sheet, 19.4% beta-turns, and 31.6% unordered structure. Among a battery of sugars investigated, TDSL was inhibited strongly by beta-d-galactopyranosides, with 4-methylumbelliferyl-beta-d-galactopyranoside being the best ligand. Chemical modification studies indicate that tyrosine residues are important for the carbohydrate-binding and hemagglutinating activities of the lectin. A partial protection was observed when the tyrosine modification was performed in the presence of 0.2 M lactose. The tryptophan residues of TDSL appear to be buried in the protein interior as they could not be modified under native conditions, whereas upon denaturation with 8 M urea two Trp residues could be selectively modified by N-bromosuccinimide. The subunit composition and size, secondary structure, and sugar specificity of this lectin are similar to those of type-2 ribosome inactivating proteins, suggesting that TDSL may belong to this protein family.
authors.affiliation: School of Chemistry, University of Hyderabad, Hyderabad 500 046, India.
authors.forename: Nabil Ali Mohammed
authors.initials: NA
authors.lastname: Sultan
authors.forename: Roopa
authors.initials: R
authors.lastname: Kenoth
authors.forename: Musti J
authors.initials: MJ
authors.lastname: Swamy
mesh_terms: D019943:Amino Acid Substitution; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000737:chemistry*; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002241:Carbohydrates / Q000737:chemistry; D019281:Dimerization; D004187:Disaccharides / Q000737:chemistry; D005690:Galactose / Q000737:chemistry*; D046911:Macromolecular Substances / Q000737:chemistry; D008970:Molecular Weight; D037121:Plant Lectins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012639:Seeds / Q000378:metabolism*; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D029778:Trichosanthes / Q000145:classification / Q000378:metabolism*
chemical_list: D015295:Antigens, Tumor-Associated, Carbohydrate; D002241:Carbohydrates; D004187:Disaccharides; D046911:Macromolecular Substances; D037121:Plant Lectins; C016467:Thomsen-Friedenreich antigen; D005690:Galactose