Modulation of Carbohydrate-Binding Characteristics and Conformational Features of the Tumor-Specific Lectin, Jacalin by Macromolecular Crowding

Protein-carbohydrate interactions play crucial roles in important biological processes, including cellular differentiation, cell-cell adhesion, mitogenicity, and microbial and viral infections. Our present understanding in this area is largely due to lectins, a unique class of carbohydrate-binding proteins. In view of their ability to differentiate between normal and tumor cells, as well as their potential applications in cancer diagnosis, prognosis, and therapy, it is important to comprehend how the <i>in vivo</i> crowding milieu can modulate the structural features and carbohydrate-binding properties of lectins. In this regard, we investigated the effect of macromolecular crowding on the structure, conformation, stability, and carbohydrate-binding characteristics of jacalin, an α-galactose-binding lectin obtained from the seeds of jackfruit, which exhibits high specificity for the tumor-specific T-antigen. Conformational features of jacalin and binding of methyl-α-d-galactopyranoside and 4-methylumbelliferyl-α-d-galactopyranoside to it were investigated in the presence of polymeric dextrans of different sizes (D6, D20, D40, and D70, with molecular weights of 6, 20, 40, and 70 kDa, respectively) used as crowders at different concentrations (50-200 g/L). The results showed that the addition of crowders to native jacalin induced changes in the β-sheet content by up to 50%, as assessed by CD spectroscopy. An 8 nm red shift and 60% quenching of the protein intrinsic fluorescence by D6, and a 2 nm blue shift with 4% quenching by D70, revealed that D6 induces maximum perturbation of the protein structure, whereas D70 has the least effect, with D20 and D40 showing intermediate effects. A similar trend was observed in the carbohydrate-binding affinity of jacalin, with a maximum decrease being observed in the presence of D6, whereas D70 exhibited only a marginal effect, and intermediate effects were seen in the presence of D20 and D40. DSC and steady-state fluorescence spectroscopic studies revealed that all 4 dextrans stabilize jacalin against thermal and urea-induced denaturation, with D6 exhibiting the maximum effect. These results show that the conformational/structural features, stability, and carbohydrate-binding characteristics of jacalin are significantly influenced by macromolecular crowding.