Glycosylation, a fundamental biochemical process, entails the covalent attachment of sugar molecules to proteins, DNA, or RNA. Beginning with an overview of the pathophysiological features of asthma, this review proceeds to elucidate various facets of glycosylation in asthma pathology, specifically in T2 high asthma and Th17-mediated responses. We examined glycosylation's involvement in regulating airway inflammation, encompassing the modulation of pro-inflammatory cytokine release such as IL-4, IL-5, and IL-13, key components of T2 inflammation, as well as its significance in modulating immune cell functionality, notably T cells and dendritic cells. Moreover, we explored glycosylation's impact on airway remodeling processes, including its regulation of airway smooth muscle cell proliferation and migration. Addressing molecular mechanisms, this review delved into several glycosylation modifications of proteins and genes implicated in asthma pathogenesis, including IgE, IL-4 receptor, TGF-β, and the regulation of select glycosylation enzymes. Additionally, the review highlights the role of Th17 cells in T2 high asthma and their modulation through glycosylation. We underscored future research imperatives, including biomarker discovery, therapeutic realization, and the potential utility of glycosylation modifications in asthma prevention and management. In short, this review provides an in-depth analysis of the critical role of glycosylation in the pathogenesis of T2 high asthma and Th17 responses.

Natural killer (NK) cells are critical components of the innate immune system. Their primary role is to induce apoptosis in target cells, such as cancerous or virally infected cells. These targets are recognized through interactions between activating or inhibitory receptors on the NK cell surface. Among the activating receptors is the natural cytotoxicity receptor NKp46. Several ligands for this receptor have been identified, including the epithelial adhesin Epa1 from the yeast Candida glabrata. Invasive candidiasis caused by this yeast is a significant complication for patients with hematological diseases. The interaction between NKp46 and Epa1 is thought to depend specifically on an O-glycan at threonine 225 of NKp46. To elucidate the molecular details of this interaction, we optimized the recombinant production of soluble NKp46 and Epa1, generated glycosylation variants of multiple NKp46 mutants, and evaluated the role of NKp46 glycosylation in Epa1 binding using microscale thermophoresis and isothermal titration calorimetry. Additionally, for the first time, we provide a comprehensive glycosylation profile of NKp46, determined through mass spectrometry of intact glycopeptides obtained by O-glycoprotease and trypsin proteolysis. Our findings demonstrate that the NKp46 stalk is glycosylated at multiple sites, involving both an N-glycan and more than one O-glycan. These glycans are critical for the interaction with Epa1, providing NK cells with enhanced sensitivity to Candida glabrata epitopes.

Glycosylation is an essential enzymatic process of building glycan structures that occur mainly within the cell and gives rise to a diversity of cell surface and secreted glycoconjugates. These glycoconjugates play vital roles, for instance in cellcell adhesion, interaction and communication, activation of cell surface receptors, inflammatory response and immune recognition. This controlled and wellcoordinated enzymatic process is altered in cancer, leading to the biosynthesis of cancerassociated glycans, which impact glycandependent biological roles.

In this review, the authors discuss the importance of targeting cancerassociated glycans through potent glycan biosynthesis inhibitors. It focuses on the use of analogs, providing an overview of findings involving these in cancer. The highly explored fluorinated monosaccharide analogs targeting aberrant glycosylation are described, aiming to inspire advances in the field.

Altered glycosylation, such as increased sialylation and fucosylation, is a feature in cancer and has been shown to play key roles in several malignant properties of cancer cells. Strategies aiming at remodeling cancer cells´ glycome are emerging and present a huge potential for cancer therapy. Fluorinated monosaccharides have been gathering promising preclinical results as novel cancer drugs. Nevertheless, cancer specific targeting strategies must be considered to avoid significant sideeffects.

Deciphering the pattern and abundance of O-glycosylation of mucin domain proteins, glycoproteins heavily implicated in cancer and other diseases, remains an ongoing challenge. Both the macro- and microheterogeneity of glycosylation complicates the analysis, motivating the development of new strategies for structural characterization of this diverse class of glycoproteins. Here we combine digestion of mucin domain proteins using a targeted protease, Enhancin from Serratia marcescens (SmE), with ultraviolet photodissociation (UVPD) mass spectrometry to advance glycan mapping and elucidation of O-glycosylation trends of densely glycosylated mucin proteins. UVPD facilitates identification of O-glycoforms of mucin domain proteins TIM-1, MUC-1 and MUC-16. Additionally, UVPD elucidates several glycoforms of MUC-16 and contributes to the discovery of O-glycosylation across tandem repeats of MUC-1.

Access to defined glycans and glycoconjugates is pivotal for discovery, dissection, and harnessing of a range of biological functions orchestrated by cellular glycosylation processes and the glycome. We previously employed genetic glycoengineering by nuclease-based gene editing to develop sustainable production of designer glycoprotein therapeutics and cell-based glycan arrays that display glycans in their natural context at the cell surface. However, access to human glycans in formats and quantities that allow structural studies of molecular interactions and use of glycans in biomedical applications currently rely on chemical and chemoenzymatic syntheses associated with considerable labor, waste, and costs. Here, we develop a sustainable and scalable method for production of glycans in glycoengineered mammalian cells by employing secreted Glycocarriers with repeat glycosylation acceptor sequence motifs for different glycans. The Glycocarrier technology provides a flexible production platform for glycans in different formats, including oligosaccharides, glycopeptides, and multimeric glycomodules, and offers wide opportunities for use in bioassays and biomedical applications.

