Glycan-costumed virus-like particles promote type 1 anti-tumor immunity.

Cancer vaccine development is inhibited by a lack of strategies for directing dendritic cell (DC) induction of effective tumor-specific cellular immunity. Pathogen engagement of DC lectins and toll-like receptors (TLRs) shapes immunity by directing T cell function. Strategies to activate specific DC signaling pathways via targeted receptor engagement are crucial to unlocking type 1 cellular immunity. Here, we engineered a glycan-costumed virus-like particle (VLP) vaccine that delivers programmable peptide antigens to induce tumor-specific cellular immunity in vivo. VLPs encapsulating TLR7 agonists and decorated with a selective mannose-derived ligand for the lectin DC-SIGN induced robust DC activation and type 1 cellular immunity, whereas VLPs lacking this key DC-SIGN ligand failed to promote DC-mediated immunity. Vaccination with glycan-costumed VLPs generated tumor antigen-specific Th1 CD4+ and CD8+ T cells that infiltrated solid tumors, inhibiting tumor growth in a murine melanoma model. Thus, VLPs employing lectin-driven immune reprogramming provide a framework for advancing cancer immunotherapies.

MUC1 Glycopeptide Vaccine Modified with a GalNAc Glycocluster Targets the Macrophage Galactose C-type Lectin on Dendritic Cells to Elicit an Improved Humoral Response.

Mucin expression and glycosylation patterns on cancer cells differ markedly from healthy cells. Mucin 1 (MUC1) is overexpressed in several solid tumors and presents high levels of aberrant, truncated O-glycans (e.g., Tn antigen). Dendritic cells (DCs) express lectins that bind to these tumor-associated carbohydrate antigens (TACAs) to modulate immune responses. Selectively targeting these receptors with synthetic TACAs is a promising strategy to develop anticancer vaccines and to overcome TACA tolerance. In this work, we prepared, via a solid phase peptide synthesis approach, a modular tripartite vaccine candidate, incorporating a high-affinity glycocluster based on a tetraphenylethylene scaffold, to target the macrophage galactose-type lectin (MGL) on antigen presenting cells. MGL is a C-type lectin receptor that binds Tn antigens and can route them to human leukocyte antigen class II or I, making it an attractive target for anticancer vaccines. Conjugation of the glycocluster to a library of MUC1 glycopeptides bearing the Tn antigen is shown to promote uptake and recognition of the TACA by DCs via MGL. In vivo testing revealed that immunization with the newly designed vaccine construct bearing the GalNAc glycocluster induced a higher titer of anti-Tn-MUC1 antibodies compared to the TACAs alone. Additionally, the antibodies obtained bind a library of tumor-associated saccharide structures on MUC1 and MUC1-positive breast cancer cells. Conjugation of a high-affinity ligand for MGL to tumor-associated MUC1 glycopeptide antigens has a synergistic impact on antibody production.

Exploring the Galectin Network by Light and Fluorescence Microscopy.

Dynamic changes of a cell's glycophenotype are increasingly interpreted as shifts in the capacity to interact with tissue (endogenous) lectins. The status of glycan branching or chain length (e.g., core 1 vs core 2 mucin-type O-glycans and polyLacNAc additions) as well as of sialylation/sulfation has been delineated to convey signals. They are "read" by galectins, for example regulating lattice formation on the membrane and cell growth. Owing to the discovery of the possibility that these effectors act in networks physiologically resulting in functional antagonism or cooperation, their detection and distribution profiling need to be expanded from an individual (single) protein to the-at best-entire family. How to work with non-cross-reactive antibodies and with the labeled tissue-derived proteins (used as probes) is exemplarily documented for chicken and human galectins including typical activity and specificity controls. This description intends to inspire the systematic (network) study of members of a lectin family and also the application of tissue proteins beyond a single lectin category in lectin histochemistry.

The Development of Vaccines from Synthetic Tumor-Associated Mucin Glycopeptides and their Glycosylation-Dependent Immune Response.

Tumor-associated carbohydrate antigens are overexpressed as altered-self in most common epithelial cancers. Their glycosylation patterns differ from those of healthy cells, functioning as an ID for cancer cells. Scientists have been developing anti-cancer vaccines based on mucin glycopeptides, yet the interplay of delivery system, adjuvant and tumor associated MUC epitopes in the induced immune response is not well understood. The current state of the art suggests that the identity, abundancy and location of the glycans on the MUC backbone are all key parameters in the cellular and humoral response. This review shares lessons learned by us in over two decades of research in glycopeptide vaccines. By bridging synthetic chemistry and immunology, we discuss efforts in designing synthetic MUC1/4/16 vaccines and focus on the role of glycosylation patterns. We provide a brief introduction into the mechanisms of the immune system and aim to promote the development of cancer subunit vaccines.

