Molecular recognition of tumor-associated antigens by lectins and antibodies

Abstract

Every living cell on Earth is covered by glycans. They are inserted in proteins and lipids by a posttranslational modification called glycosylation. Their recognition by specific receptors is translated into distinct biological signals. In cancer cells, a misregulation in expression and/or activity of glycosyltransferases, alters the mechanism of glycosylation, creating new glycan epitopes dubbed tumor-associated carbohydrate antigens (TACAs). These are recognized by various receptors, playing a major role in tumor immune responses and metastasis. To target cancer-associated glycan phenotype is crucial to disentangle the molecular recognition process that involves TACAs recognition and biosynthesis. Therefore, NMR techniques were employed to investigate distinct glycan-protein systems: i) the molecular interactions between a mucin-1 (MUC1) related Tn-glycopeptide mimetic containing a non-natural amino acid and distinct antibodies by saturation transfer-difference (STD-NMR); ii) the molecular interactions between galectin-3 (Gal-3) and TF-antigen (TF-Thr and TF-peptide), by heteronuclear single quantum coherence 1H,15N-HSQC titrations, STD-NMR and line broadening analysis and iii) the glycosylation of MUC1 tandem repeated protein (G1VT3S4APDT8RPAPGS14T15APPAH20)4 by GalNAc-T3 using 1H,15N-HSQC and STD-NMR. In i), the STD-NMR binding experiments show that all antibodies under study recognize the Tn-glycopeptide mimetic and point out structural differences that explain antibodies’ binding preferences. In ii), the 1H,15N-HSQC titrations experiments indicate that Gal-3 binds both TF-derivatives. The dissociation constant KD estimated for both through chemical shift analysis also shows the same range of affinity (275 μM and 413 μM for TF-antigen and TF-peptide, respectively). STD-NMR results demonstrate that the protons from galactose in the TF-moiety govern the recognition process of Gal-3. In iii), the 1H,15N-HSQC experiments of MUC1 in presence of GalNAc-T3 show that the enzyme has preference to glycosylate first the Thr at –GVTS-, followed by the residue Thr at –GSTA-. STD-NMR confirms the cooperative mechanism between the lectin and catalytic domain of GalNAc-T3.