Galectins Bind to the Multivalent Glycoprotein Asialofetuin with Enhanced Affinities and a Gradient of Decreasing Binding Constants

Abstract

Our previous isothermal titration microcalorimetry (ITC) studies of the binding of synthetic multivalent carbohydrates to the Man/Glc-specific lectins concanavalin A (ConA) and Dioclea grandiflora lectin (DGL) showed negative binding cooperativity that was due to the carbohydrate ligands and not the proteins [Dam, T. K., et al. (2002) Biochemistry 41, 1351-1358]. The negative cooperativity was associated with the decreasing functional valence of the carbohydrates upon progressive binding of their epitopes. The present study also shows negative cooperativity in the ITC binding data of asialofetuin (ASF), a glycoprotein that possesses nine LacNAc epitopes, to galectin-1, -2, -3, -4, -5, and -7, and truncated, monomer versions of galectin-3 and -5, which are members of a family of animal lectins. Although the observed K(a) values for binding of ASF to the galectins and two truncated forms are only 50-80-fold greater than that of LacNAc, analysis of the data in terms of the relationship between the observed macroscopic free energy of binding and the decreasing microscopic free energies of binding of the epitopes shows that the first LacNAc epitope of ASF binds with approximately 6000-fold higher affinity than the last epitope. Thus, the microscopic binding constants of the galectins for the first epitope(s) of ASF are in the nanomolar range, with a gradient of decreasing binding constants of the remaining epitopes. The results indicate that the above galectins bind with fractional, high affinities to multivalent glycoproteins such as ASF, independent of the quaternary structures of the galectins. These findings have important implications for the binding of galectins to multivalent carbohydrate receptors.

Assessing the inhibitory potency of galectin ligands identified from combinatorial (glyco)peptide libraries using surface plasmon resonance spectroscopy

“…Therefore, an SPR-based competition assay was set up with asialofetuin as ligand to establish that the (glyco)peptides identified from the library screenings are able to inhibit carbohydrate-dependent binding. Note that asialofetuin is a pan-galectin ligand, with its reactivity being dependent on binding site occupancy exhibiting a gradient of decreasing binding constants [23]. The glycoprotein was immobilized on a flow cell of a CM5 chip (106 RU), and an untreated flow cell was used as the reference surface for h-Gal-1.…”

Functions of cell surface galectin-glycoprotein lattices

“…Galectins preferentially bind β-galactoside-containing glycans comprised of repeating units of N-acetyllactosamine (Galβ1,4GlcNAc; LacNAc), either as disaccharide units at the termini of complex N-glycans, or as repeating units in a poly-N-acetyllactosamine chain on N-or O-glycans [5,7,8]. Galectin binding affinities to complex N-glycans are proportional to their LacNAc content and to their GlcNAc branching [5,[7][8][9][10][11].…”

“…While galectin binding to a single saccharide ligand is typically a low-affinity interaction (association constants ~10 4 M −1 ), the multivalent nature of galectin-saccharide interactions results in high overall avidity (association constants ~10 6 M −1 ) [5,9]. This multivalency also allows the formation of lectin-carbohydrate lattices.…”

Evaluation of Riproximin Binding Properties Reveals a Novel Mechanism for Cellular Targeting

“…When the interactions of riproximin with each of the proteins ASF and aBSM is considered, the main features of the mechanism of lectins binding to multivalent targets apply (19,20). On multivalent structures, several equivalent epitopes are available for the so-called bind-and-jump effects; the lectin can be recaptured by each of the remaining free epitopes, leading to decreased dissociation.…”

“…The mechanism of binding of lectins to multivalent globular and linear glycoproteins was recently elucidated (18). Both the interaction of Gal-binding galectins with multiple NA3 structures on ASF (19) and that of GalNAc-binding soybean agglutinin with Tn on Tn-rich porcine submaxillary mucin (20) were described as depending on bind-and-jump and negative cooperativity effects. Using similar model glycoproteins, the mechanism of riproximin interaction with its glycotargets and implications for its cytotoxicity were investigated.…”