Preformed mincle dimers stabilized by an interchain disulfide bond in the neck region.

The sugar-binding receptor mincle stimulates macrophages when it encounters surface glycans on pathogens, such as trehalose dimycolate glycolipid in the outer membrane of mycobacteria. Binding of oligosaccharide ligands to the extracellular C-type carbohydrate-recognition domain (CRD) in mincle initiates intracellular signaling through the common Fc receptor γ (FcRγ) adapter molecule associated with mincle. One potential mechanism for initiation of signaling involves clustering of receptors, so it is important to understand the oligomeric state of mincle. Affinity purification of mincle from transfected mammalian cells has been used to show that mincle exists as a pre-formed, disulfide-linked dimer. Deletion of cysteine residues and chemical crosslinking further demonstrate that the dimers of mincle are stabilized by a disulfide bond between cysteine residues in the neck sequence that links the CRD to the membrane. In contrast, cysteine residues in the transmembrane region of mincle are not required for dimer formation or association with FcRγ. A protocol has been developed for efficient production of a disulfide-linked extracellular domain fragment of mincle in a bacterial expression system by appending synthetic dimerization domains to guide dimer formation in the absence of the membrane anchor.

A human lectin array for characterizing host-pathogen interactions.

A human lectin array has been developed to probe the interactions of innate immune receptors with pathogenic and commensal micro-organisms. Following the successful introduction of a lectin array containing all of the cow C-type carbohydrate-recognition domains (CRDs), a human array described here contains the C-type CRDs as well as CRDs from other classes of sugar-binding receptors, including galectins, siglecs, R-type CRDs, ficolins, intelectins and chitinase-like lectins. The array is constructed with CRDs modified with single-site biotin tags, ensuring that the sugar-binding sites in CRDs are displayed on a streptavidin-coated surface in a defined orientation and are accessible to the surfaces of microbes. A common approach used for expression and display of CRDs from all of the different structural categories of glycan-binding receptors allows comparisons across lectin families. In addition to previously documented protocols for binding of fluorescently-labeled bacteria, methods have been developed for detecting unlabeled bacteria bound to the array by counter-staining with DNA-binding dye. Screening has also been undertaken with viral glycoproteins and bacterial and fungal polysaccharides. The array provides an unbiased screen for sugar ligands that interact with receptors and many show binding not anticipated from earlier studies. For example, some of the galectins bind with high affinity to bacterial glycans that lack lactose or N-acetyllactosamine. The results demonstrate the utility of the human lectin array for providing a unique overview of the interactions of multiple classes of glycan-binding proteins in the innate immune system with different types of micro-organisms.

Expression and immobilization of novel N-glycan-binding protein for highly efficient purification and enrichment of N-glycans, N-glycopeptides, and N-glycoproteins.

Comprehensive and selective enrichment of N-glycans, N-glycopeptides, and N-glycoproteins prior to analysis is of great significance in N-glycomics research, reducing sample complexity, removing impurity interference, increasing sample abundance and enhancing signal intensity. However, only an Fbs1 (F-box protein that recognizes sugar chain 1) GYR variant (Fg) can enrich these N-glycomolecules solely due to its substantial binding affinity for the core pentasaccharide motif of N-glycans. Stationary phase separation is commonly used to enrich N-glycomolecules efficiently. Herein, DNA encoding the Fg was cloned into pGEX-4T-1, and the protein was expressed with a GST tag, which facilitates the convenient and efficient immobilization of recombinant GST-tagged Fg to GSH agarose resin. The yield of the GST-tagged Fg reached to 0.05 g/L after optimization of the induction condition, and the purified protein exhibited good identification ability and excellent stability for months. In particular, the immobilized GST-tagged Fg can enrich N-glycans released by PNGase F and capture derivatized N-glycans possessing an intact terminal N-acetyl glucosamine (GlcNAc). Validation of immobilized GST-tagged Fg with standard N-glycopeptides and N-glycoproteins revealed its high loading capacity, sensitivity, and selectivity. The novel immobilized GST-tagged Fg is a convenient and efficient enrichment material specific for N-glycans, N-glycopeptides, and N-glycoproteins, suggesting excellent performance and prospects for industrial application.

Insights into Glycobiology and the Protein-Glycan Interactome Using Glycan Microarray Technologies.

