The transformation from normal to malignant phenotype in human cancers is associated with aberrant cell-surface glycosylation. It has frequently been reported that MUC1, the heavily glycosylated cell-surface mucin, is altered in both, expression and glycosylation pattern, in human carcinomas of the epithelium. The presence of incomplete or truncated glycan structures, often capped by sialic acid, commonly known as tumor-associated carbohydrate antigens (TACAs), play a key role in tumor initiation, progression, and metastasis. Accumulating evidence suggests that expression of TACAs is associated with tumor escape from immune defenses. In this report, we will give an overview of the oncogenic functions of MUC1 that are exerted through. TACA interactions with endogenous carbohydrate-binding proteins (lectins). These interactions often lead to creation of a protumor microenvironment, favoring tumor progression and metastasis, and tumor evasion. In addition, we will describe current efforts in the design of cancer vaccines with special emphasis on the synthetic MUC1 glycopeptide vaccines. Analysis of key factors that govern structure-based design of immunogenic MUC1 glycopeptide epitopes are described. The role of TACA type, position, and density on observed humoral and cellular immune responses is evaluated.

The concept of biomedical significance of the functional pairing between tissue lectins and their glycoconjugate counterreceptors has reached the mainstream of research on the flow of biological information. A major challenge now is to identify the principles of structure–activity relationships that underlie specificity of recognition and the ensuing post-binding processes. Toward this end, we focus on a distinct feature on the side of the lectin, i.e. its architecture to present the carbohydrate recognition domain (CRD). Working with a multifunctional human lectin, i.e. galectin-3, as model, its CRD is used in protein engineering to build variants with different modular assembly. Hereby, it becomes possible to compare activity features of the natural design, i.e. CRD attached to an N-terminal tail, with those of homo- and heterodimers and the tail-free protein. Thermodynamics of binding disaccharides proved full activity of all proteins at very similar affinity. The following glycan array testing revealed maintained preferential contact formation with N-acetyllactosamine oligomers and histo-blood group ABH epitopes irrespective of variant design. The study of carbohydrate-inhibitable binding of the test panel disclosed up to qualitative cell-type-dependent differences in sections of fixed murine epididymis and especially jejunum. By probing topological aspects of binding, the susceptibility to inhibition by a tetravalent glycocluster was markedly different for the wild-type vs the homodimeric variant proteins. The results teach the salient lesson that protein design matters: the type of CRD presentation can have a profound bearing on whether basically suited oligosaccharides, which for example tested positively in an array, will become binding partners in situ. When lectin-glycoconjugate aggregates (lattices) are formed, their structural organization will depend on this parameter. Further testing (ga)lectin variants will thus be instrumental (i) to define the full range of impact of altering protein assembly and (ii) to explain why certain types of design have been favored during the course of evolution, besides opening biomedical perspectives for potential applications of the novel galectin forms.

Functional pairing between cellular glycoconjugates and tissue lectins like galectins has wide (patho)physiological significance.T heir study is facilitated by nonhydrolysable derivatives of the natural O-glycans, such as Sand Se-glycosides.T he latter enable extensive analyses by specific 77 Se NMR spectroscopy,b ut stillr emain underexplored. By using the example of selenodigalactoside (SeDG) and the human galectin-1 and-3, we have evaluated diverse 77 Se NMR detection methods and propose selective 1 H, 77 Se heteronuclear Hartmann-Hahn transfer for efficient use in competitive NMR screening against as elenoglycoside spy ligand.B yf luorescence anisotropy,c ircular dichroism, and isothermal titration calorimetry (ITC), we show that the affinity and thermodynamics of SeDGb inding by galectins are similart ot hiodigalactoside (TDG) and N-acetyllactosamine (LacNAc), confirming that Se substitution has no major impact.I TC data in D 2 Ov ersus H 2 Oa re similar for TDG and LacNAcb inding by both galectins, but as olvent effect, indicating solvent rearrangement at the binding site, is hinted at for SeDG and clearly observed for LacNAcd imers with extendedc hain length.

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.

Human macrophage galactose-type lectin (hMGL, HML, CD301, CLEC10A), a C-type lectin expressed by dendritic cells and macrophages, is a receptor for N -acetylgalactosamine α-linked to serine/threonine residues (Tn antigen, CD175) and its α2,6-sialylated derivative (sTn, CD175s). Because these two epitopes are among malignant cell glycan displays, particularly when presented by mucin-1 (MUC1), assessing the influence of the site and frequency of glycosylation on lectin recognition will identify determinants governing this interplay. Thus, chemical synthesis of the tandem-repeat O -glycan acceptor region of MUC1 and site-specific threonine glycosylation in all permutations were carried out. Isothermal titration calorimetry (ITC) analysis of the binding of hMGL to this library of MUC1 glycopeptides revealed an enthalpy-driven process and an affinity enhancement of an order of magnitude with an increasing glycan count from 6–8 μM for monoglycosylated peptides to 0.6 μM for triglycosylated peptide. ITC measurements performed in D 2 O permitted further exploration of the solvation dynamics during binding. A shift in enthalpy–entropy compensation and contact position-specific effects with the likely involvement of the peptide surroundings were detected. KinITC analysis revealed a prolonged lifetime of the lectin–glycan complex with increasing glycan valency and with a change in the solvent to D 2 O.