Imitating evolution's tinkering by protein engineering reveals extension of human galectin-7 activity.

Wild-type lectins have distinct types of modular design. As a step to explain the physiological importance of their special status, hypothesis-driven protein engineering is used to generate variants. Concerning adhesion/growth-regulatory galectins, non-covalently associated homodimers are commonly encountered in vertebrates. The homodimeric galectin-7 (Gal-7) is a multifunctional context-dependent modulator. Since the possibility of conversion from the homodimer to hybrids with other galectin domains, i.e. from Gal-1 and Gal-3, has recently been discovered, we designed Gal-7-based constructs, i.e. stable (covalently linked) homo- and heterodimers. They were produced and purified by affinity chromatography, and the sugar-binding activity of each lectin unit proven by calorimetry. Inspection of profiles of binding of labeled galectins to an array-like platform with various cell types, i.e. sections of murine epididymis and jejunum, and impact on neuroblastoma cell proliferation revealed no major difference between natural and artificial (stable) homodimers. When analyzing heterodimers, acquisition of altered properties was seen. Remarkably, binding properties and activity as effector can depend on the order of arrangement of lectin domains (from N- to C-termini) and on the linker length. After dissociation of the homodimer, the Gal-7 domain can build new functionally active hybrids with other partners. This study provides a clear direction for research on defining the full range of Gal-7 functionality and offers the perspective of testing applications for engineered heterodimers.

Crystal Structure of the Carbohydrate Recognition Domain of the Human Macrophage Galactose C-Type Lectin Bound to GalNAc and the Tumor-Associated Tn Antigen.

The human macrophage galactose lectin (MGL) is an endocytic type II transmembrane receptor expressed on immature monocyte-derived dendritic cells and activated macrophages and plays a role in modulating the immune system in response to infections and cancer. MGL contains an extracellular calcium-dependent (C-type) carbohydrate recognition domain (CRD) that specifically binds terminal N-acetylgalactosamine glycan residues such as the Tn and sialyl-Tn antigens found on tumor cells, as well as other N- and O-glycans displayed on certain viruses and parasites. Even though the glycan specificity of MGL is known and several binding glycoproteins have been identified, the molecular basis for substrate recognition has remained elusive due to the lack of high-resolution structures. Here we present crystal structures of the MGL CRD at near endosomal pH and in several complexes, which reveal details of the interactions with the natural ligand, GalNAc, the cancer-associated Tn-Ser antigen, and a synthetic GalNAc mimetic ligand. Like the asialoglycoprotein receptor, additional calcium atoms are present and contribute to stabilization of the MGL CRD fold. The structure provides the molecular basis for preferential binding of N-acetylgalactosamine over galactose and prompted the re-evaluation of the binding modes previously proposed in solution. Saturation transfer difference nuclear magnetic resonance data acquired using the MGL CRD and interpreted using the crystal structure indicate a single binding mode for GalNAc in solution. Models of MGL1 and MGL2, the mouse homologues of MGL, explain how these proteins might recognize LewisX and GalNAc, respectively.

Probing sulfatide-tissue lectin recognition with functionalized glycodendrimersomes.

The small 3-O-sulfated galactose head group of sulfatides, an abundant glycosphingolipid class, poses the (sphinx-like) riddle on involvement of glycan bridging by tissue lectins (sugar code). First, synthesis of head group derivatives for functionalization of amphiphilic dendrimers is performed. Aggregation of resulting (biomimetic) vesicles, alone or in combination with lactose, demonstrates bridging by a tissue lectin (galectin-4). Physiologically, this can stabilize glycolipid-rich microdomains (rafts) and associate sulfatide-rich regions with specific glycoproteins. Further testing documents importance of heterobivalency and linker length. Structurally, sulfatide recognition by galectin-8 is shown to involve sphingosine's OH group as substitute for the 3'-hydroxyl of glucose of lactose. These discoveries underscore functionality of this small determinant on biomembranes intracellularly and on the cell surface. Moreover, they provide a role model to examine counterreceptor capacity of more complex glycans of glycosphingolipids and to start their bottom-up glycotope surface programming.