Glycans linked to proteins and lipids and also occurring in free forms have many functions, and these are partly elicited through specific interactions with glycan-binding proteins (GBPs). These include lectins, adhesins, toxins, hemagglutinins, growth factors, enzymes, but antibodies can also bind glycans. While humans and other animals generate a vast repertoire of GBPs and different glycans in their glycomes, other organisms, including phage, microbes, protozoans, fungi, and plants also express glycans and GBPs, and these can also interact with their host glycans. This can be termed the protein-glycan interactome, and in nature is likely to be vast, but is so far very poorly described. Understanding the breadth of the protein-glycan interactome is also a key to unlocking our understanding of infectious diseases involving glycans, and immunology associated with antibodies binding to glycans. A key technological advance in this area has been the development of glycan microarrays. This is a display technology in which minute quantities of glycans are attached to the surfaces of slides or beads. This allows the arrayed glycans to be interrogated by GBPs and antibodies in a relatively high throughput approach, in which a protein may bind to one or more distinct glycans. Such binding can lead to novel insights and hypotheses regarding both the function of the GBP, the specificity of an antibody, and the function of the glycan within the context of the protein-glycan interactome. This article focuses on the types of glycan microarray technologies currently available to study animal glycobiology and examples of breakthroughs aided by these technologies. (254 words).

The molecular basis of cereal mixed-linkage ß-glucan utilization by the human gut bacterium Segatella copri.

Mixed-linkage ß(1,3)/ß(1,4)-glucan (MLG) is abundant in the human diet through the ingestion of cereal grains and is widely associated with healthful effects on metabolism and cholesterol levels. MLG is also a major source of fermentable glucose for the human gut microbiota (HGM). Bacteria from the family Prevotellaceae are highly represented in the HGM of individuals who eat plant-rich diets, including certain indigenous people and vegetarians in postindustrial societies. Here, we have defined and functionally characterized an exemplar Prevotellaceae MLG polysaccharide utilization locus (MLG-PUL) in the type-strain Segatella copri (syn. Prevotella copri) DSM 18205 through transcriptomic, biochemical, and structural biological approaches. In particular, structure-function analysis of the cell-surface glycan-binding proteins and glycoside hydrolases of the S. copri MLG-PUL revealed the molecular basis for glycan capture and saccharification. Notably, syntenic MLG-PULs from human gut, human oral, and ruminant gut Prevotellaceae are distinguished from their counterparts in Bacteroidaceae by the presence of a ß(1,3)-specific endo-glucanase from glycoside hydrolase family 5, subfamily 4 (GH5_4) that initiates MLG backbone cleavage. The definition of a family of homologous MLG-PULs in individual species enabled a survey of nearly 2000 human fecal microbiomes using these genes as molecular markers, which revealed global population-specific distributions of Bacteroidaceae- and Prevotellaceae-mediated MLG utilization. Altogether, the data presented here provide new insight into the molecular basis of ß-glucan metabolism in the HGM, as a basis for informing the development of approaches to improve the nutrition and health of humans and other animals.

"Glycans in Trained Immunity: Educators of innate immune memory in homeostasis and disease".

Trained Immunity is defined as a biological process normally induced by exogenous or endogenous insults that triggers epigenetic and metabolic reprogramming events associated with long-term adaptation of innate immune cells. This trained phenotype confers enhanced responsiveness to subsequent triggers, resulting in an innate immune "memory" effect. Trained Immunity, in the past decade, has revealed important benefits for host defense and homeostasis, but can also induce potentially harmful outcomes associated with chronic inflammatory disorders or autoimmune diseases. Interestingly, evidence suggest that the "trainers" prompting trained immunity are frequently glycans structures. In fact, the exposure of different types of glycans at the surface of pathogens is a key driver of the training phenotype, leading to the reprogramming of innate immune cells through the recognition of those glycan-triggers by a variety of glycan-binding proteins (GBPs) expressed by the immune cells. ß-glucan or mannose-enriched structures in Candida albicans are some of the examples that highlight the potential of glycans in trained immunity, both in homeostasis and in disease. In this review, we will discuss the relevance of glycans exposed by pathogens in establishing key immunological hubs with glycan-recognizing receptors expressed in immune cells, highlighting how this glycan-GBP network can impact trained immunity. Finally, we discuss the power of glycans and GBPs as potential targets in trained immunity, envisioning potential therapeutic applications.

Recent advances in photoaffinity labeling strategies to capture Glycan-Protein interactions.

