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 β-mercaptoethanol, two non-identical bands of M r 24 and 37 kDa were observed, whereas in the absence of β-mercaptoethanol, the lectin yielded a single band corresponding to ∼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% α-helix, 36.7% β-sheet, 19.4% β-turns, and 31.6% unordered structure. Among a battery of sugars investigated, TDSL was inhibited strongly by β-d-galactopyranosides, with 4-methylumbelliferyl-β-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. © 2004 Elsevier Inc. All rights reserved.