Glycosaminoglycan (GAG) binding proteins (GAGBPs), including growth factors, cytokines, morphogens, and extracellular matrix proteins, interact with both free GAGs and those covalently linked to proteoglycans. Such interactions modulate a variety of cellular and extracellular events, such as cell growth, metastasis, morphogenesis, neural development, and inflammation. GAGBPs are structurally and evolutionarily unrelated proteins that typically recognize internal sequences of sulfated GAGs. GAGBPs are distinct from the other major group of glycan binding proteins, lectins. The multifunctional human galectin-3 (Gal-3) is a β-galactoside binding lectin that preferentially binds to N-acetyllactosamine moieties on glycoconjugates. Here, we demonstrate through microcalorimetric and spectroscopic data that Gal-3 possesses the characteristics of a GAGBP. Gal-3 interacts with unmodified heparin, chondroitin sulfate-A (CSA), -B (CSB), and -C (CSC) as well as chondroitin sulfate proteoglycans (CSPGs). While heparin, CSA, and CSC bind with micromolar affinity, the affinity of CSPGs is nanomolar. Significantly, CSA, CSC, and a bovine CSPG were engaged in multivalent binding with Gal-3 and formed noncovalent cross-linked complexes with the lectin. Binding of sulfated GAGs was completely abolished when Gal-3 was preincubated with β-lactose. Cross-linking of Gal-3 by CSA, CSC, and the bovine CSPG was reversed by β-lactose. Both observations strongly suggest that GAGs primarily occupy the lactose/LacNAc binding site of Gal-3. Hill plot analysis of calorimetric data reveals that the binding of CSA, CSC, and a bovine CSPG to Gal-3 is associated with progressive negative cooperativity effects. Identification of Gal-3 as a GAGBP should help to reveal new functions of Gal-3 mediated by GAGs and proteoglycans.

The glycan epitopes of natural and synthetic glycoconjugates exist as covalent attachments of well-defined inner structures or scaffolds. Macromolecules such as proteins, peptides, lipids, and saccharides and synthetic structures serve as scaffolds of glycoconjugates. It is generally perceived that the biological activities of glycoconjugates are determined mainly by the attached glycans, while the seemingly inert inner scaffolds play a passive role by providing physical support to the attached glycan epitopes. However, our data show that scaffolds actively influence lectin recognition and can potentially modulate lectin-mediated signaling properties of glycoconjugates. Through in vitro experiments, we found that the scaffolds significantly altered the thermodynamic binding properties of the covalently attached glycan epitopes. When a free glycan was attached to a scaffold, its lectin binding entropy became more positive. The level of positive entropic gain was dependent on the types of scaffolds tested. For example, protein scaffolds of glycoproteins were found to generate more positive entropy of binding than synthetic scaffolds. Certain scaffolds were found to have limiting effects on glycoconjugate affinity. We also found that scaffold-bearing glycans with a similar affinity or an identical valence demonstrated different kinetics of lattice formation with lectins, when the scaffold structures were different. Our data support the view that scaffolds of glycoconjugates (i) help the covalently attached glycans become more spontaneous in lectin binding and (ii) help diversify the lattice forming or cross-linking properties of glycoconjugates.

Complex formation between Tg and Gal-3 through high affinity binding and the sensitivity of the complex to free Tg and Gal-3 can potentially influence their biological functions. Interactions between Tg and Gal-3 might also interfere with their clinical detection, the same way Tg autoantibody (TgAb) is reported to interfere with Tg assays. The data support a model of Gal-3-mediated homeostatic process of Tg.

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Glycan‐binding proteins are classified in two major but distinct groups: lectins and glycosaminoglycan (GAG)‐binding proteins (GAGBPs). The multifunctional human lectin galectin‐3 (Gal‐3) is a member of the first group. GAGBPs, on the other hand, include a variety of non‐lectin proteins such as growth factors, cytokines, morphogens and ECM (extracellular matrix) proteins. The general structures of the binding sites of lectins, their preferred glycan structures and their mode of action are fundamentally different from those of GAGBPs. A member of one group rarely possesses the characteristics of both groups. Our data, however, show that Gal‐3 is a notable exception to that rule. In the present study, we report several significant findings. First, calorimetric and spectroscopic data show for the first time that Gal‐3 behaves like a GAGBP. The lectin interacts with unmodified sulfated GAGs (heparin and chondroitin sulfates) and chondroitin sulfate proteoglycans (CSPGs) at physiological pH and NaCl concentration. Second, the binding of sulfated GAGs and CSPGs to Gal‐3 is not a result of non‐specific interactions, rather sulfated GAGs and CSPGs specifically bind to Gal‐3, apparently, via the LacNAc/lactose binding site of Gal‐3. Finally, while heparin and chondroitin sulfate B (dermatan sulfate) are found to be monovalent ligands of Gal‐3, chondroitin sulfate A (CSA), chondroitin sulfate C (CSC) and bovine CSPG engage in multivalent binding with Gal‐3. Such multivalent binding leads to the formation of noncovalent cross‐linked complexes, which is reversible by lactose and different sulfated GAGs. Hill plot analysis of calorimetric data show that the binding of CSA, CSC and bovine CSPG to Gal‐3 is associated with progressive negative cooperativity effects. The present study provides a foundation for discovering new functions of GAGs and CSPGs mediated by Gal‐3 [1].Support or Funding InformationNational Science FoundationMichigan Technological University