The thermodynamics of carbohydrate binding to the 14 kDa dimeric beta-galactoside-binding lectin galectin-1 (Gal-1) from Chinese hamster ovary cells and four galactose-specific plant lectins were investigated by isothermal titration microcalorimetry. Recombinant Gal-1 from Escherichia coli, a Cys-->Ser mutant with enhanced stability (C2S-Gal-1), and a monomeric mutant of the lectin (N-Gal-1) were studied along with the soybean agglutinin and the lectins from Erythrina indica, Erythrina crystagalli, and Erythrina corollodendrum. Although the pattern of association constants of the Erythrina lectins was similar for mono- and disaccharides, variations exist in their enthalpy of binding (-delta H) values for individual carbohydrates. While the Erythrina lectins show greater affinities and -delta H values for lactose and N-acetyllactosamine, the soybean agglutinin possesses similar affinities for methyl beta-galactopyranoside, lactose, and N-acetyllactosamine and a greater -delta H value for the monosaccharide. Gal-1 and the plant lectins possess essentially the same affinities for N-acetyllactosamine; however, the animal lectin shows a lower -delta H value and more favorable binding entropy for the disaccharide. While Gal-1, C2S-Gal-1, and N-Gal-1 all possess essentially the same affinities for N-acetyllactosamine, the two mutants possess much lower -delta H values, even though the mutation site(s) are far removed from the carbohydrate binding site. These results indicate that there are different energetic mechanisms of carbohydrate binding between galectin-1, its two mutants, and the Gal-specific plant lectins.

The existence of two conformational states of concanavalin A (Con A) with different metal ion binding properties has been recently demonstrated (Brown, R. D., Brewer, C. F., & Koenig, S. H. (1977) Biochemistry 16, 3883). Introduction of Mn2+ to the S1 site and Ca2+ to the S2 site of apo-Con A was shown to induce a conformational change in the protein, ascribed to a cis-trans isomerization of a peptide bond in the secondary structure, which results in extremely tight binding of the metal ions. This induced conformation is referred to as "locked" and the initial conformation as "unlocked". The locked ternary complex is identical with the native protein. In the present paper, we report evidence for the formation of a relatively stable, locked, ternary Ca2+-Con A complex that possesses properties similar to those of native Ca2+-Mn2+Con A. The experimental technique involves measurement of the magnetic field and time dependence of the nuclear magnetic relaxation rate (1/T1) of solvent water protons in solutions of Ca2+-Con A, after the addition of Mn2+ ion which slowly bind to the protein. The kinetic data can be fit by a model for Ca2+ interactions with Con A which indicates that Ca2+, in the absence of Mn2+, can bind at both the S1 and S2 sites of the protein and, furthermore, can induce the protein to undergo the unlocked to locked conformational transition. In terms of this model, the time-dependent binding of the Mn2+ ions is due to replacement of Ca2+ ions at the S1 sites in the locked protein. The off-rate of Ca2+ from the S2 site of the locked ternary Ca2+-Con A complex is much greater than that from the locked Ca2+-Mn2+-Con A complex. From the effects of added alpha-methyl D-mannopyranoside on the rate of replacement of Ca2+ by Mn2+ at the S1 site of the locked ternary Ca2+-Con A complex, it is concluded that the latter complex binds saccharides as strongly as the locked Ca2+-Mn2+-Con A complex. In addition, analysis of the data indicates that apo-Con A in the locked conformation binds alpha -methyl D-mannopyranoside with approximately 7% of the affinity of the fully metallized locked form of the protein. This strong saccharide-binding activity of locked apo-Con A, compared with that of the unlocked apo-Con A, was further demonstrated by equilibration of unlocked apo-Con A with alpha-methyl D-mannopyranoside, which resulted in the formation of the locked apo-Con A-saccharide complex. These results demonstrate that it is the locked conformation of Con A that is primarily responsible for saccharide-binding activity, and that the function of the bound metals is primarily to maintain the protein in the locked conformation.

We have recently demonstrated that certain oligomannose and bisected hybrid type glycopeptides and bisected complex type oligosaccharides are bivalent for binding to concanavalin A and can precipitate the lectin [Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., & Brewer, C.F. (1987) J. Biol. Chem. 262, 1288-1293; Bhattacharyya, L., Haraldsson, M., & Brewer, C.F. (1987) J. Biol. Chem. 262, 1294-1299]. The present results show that tri- and tetraantennary complex type oligosaccharides containing nonreducing terminal galactose residues, and a related triantennary glycopeptide, precipitate the D-galactose-specific lectins from Ricinus communis (agglutinin I) (RCA-I), Erythrina indica (EIL), Erythrina arborescens (EAL), and Glycine max (soybean) (SBA). Nonbisected and bisected biantennary complex type oligosaccharides can precipitate SBA, which is a tetrameric lectin, but not RCA-I, EIL, or EAL, which are dimeric lectins. The relative affinities of the oligosaccharides and glycopeptide were determined by hemagglutination inhibition measurements and their valencies by quantitative precipitin analyses. The equivalence points of the precipitin curves indicate that the tri- and tetraantennary oligosaccharides are tri- and tetravalent, respectively, for EIL, EAL, and SBA binding. However, the oligosaccharides are all trivalent for RCA-I binding due apparently to the larger size of the monomeric subunit of the lectin. The triantennary glycopeptide was also trivalent for RCA-I and EIL binding. Biantennary oligosaccharides with adequate chain lengths were found to be bivalent for binding to SBA; those with shorter chains did not precipitate the lectin.(ABSTRACT TRUNCATED AT 250 WORDS)

We have previously demonstrated that the interactions between branched chain oligosaccharides and glycopeptides isolated from glycoproteins and glycolipids with specific lectins lead to the formation of homopolymeric carbohydrate-protein cross-linked complexes, even in the presence of mixtures of the carbohydrates or lectins [cf. Bhattacharyya, L., Fant, J., Lonn, H., & Brewer, C. F. (1990) Biochemistry 29, 7523-7530]. Recently, we have shown that highly ordered cross-linked lattices are formed between the tetrameric glycoprotein soybean agglutinin (SBA), which possesses a Man9 oligomannose chain per monomer, and the Glc/Man-specific plant lectin concanavalin A (Con A) [Khan, M. I., Mandal, D. K., & Brewer, C. F. (1991) Carbohydr. Res. 213, 69-77]. Using radiolabeling and quantitative precipitation techniques, we show in the present study that Con A binds and forms unique cross-linked complexes with four different glycoproteins having different numbers and types of carbohydrate chains as well as different quaternary structures. The glycoproteins include quail ovalbumin, Lotus tetragonolobus isolectin A (LTL-A), Erythrina cristagalli lectin (ECL), and Erythrina corallodendron lectin (EcorL). The results show that a preparation of quail ovalbumin containing either one Man7 or Man8 oligomannose chain per molecule forms a 1:2 cross-linked complex with tetrameric Con A, thereby demonstrating bivalency of the single carbohydrate chain(s) on the glycoprotein. Tetrameric LTL-A and dimeric ECL, which possess two xylose-containing carbohydrate chains per monomer, both form 1:2 and 1:1 cross-linked complexes (per monomer) of glycoprotein to lectin, depending on their relative ratios in solution. However, dimeric EcorL, which has the same carbohydrate structure and number of chains as ECL, forms only a 1:2 cross-linked complex with tetrameric Con A.(ABSTRACT TRUNCATED AT 250 WORDS)