Glycans regulate multiple physiological processes, including immune recognition and cancer progression. In disease, altered glycan landscapes are interpreted by human lectins. Functional glycan-lectin interactions are difficult to profile because glycans are not genome-encoded and their changes are poorly captured by existing multimodal methods. We present two platforms, single-cell outlining and transcriptome sequencing (scGOAT-seq) and GlycoScope, which use human lectins to enable functional glycan accessibility into single-cell and spatial multiomic measurements. ScGOAT-seq quantifies lectin-accessible glycan states with gene expression, while GlycoScope enables multiplexed in situ co-detection of glycans and proteins in tissues. Applying these approaches to immune cells, we identify stimulus-specific glycan remodeling and show that distinct Siglec-ligand-defined programs stratify immune activation states not captured by traditional methods; in follicular lymphoma, GlycoScope, resolves spatial glycan programs associated with malignant B cells and localized immune microenvironments. The presented methods provide a general framework for integrating functional glycan accessibility into single-cell and spatial multiomics.

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) is thought to shape immunity by directing T cell function. Controlling downstream responses, however, remains a major challenge. A critical goal in advancing vaccine development involves the identification of receptors that drive type 1 cellular immunity. The immune system monitors cells for aberrant glycosylation (a sign of a foreign entity), but potent activation occurs when a second signal, such as single-stranded RNA or lipopolysaccharide, is present to activate TLR signaling. To exploit dual signaling, we engineered a glycan-costumed virus-like particle (VLP) vaccine that displays a DC-SIGN-selective aryl mannose ligand and encapsulates TLR7 agonists. These VLPs deliver programmable peptide antigens to induce robust DC activation and type 1 cellular immunity. In contrast, VLPs lacking this critical DC-SIGN ligand promoted DC-mediated humoral immunity, offering limited tumor control. Vaccination with glycan-costumed VLPs generated tumor antigen-specific Th1 CD4+ and CD8+ T cells that infiltrated solid tumors, significantly inhibiting tumor growth in a murine melanoma model. The tailored VLPs also afforded protection against the reintroduction of tumor cells. Thus, DC lectin-driven immune reprogramming, combined with the modular programmability of VLP platforms, provides a promising framework for directing cellular immunity to advance cancer immunotherapies and vaccines.

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.

ADVERTISEMENT RETURN TO ARTICLES ASAPViewpointNEXTBeyond the Crystal Structure of Human Macrophage C-Type LectinAdele Gabba*Adele GabbaDepartment of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United StatesSchool of Biological and Chemical Sciences, University of Galway, University Rd., Galway H91 TK33, Ireland*Email: [email protected]More by Adele Gabbahttps://orcid.org/0000-0001-8240-6482, Paul V. MurphyPaul V. MurphySchool of Biological and Chemical Sciences, University of Galway, University Rd., Galway H91 TK33, IrelandSSPC, The Science Foundation Ireland Research Centre for Pharmaceuticals, University of Galway, University Rd., Galway H91 TK33, IrelandMore by Paul V. Murphyhttps://orcid.org/0000-0002-1529-6540, Laura L. KiesslingLaura L. KiesslingDepartment of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United StatesMore by Laura L. Kiesslinghttps://orcid.org/0000-0001-6829-1500, and Gabriel BirraneGabriel BirraneDivision of Experimental Medicine, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, United StatesMore by Gabriel Birranehttps://orcid.org/0000-0002-1759-5499Cite this: Biochemistry 2024, XXXX, XXX, XXX-XXXPublication Date (Web):January 2, 2024Publication History Received16 November 2023Published online2 January 2024https://doi.org/10.1021/acs.biochem.3c00642© 2024 American Chemical SocietyRequest reuse permissions This publication is free to access through this site. Learn MoreArticle Views-Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-AlertscloseSUBJECTS:Antigens,Carbohydrates,Immunology,Peptides and proteins,Receptors Get e-Alerts

Mucin expressionand glycosylation patterns on cancercells differmarkedly from healthy cells. Mucin 1 (MUC1) is overexpressed in severalsolid tumors and presents high levels of aberrant, truncated O-glycans(e.g., Tn antigen). Dendritic cells (DCs) express lectins that bindto these tumor-associated carbohydrate antigens (TACAs) to modulateimmune responses. Selectively targeting these receptors with syntheticTACAs is a promising strategy to develop anticancer vaccines and toovercome TACA tolerance. In this work, we prepared, via a solid phasepeptide synthesis approach, a modular tripartite vaccine candidate,incorporating a high-affinity glycocluster based on a tetraphenylethylenescaffold, to target the macrophage galactose-type lectin (MGL) onantigen presenting cells. MGL is a C-type lectin receptor that bindsTn antigens and can route them to human leukocyte antigen class IIor I, making it an attractive target for anticancer vaccines. Conjugationof the glycocluster to a library of MUC1 glycopeptides bearing theTn antigen is shown to promote uptake and recognition of the TACAby DCs via MGL. In vivo testing revealed that immunization with thenewly designed vaccine construct bearing the GalNAc glycocluster induceda higher titer of anti-Tn-MUC1 antibodies compared to the TACAs alone.Additionally, the antibodies obtained bind a library of tumor-associatedsaccharide structures on MUC1 and MUC1-positive breast cancer cells.Conjugation of a high-affinity ligand for MGL to tumor-associatedMUC1 glycopeptide antigens has a synergistic impact on antibody production.

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 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 Lewis(x) and GalNAc, respectively.

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.

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.

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 bio-membranes intracellularly and on the cell surface. Moreover, they provide a role model to examine counterreceptor capacity of more complex glycans of gly-cosphingolipids and to start their bottom-up glycotope surface programming.

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

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.

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.

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.

The synthesis of three macrotriazoles that are analogues of migrastatin is reported. The synthesis is based on copper(I)- and ruthenium-catalyzed azide–alkyne cycloaddition reactions. The enantiopure terminal alkyne derivatives were prepared by using the Trost desymmetrization, Brown alkoxyallylation and an efficient Colvin reaction. Biological evaluation of the products revealed a promising efficiency in reducing the ability of MDA-MB-361 cell lines to migrate.