Correction to: Influence of protein (human galectin-3) design on aspects of lectin activity.

In the original publication of the article, the "Result" section has been published.

Influence of protein (human galectin-3) design on aspects of lectin activity.

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.

How altering the modular architecture affects aspects of lectin activity: case study on human galectin-1.

Discoveries on involvement of glycan-protein recognition in many (patho)physiological processes are directing attention to exploring the significance of a fundamental structural aspect of sugar receptors beyond glycan specificity, i.e., occurrence of distinct types of modular architecture. In order to trace clues for defining design-functionality relationships in human lectins, a lectin's structural unit has been used as source material for engineering custom-made variants of the wild-type protein. Their availability facilitates comparative analysis toward the stated aim. With adhesion/growth-regulatory human galectin-1 as example, the strategy of evaluating how changes of its design (here, from the homodimer of non-covalently associated domains to (i) linker-connected di- and tetramers and (ii) a galectin-3-like protein) affect activity is illustrated by using three assay systems of increasing degree of glycan complexity. Whereas calorimetry with two cognate disaccharides and array testing with 647 (glyco)compounds disclosed no major changes, galectin histochemical staining profiles of tissue sections that present natural glycome complexity revealed differences between wild-type and linker-connected homo-oligomers as well as between the galectin-3-like variant and wild-type galectin-3 for cell-type positivity, level of intensity at the same site and susceptibility for inhibition by a bivalent glycocompound. These results underscore the strength of the documented approach. Moreover, they give direction to proceed to (i) extending its application to other members of this lectin family, especially galectin-3 and (ii) then analyzing impact of architectural alterations on cell surface lattice formation and ensuing biosignaling systematically, considering the variants' potential for translational medicine.

Anomer Preferences for Glucuronic and Galacturonic Acid and Derivatives and Influence of Electron-Withdrawing Substituents.

Equilibrium anomeric ratios are reported for pyranoses (hemiacetals) of glucuronic and galacturonic acid and their derivatives. These are compared to related gluco- and galactopyranoses and to deoxyfluorogluco- and deoxyfluorogalactopyranoses. An association between axial anomer stability and the sum of 1H NMR downfield chemical shifts for protons H-3 and H-5 was observed in D2O with gluco- and galactopyranoses as reference compounds. When compared to 2-hydroxytetrahydropyran in water, introduction of three OAc substituents and one carboxylic acid substituent leads to an increase in stability of the axial anomer by 0.89-1.05 kcal/mol. This is interpreted as the electron-withdrawing substituents causing a reduction in the steric (gauche) interaction and an increase in favourable Coulombic interaction between CH groups of the pyranose and the anomeric group through substituent deshielding effects. Anomer preferences for galacturonic acid and its derivatives were more sensitive to solvent polarity compared to other pyranoses, and this may be linked to the electrostatic potential and reduced stabilization of the equatorial anomeric OH group due to reduced hydrogen bonding. The latter is more notable in nonpolar chloroform. Analysis of crystal structures combined with molecular dynamics indicated there are conformational distinctions between galacturonic acid and glucuronic acid that could influence properties.

Revealing biomedically relevant cell and lectin type-dependent structure-activity profiles for glycoclusters by using tissue sections as an assay platform.

The increasing realization of the involvement of lectin-glycan recognition in (patho)physiological processes inspires envisioning therapeutic intervention by high-avidity/specificity blocking reagents. Synthetic glycoclusters are proving to have potential for becoming such inhibitors but the commonly used assays have their drawbacks to predict in vivo efficacy. They do not represent the natural complexity of (i) cell types and (ii) spatial and structural complexity of glycoconjugate representation. Moreover, testing lectins in mixtures, as present in situ, remains a major challenge, giving direction to this work. Using a toolbox with four lectins and six bi- to tetravalent glycoclusters bearing the cognate sugar in a model study, we here document the efficient and versatile application of tissue sections (from murine jejunum as the model) as a platform for routine and systematic glycocluster testing without commonly encountered limitations. The nature of glycocluster structure, especially core and valency, and of protein features, i.e. architecture, fine-specificity and valency, are shown to have an influence, as cell types can differ in response profiles. Proceeding from light microscopy to monitoring by fluorescence microscopy enables grading of glycocluster activity on individual lectins tested in mixtures. This work provides a robust tool for testing glycoclusters prior to considering in vivo experiments.