Glycans decorate all cells and are critical mediators of cellular processes through recognition by glycan-binding proteins (GBPs). While targeting glycan-protein interactions has great therapeutic potential, these interactions are challenging to study as they are generally transient and exhibit low binding affinities. Glycan-based photo-crosslinkable probes have enabled covalent capture and identification of unknown GBP receptors and glycoconjugate ligands. Here, we review recent progress in photo-crosslinking approaches targeting glycan-mediated interactions. We discuss two prominent emerging strategies: 1) development of photo-crosslinkable oligosaccharide ligands to identify GBP receptors; and 2) cell-surface glyco-engineering to identify glycoconjugate ligands of GBPs. Overall, photoaffinity labeling affords valuable insights into complex glycan-protein networks and is poised to help elucidate the glycan-protein interactome, providing novel targets for therapeutic intervention.

Reading and Writing the Human Glycocode.

The complex carbohydrate structures decorating human proteins and lipids, also called glycans, are abundantly present at cell surfaces and in the secretome. Glycosylation is vital for biological processes including cell-cell recognition, immune responses, and signaling pathways. Therefore, the structural and functional characterization of the human glycome is gaining more and more interest in basic biochemistry research and in the context of developing new therapies, diagnostic tools, and biotechnology applications. For glycomics to reach its full potential in these fields, it is critical to appreciate the specific factors defining the function of the human glycome. Here, we review the glycosyltransferases (the writers) that form the glycome and the glycan-binding proteins (the readers) with an essential role in decoding glycan functions. While abundantly present throughout different cells and tissues, the function of specific glycosylation features is highly dependent on their context. In this review, we highlight the relevance of studying the glycome in the context of specific carrier proteins, cell types, and subcellular locations. With this, we hope to contribute to a richer understanding of the glycome and a more systematic approach to identifying the roles of glycosylation in human physiology.

A Complex-Type N-Glycan-Specific Lectin Isolated from Green Alga Halimeda borneensis Exhibits Potent Anti-Influenza Virus Activity.

Marine algal lectins specific for high-mannose N-glycans have attracted attention because they strongly inhibit the entry of enveloped viruses, including influenza viruses and SARS-CoV-2, into host cells by binding to high-mannose-type N-glycans on viral surfaces. Here, we report a novel anti-influenza virus lectin (named HBL40), specific for complex-type N-glycans, which was isolated from a marine green alga, Halimeda borneensis. The hemagglutination activity of HBL40 was inhibited with both complex-type N-glycan and O-glycan-linked glycoproteins but not with high-mannose-type N-glycan-linked glycoproteins or any of the monosaccharides examined. In the oligosaccharide-binding experiment using 26 pyridylaminated oligosaccharides, HBL40 only bound to complex-type N-glycans with bi- and triantennary-branched sugar chains. The sialylation, core fucosylation, and the increased number of branched antennae of the N-glycans lowered the binding activity with HBL40. Interestingly, the lectin potently inhibited the infection of influenza virus (A/H3N2/Udorn/72) into NCI-H292 cells at IC50 of 8.02 nM by binding to glycosylated viral hemagglutinin (KD of 1.21 × 10-6 M). HBL40 consisted of two isolectins with slightly different molecular masses to each other that could be separated by reverse-phase HPLC. Both isolectins shared the same 16 N-terminal amino acid sequences. Thus, HBL40 could be useful as an antivirus lectin specific for complex-type N-glycans.

Enzymatic modular synthesis of asymmetrically branched human milk oligosaccharides.

Human milk oligosaccharides (HMOs) are intricate glycans that promote healthy growth of infants and have been incorporated into infant formula as food additives. Despite their importance, the limited availability of asymmetrically branched HMOs hinders the exploration of their structure and function relationships. Herein, we report an enzymatic modular strategy for the efficient synthesis of these HMOs. The key branching enzyme for the assembly of branched HMOs, human ß1,6-N-acetylglucosaminyltransferase 2 (GCNT2), was successfully expressed in Pichia pastoris for the first time. Then, it was integrated with six other bacterial glycosyltransferases to establish seven glycosylation modules. Each module comprises a one-pot multi-enzyme (OPME) system for in-situ generation of costly sugar nucleotide donors, combined with a glycosyltransferase for specific glycosylation. This approach enabled the synthesis of 31 branched HMOs and 13 linear HMOs in a stepwise manner with well-programmed synthetic routes. The binding details of these HMOs with related glycan-binding proteins were subsequently elucidated using glycan microarray assays to provide insights into their biological functions. This comprehensive collection of synthetic HMOs not only serves as standards for HMOs structure identification in complex biological samples but also significantly enhances the fields of HMOs glycomics, opening new avenues for biomedical applications.

