Presentation Is Essential for Glycan-Lectin Recognition at the Molecular and Cellular Levels: The Interaction of Tumor-Associated ‑Glycans with the Macrophage Galactose-Type Lectin.

The human macrophage galactose-type lectin (MGL) recognizes exposed GalNAc residues abundantly found in tumor -glycans. Herein, we have used an integrative chemical, structural, and functional approach to unravel the intricate specificity and molecular determinants that underlie the recognition of Thomsen-nouveau (Tn), the sialylated variant (STn), and Thomsen-Friedenreich (TF) -glycans by the carbohydrate recognition domain of the MGL (MGL-CRD) at the molecular and cellular levels. The MGL-CRD prefers binding to Tn > STn ≫ TF -glycans. In this molecular context, NMR, isothermal titration calorimetry, and molecular dynamics simulations revealed quantitative key structural and dynamic differences in binding, depending on the -glycan. Interestingly, the density of Tn epitopes was critical for engaging multiple MGL-CRDs to MUC1 Tn-glycopeptides; however, the enthalpy-entropy balance strongly influenced the affinity, and a higher Tn density did not improve the binding. Cell-based mucin arrays recapitulated the MGL-CRD binding preference (Tn > STn ≫TF), but no preference for a specific -glycan pattern in mucins was observed. The MGL-CRD also selectively recognizes glycoengineered gastric cancer cells expressing Tn/STn. Conversely, in the cellular context, employing CHO cells expressing the full-length MGL (CHO) allowed analysis of the MGL binding properties in its native presentation toward tagged isolated mucin reporters. Specificity for short tumor-associated -glycans without any preference for a specific mucin was confirmed. Stunningly, the CHO cells revealed that the MGL shows similar binding to the STn and TF mucin reporters, suggesting that its natural oligomeric state displays promiscuous binding to simple -glycans. Conceptually, the key role of glycan and lectin presentations for binding is thus highlighted. Moreover, this suggests the compelling scenario that the MGL serves as a universal receptor for truncated cancer-associated -glycans.