Purification, Physicochemical Characterization, Saccharide Specificity, and Chemical Modification of a Gal/GalNAc Specific Lectin From the Seeds of Trichosanthes Dioica

Arch Biochem Biophys. 2004 Dec 15;432(2):212-21. doi: 10.1016/j.abb.2004.09.016.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Antigens, Tumor-Associated, Carbohydrate / chemistry*
  • Binding Sites
  • Carbohydrate Conformation
  • Carbohydrates / chemistry
  • Dimerization
  • Disaccharides / chemistry
  • Galactose / chemistry*
  • Macromolecular Substances / chemistry
  • Molecular Weight
  • Plant Lectins / analysis
  • Plant Lectins / chemistry*
  • Plant Lectins / isolation & purification
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Tertiary
  • Seeds / metabolism*
  • Species Specificity
  • Structure-Activity Relationship
  • Substrate Specificity
  • Trichosanthes / classification
  • Trichosanthes / metabolism*

Substances

  • Antigens, Tumor-Associated, Carbohydrate
  • Carbohydrates
  • Disaccharides
  • Macromolecular Substances
  • Plant Lectins
  • Thomsen-Friedenreich antigen
  • Galactose