CD301 and LSECtin glycan-binding receptors of innate immune cells serve as prognostic markers and potential predictors of immune response in breast cancer subtypes.

Glycosylation is a prominent posttranslational modification, and alterations in glycosylation are a hallmark of cancer. Glycan-binding receptors, primarily expressed on immune cells, play a central role in glycan recognition and immune response. Here, we used the recombinant C-type glycan-binding receptors CD301, Langerin, SRCL, LSECtin, and DC-SIGNR to recognize their ligands on tissue microarrays (TMA) of a large cohort (n = 1859) of invasive breast cancer of different histopathological types to systematically determine the relevance of altered glycosylation in breast cancer. Staining frequencies of cancer cells were quantified in an unbiased manner by a computer-based algorithm. CD301 showed the highest overall staining frequency (40%), followed by LSECtin (16%), Langerin (4%) and DC-SIGNR (0.5%). By Kaplan-Meier analyses, we identified LSECtin and CD301 as prognostic markers in different breast cancer subtypes. Positivity for LSECtin was associated with inferior disease-free survival in all cases, particularly in estrogen receptor positive (ER+) breast cancer of higher histological grade. In triple negative breast cancer, positivity for CD301 correlated with a worse prognosis. Based on public RNA single-cell sequencing data of human breast cancer infiltrating immune cells, we found CLEC10A (CD301) and CLEC4G (LSECtin) exclusively expressed in distinct subpopulations, particularly in dendritic cells and macrophages, indicating that specific changes in glycosylation may play a significant role in breast cancer immune response and progression.

Reusable glycan microarrays using a microwave assisted wet-erase (MAWE) process.

Modern studies on binding of proteins to glycans commonly involve the use of synthetic glycans and their derivatives in which a small amount of the material is covalently printed onto a functionalized slide in a glycan microarray format. While incredibly useful to explore binding interactions with many types of samples, the common techniques involve drying the slides, which leads to irreversible association of the protein to the spots on slides to which they bound, thus limiting a microarray to a single use. We have developed a new technique which we term Microwave Assisted Wet-Erase (MAWE) glycan microarrays. In this approach we image the slides under wet conditions to acquire the data, after which the slides are cleaned of binding proteins by treatment with a denaturing SDS solution along with microwave treatment. Slides cleaned in this way can be reused multiple times, and an example here shows the reuse of a single array 15 times. We also demonstrate that this method can be used for a single-array per slide or multi-array per slide platforms. Importantly, the results obtained using this technique for a variety of lectins sequentially applied to a single array, are concordant to those obtained via the classical dry approaches on multiple slides. We also demonstrate that MAWE can be used for different types of samples, such as serum for antibody binding, and whole cells, such as yeast. This technique will greatly conserve precious glycans and prolong the use of existing and new glycan microarrays.

The Pseudomonas aeruginosa lectin LecB modulates intracellular reactive oxygen species production in human neutrophils.

Pseudomonas aeruginosa is a Gram-negative bacterium and an opportunistic pathogen ubiquitously present throughout nature. LecB, a fucose-, and mannose-binding lectin, is a prominent virulence factor of P. aeruginosa, which can be expressed on the bacterial surface but also be secreted. However, the LecB interaction with human immune cells remains to be characterized. Neutrophils comprise the first line of defense against infections and their production of reactive oxygen species (ROS) and release of extracellular traps (NETs) are critical antimicrobial mechanisms. When profiling the neutrophil glycome we found several glycoconjugates on granule and plasma membranes that could potentially act as LecB receptors. In line with this, we here show that soluble LecB can activate primed neutrophils to produce high levels of intracellular ROS (icROS), an effect that was inhibited by methyl fucoside. On the other hand, soluble LecB inhibits P. aeruginosa-induced icROS production. In support of that, during phagocytosis of wild-type and LecB-deficient P. aeruginosa, bacteria with LecB induced less icROS production as compared with bacteria lacking the lectin. Hence, LecB can either induce or inhibit icROS production in neutrophils depending on the circumstances, demonstrating a novel and potential role for LecB as an immunomodulator of neutrophil functional responses.

