Presentation Is Essential for Glycan-Lectin Recognition at the Molecular and Cellular Levels: The Interaction of Tumor-Associated <i>O</i> -Glycans with the Macrophage Galactose-Type Lectin

High Resolution Image Download MS PowerPoint Slide The human macrophage galactose-type lectin (MGL) recognizes exposed GalNAc residues abundantly found in tumor O -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) O -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 O -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 O -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 O -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 +MGL ) allowed analysis of the MGL binding properties in its native presentation toward tagged isolated mucin reporters. Specificity for short tumor-associated O -glycans without any preference for a specific mucin was confirmed. Stunningly, the CHO +MGL 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 O -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 O -glycans.