Amadori and Heyns rearrangement products of bioactive peptides as potential new ligands of galectin-3.

Non-enzymatic cascade reactions between amines and reducing sugars are known as Maillard reaction. The late phase of these reactions consists of advanced glycation end products (AGEs), which have been implicated in the pathogenesis of numerous human diseases. Recent evidence suggests that galectin-3 acts as a receptor for AGEs and some early products of the Maillard reaction. The early phase of the Maillard reaction, which consists of 1-amino-1-deoxyketoses (Amadori compounds) and 2-amino-2-deoxyaldoses (Heyns compounds), was the subject of our study. The binding interactions between galectin-3 and the Amadori and Heyns compounds of leucine-enkephalin (YGGFL), leucine-enkephalin methyl ester (YGGFL-OMe), truncated enkephalin (YGG and Y) and tetrapeptide (LSKL) were measured using the AlphaScreen competitive binding assay. The affinity of galectin-3 for Amadori and Heyns compounds depends on both the sugar moiety and the amino acid sequence of the model compounds. The best results were obtained with Leu-enkephalin derivatives of Amadori (IC50 = 6.06 µm) and Heyns (IC50 = 8.6 µm) compound, respectively.

Synthesis and Thermodynamic Evaluation of Sialyl-Tn MUC1 Glycopeptides Binding to Macrophage Galactose-Type Lectin.

Interactions between the tumor-associated carbohydrate antigens of Mucin 1 (MUC1) and the carbohydrate-binding proteins, lectins, often lead to the creation of a pro-tumor microenvironment favoring tumor initiation, progression, metastasis, and immune evasion. Macrophage galactose binding lectin (MGL) is a C-type lectin receptor found on antigen-presenting cells that facilitates the uptake of carbohydrate antigens for antigen presentation, modulating the immune response homeostasis, autoimmunity, and cancer. Considering the crucial role of tumor-associated forms of MUC1 and MGL in tumor immunology, a thorough understanding of their binding interaction is essential for it to be exploited for cancer vaccine strategies. The synthesis of MUC1 glycopeptide models carrying a single or multiple Tn and/or sialyl-Tn antigen(s) is described. A novel approach for the sialyl-Tn threonine building block suitable for the solid phase peptide synthesis was developed. The thermodynamic profile of the binding interaction between the human MGL and MUC1 glycopeptide models was analyzed using isothermal titration calorimetry. The measured dissociation constants for the sialyl-Tn-bearing peptide epitopes were consistently lower compared to the Tn antigen and ranged from 10 µM for mono- to 1 µM for triglycosylated MUC1 peptide, respectively. All studied interactions, regardless of the glycan's site of attachment or density, exhibited enthalpy-driven thermodynamics.

Targeting Siglec-Sialylated MUC1 Immune Axis in Cancer.

Siglecs play a key role in mediating cell-cell interactions via the recognition of different sialylated glycoconjugates, including tumor-associated MUC1, which can lead to the activation or inhibition of the immune response. The activation occurs through the signaling of Siglecs with the cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM)-containing proteins, while the inhibition signal is a result of the interaction of intracellular immunoreceptor tyrosine-based inhibition motif (ITIM)-bearing receptors. The interaction of tumor-associated MUC1 sialylated glycans with Siglecs via ITIM motifs decreases antitumor immunity. Consequently, these interactions are expected to play a key role in tumor evasion. Efforts to modulate the response of immune cells by blocking the immune-suppressive effects of inhibitory Siglecs, driving immune-activating Siglecs, and/or altering the synthesis and expression of the sialic acid glycocalyx are new therapeutic strategies deserving further investigation. We will highlight the role of Siglec's family receptors in immune evasion through interactions with glycan ligands in their natural context, presented on the protein such as MUC1, factors affecting their fine binding specificities, such as the role of multivalency either at the ligand or receptor side, their spatial organization, and finally the current and future therapeutic interventions targeting the Siglec-sialylated MUC1 immune axis in cancer.

Positional Scanning MUC1 Glycopeptide Library Reveals the Importance of PDTR Epitope Glycosylation for Lectin Binding.

One of the main barriers to explaining the functional significance of glycan-based changes in cancer is the natural epitope heterogeneity found on the surface of cancer cells. To help address this knowledge gap, we focused on designing synthetic tools to explore the role of tumor-associated glycans of MUC1 in the formation of metastasis via association with lectins. In this study, we have synthesized for the first time a MUC1-derived positional scanning synthetic glycopeptide combinatorial library (PS-SGCL) that vary in number and location of cancer-associated Tn antigen using the "tea bag" approach. The determination of the isokinetic ratios necessary for the equimolar incorporation of (glyco)amino acids mixtures to resin-bound amino acid was determined, along with developing an efficient protocol for on resin deprotection of O-acetyl groups. Enzyme-linked lectin assay was used to screen PS-SGCL against two plant lectins, Glycine max soybean agglutinin and Vicia villosa. The results revealed a carbohydrate density-dependent affinity trend and site-specific glycosylation requirements for high affinity binding to these lectins. Hence, PS-SGCLs provide a platform to systematically elucidate MUC1-lectin binding specificities, which in the long term may provide a rational design for novel inhibitors of MUC1-lectin interactions involved in tumor spread and glycopeptide-based cancer vaccines.