The functional roles of protein glycosylation in human maternal-fetal crosstalk.

BACKGROUND: The establishment of maternal-fetal crosstalk is vital to a successful pregnancy. Glycosylation is a post-translational modification in which glycans (monosaccharide chains) are attached to an organic molecule. Glycans are involved in many physiological and pathological processes. Human endometrial epithelium, endometrial gland secretions, decidual immune cells, and trophoblasts are highly enriched with glycoconjugates and glycan-binding molecules important for a healthy pregnancy. Aberrant glycosylation in the placenta and uterus has been linked to repeated implantation failure and various pregnancy complications, but there is no recent review summarizing the functional roles of glycosylation at the maternal-fetal interface and their associations with pathological processes. OBJECTIVE AND RATIONALE: This review aims to summarize recent findings on glycosylation, glycosyltransferases, and glycan-binding receptors at the maternal-fetal interface, and their involvement in regulating the biology and pathological conditions associated with endometrial receptivity, placentation and maternal-fetal immunotolerance. Current knowledge limitations and future insights into the study of glycobiology in reproduction are discussed. SEARCH METHODS: A comprehensive PubMed search was conducted using the following keywords: glycosylation, glycosyltransferases, glycan-binding proteins, endometrium, trophoblasts, maternal-fetal immunotolerance, siglec, selectin, galectin, repeated implantation failure, early pregnancy loss, recurrent pregnancy loss, preeclampsia, and fetal growth restriction. Relevant reports published between 1980 and 2023 and studies related to these reports were retrieved and reviewed. Only publications written in English were included. OUTCOMES: The application of ultrasensitive mass spectrometry tools and lectin-based glycan profiling has enabled characterization of glycans present at the maternal-fetal interface and in maternal serum. The endometrial luminal epithelium is covered with highly glycosylated mucin that regulates blastocyst adhesion during implantation. In the placenta, fucose and sialic acid residues are abundantly presented on the villous membrane and are essential for proper placentation and establishment of maternal-fetal immunotolerance. Glycan-binding receptors, including selectins, sialic-acid-binding immunoglobulin-like lectins (siglecs) and galectins, also modulate implantation, trophoblast functions and maternal-fetal immunotolerance. Aberrant glycosylation is associated with repeated implantation failure, early pregnancy loss and various pregnancy complications. The current limitation in the field is that most glycobiological research relies on association studies, with few studies revealing the specific functions of glycans. Technological advancements in analytic, synthetic and functional glycobiology have laid the groundwork for further exploration of glycans in reproductive biology under both physiological and pathological conditions. WIDER IMPLICATIONS: A deep understanding of the functions of glycan structures would provide insights into the molecular mechanisms underlying their involvement in the physiological and pathological regulation of early pregnancy. Glycans may also potentially serve as novel early predictive markers and therapeutic targets for repeated implantation failure, pregnancy loss, and other pregnancy complications.

Taxonomic Distribution and Molecular Evolution of Mytilectins.

R-type lectins are a widespread group of sugar-binding proteins found in nearly all domains of life, characterized by the presence of a carbohydrate-binding domain that adopts a ß-trefoil fold. Mytilectins represent a recently described subgroup of ß-trefoil lectins, which have been functionally characterized in a few mussel species (Mollusca, Bivalvia) and display attractive properties, which may fuel the development of artificial lectins with different biotechnological applications. The detection of different paralogous genes in mussels, together with the description of orthologous sequences in brachiopods, supports the formal description of mytilectins as a gene family. However, to date, an investigation of the taxonomic distribution of these lectins and their molecular diversification and evolution was still lacking. Here, we provide a comprehensive overview of the evolutionary history of mytilectins, revealing an ancient monophyletic evolutionary origin and a very broad but highly discontinuous taxonomic distribution, ranging from heteroscleromorphan sponges to ophiuroid and crinoid echinoderms. Moreover, the overwhelming majority of mytilectins display a chimera-like architecture, which combines the ß-trefoil carbohydrate recognition domain with a C-terminal pore-forming domain, suggesting that the simpler structure of most functionally characterized mytilectins derives from a secondary domain loss.

Immune regulatory networks coordinated by glycans and glycan-binding proteins in autoimmunity and infection.

The immune system is coordinated by an intricate network of stimulatory and inhibitory circuits that regulate host responses against endogenous and exogenous insults. Disruption of these safeguard and homeostatic mechanisms can lead to unpredictable inflammatory and autoimmune responses, whereas deficiency of immune stimulatory pathways may orchestrate immunosuppressive programs that contribute to perpetuate chronic infections, but also influence cancer development and progression. Glycans have emerged as essential components of homeostatic circuits, acting as fine-tuners of immunological responses and potential molecular targets for manipulation of immune tolerance and activation in a wide range of pathologic settings. Cell surface glycans, present in cells, tissues and the extracellular matrix, have been proposed to serve as "self-associated molecular patterns" that store structurally relevant biological data. The responsibility of deciphering this information relies on different families of glycan-binding proteins (including galectins, siglecs and C-type lectins) which, upon recognition of specific carbohydrate structures, can recalibrate the magnitude, nature and fate of immune responses. This process is tightly regulated by the diversity of glycan structures and the establishment of multivalent interactions on cell surface receptors and the extracellular matrix. Here we review the spatiotemporal regulation of selected glycan-modifying processes including mannosylation, complex N-glycan branching, core 2 O-glycan elongation, LacNAc extension, as well as terminal sialylation and fucosylation. Moreover, we illustrate examples that highlight the contribution of these processes to the control of immune responses and their integration with canonical tolerogenic pathways. Finally, we discuss the power of glycans and glycan-binding proteins as a source of immunomodulatory signals that could be leveraged for the treatment of autoimmune inflammation and chronic infection.

Structure-based neural network protein-carbohydrate interaction predictions at the residue level.

Carbohydrates dynamically and transiently interact with proteins for cell-cell recognition, cellular differentiation, immune response, and many other cellular processes. Despite the molecular importance of these interactions, there are currently few reliable computational tools to predict potential carbohydrate-binding sites on any given protein. Here, we present two deep learning (DL) models named CArbohydrate-Protein interaction Site IdentiFier (CAPSIF) that predicts non-covalent carbohydrate-binding sites on proteins: (1) a 3D-UNet voxel-based neural network model (CAPSIF:V) and (2) an equivariant graph neural network model (CAPSIF:G). While both models outperform previous surrogate methods used for carbohydrate-binding site prediction, CAPSIF:V performs better than CAPSIF:G, achieving test Dice scores of 0.597 and 0.543 and test set Matthews correlation coefficients (MCCs) of 0.599 and 0.538, respectively. We further tested CAPSIF:V on AlphaFold2-predicted protein structures. CAPSIF:V performed equivalently on both experimentally determined structures and AlphaFold2-predicted structures. Finally, we demonstrate how CAPSIF models can be used in conjunction with local glycan-docking protocols, such as GlycanDock, to predict bound protein-carbohydrate structures.

(Glycan Binding) Activity-Based Protein Profiling in Cells Enabled by Mass Spectrometry-Based Proteomics.

The presence of glycan modifications at the cell surface and other locales positions them as key regulators of cell recognition and function. However, due to the complexity of glycosylation, the annotation of which proteins bear glycan modifications, which glycan patterns are present, and which proteins are capable of binding glycans is incomplete. Inspired by activity-based protein profiling to enrich for proteins in cells based on select characteristics, these endeavors have been greatly advanced by the development of appropriate glycan-binding and glycan-based probes. Here, we provide context for these three problems and describe how the capability of molecules to interact with glycans has enabled the assignment of proteins with specific glycan modifications or of proteins that bind glycans. Furthermore, we discuss how the integration of these probes with high resolution mass spectrometry-based technologies has greatly advanced glycoscience.

Anti-glycan monoclonal antibodies: Basic research and clinical applications.

Anti-glycan monoclonal antibodies have important applications in human health and basic research. Therapeutic antibodies that recognize cancer- or pathogen-associated glycans have been investigated in numerous clinical trials, resulting in two FDA-approved biopharmaceuticals. Anti-glycan antibodies are also utilized to diagnose, prognosticate, and monitor disease progression, as well as to study the biological roles and expression of glycans. High-quality anti-glycan mAbs are still in limited supply, highlighting the need for new technologies for anti-glycan antibody discovery. This review discusses anti-glycan monoclonal antibodies with applications to basic research, diagnostics, and therapeutics, focusing on recent advances in mAbs targeting cancer- and infectious disease-associated glycans.