Mucins, the biomolecular components of mucus, are glycoproteins that form a thick physical barrier at all tissue-air interfaces, forming a first line of defense against pathogens. Structural features of mucins and their interactions with other biomolecules remain largely unexplored due to the challenges associated with their high-resolution characterization. Combining limited mass spectrometry glycomics and protein sequencing data, we present all-atom, explicitly solvated molecular dynamics simulations of a major respiratory mucin, MUC5B. We detail key forces and degrees of freedom imposed by the extensive O-glycosylation, which imbue the canonically observed bottlebrush-like structures to these otherwise intrinsically disordered protein backbones. We compare our simulation results to static structures observed in recent scanning tunneling microscopy experiments as well as other published experimental efforts. Our work represents the demonstration of a workflow applied to a mucin example, which we hope will be employed by other groups to investigate the dynamics and interactions of other mucins, which can inform on structural details currently inaccessible to experimental techniques.

Multivalent interactions between receptors and glycans play an important role in many different biological processes, including pathogen infection, self-recognition, and the immune response. The growth in the number of tools and techniques toward the assembly of multivalent glycoconjugates means it is possible to create synthetic systems that more and more closely resemble the diversity and complexity we observe in nature. In this Perspective we present the background to the recognition and binding enabled by multivalent interactions in nature, and discuss the strategies used to construct synthetic glycoconjugate equivalents. We highlight key discoveries and the current state of the art in their applications to glycan arrays, vaccines, and other therapeutic and diagnostic tools, with an outlook toward some areas we believe are of most interest for future work in this area.

Glycosylation of proteins and lipids in mammals is essential for embryogenesis and the development of all tissues. Analyses of glycosylation mutants in cultured mammalian cells and model organisms have been key to defining glycosylation pathways and the biological functions of glycans. More recently, applications of genome sequencing have revealed the breadth of rare congenital disorders of glycosylation in humans and the influence of genetics on the synthesis of glycans relevant to infectious diseases, cancer progression and diseases of the immune system. This improved understanding of glycan synthesis and functions is paving the way for advances in the diagnosis and treatment of glycosylation-related diseases, including the development of glycoprotein therapeutics through glycosylation engineering.

Goblet cells (GCs) are specialised guardians lining the intestine. They play a critical role in gut defence and immune regulation. GCs continuously secrete mucus creating a physical barrier to protect from pathogens while harbouring symbiotic gut bacteria adapted to live within the mucus. GCs also form specialised GC-associated passages in a dynamic and regulated manner to deliver luminal antigens to immune cells, promoting gut tolerance and preventing inflammation. The composition of gut bacteria directly influences GC function, highlighting the intricate interplay between these components of a healthy gut. Indeed, imbalances in the gut microbiome can disrupt GC function, contributing to various gastrointestinal diseases like colorectal cancer, inflammatory bowel disease, cystic fibrosis, pathogen infections and liver diseases. This review explores the interplay between GCs and the immune system. We delve into the underlying mechanisms by which GC dysfunction contributes to the development and progression of gastrointestinal diseases. Finally, we examine current and potential treatments that target GCs and represent promising avenues for further investigation.

The chronic airway infections with Pseudomonas aeruginosa are the major co-morbidity in people with cystic fibrosis (CF). Within CF lungs, P. aeruginosa persists in the conducting airways together with human mucins as the most abundant structural component of its microenvironment. We investigated the adhesion of 41 serial CF airway P. aeruginosa isolates to airway mucin preparations from CF sputa. Mucins and bacteria were retrieved from five modulator-naïve patients with advanced CF lung disease. The P. aeruginosa isolates from CF airways and non-CF reference strains showed a strain-specific signature in their adhesion to ovine, porcine and bovine submaxillary mucins and CF airway mucins ranging from no or low to moderate and strong binding. Serial CF clonal isolates and colony morphotypes from the same sputum sample were as heterogeneous in their affinity to mucin as representatives of other clones thus making 'mucin binding' one of the most variable intraclonal phenotypic traits of P. aeruginosa known to date. Most P. aeruginosa CF airway isolates did not adhere more strongly to CF airway mucins than to plastic surfaces. The strong binders, however, exhibited a strain-specific affinity gradient to O-glycans, CF airway and mammalian submaxillary mucins.

The intestinal anaerobic bacterium Akkermansia muciniphila is specialized in the degradation of mucins, which are heavily O-glycosylated proteins that constitute the major components of the mucus lining the intestine. Despite that adhesion to mucins is considered critical for the persistence of A. muciniphila in the human intestinal tract, our knowledge of how this intestinal symbiont recognizes and binds to mucins is still limited. Here, we first show that the mucin-binding properties of A. muciniphila are independent of environmental oxygen concentrations and not abolished by pasteurization. We then dissected the mucin-binding properties of pasteurized A. muciniphila by use of a recently developed cell-based mucin array that enables display of the tandem repeats of human mucins with distinct O-glycan patterns and structures. We found that A. muciniphila recognizes the unsialylated LacNAc (Galβ1-4GlcNAcβ1-R) disaccharide selectively on core2 and core3 O-glycans. This disaccharide epitope is abundantly found on human colonic mucins capped by sialic acids, and we demonstrated that endogenous A. muciniphila neuraminidase activity can uncover the epitope and promote binding. In summary, our study provides insights into the mucin-binding properties important for colonization of a key mucin-foraging bacterium.

Targeted protein degradation is an emerging strategy for the elimination of classically undruggable proteins. Here, to expand the landscape of targetable substrates, we designed degraders that achieve substrate selectivity via recognition of a discrete peptide and glycan motif and achieve cell-type selectivity via antigen-driven cell-surface binding. We applied this approach to mucins, O-glycosylated proteins that drive cancer progression through biophysical and immunological mechanisms. Engineering of a bacterial mucin-selective protease yielded a variant for fusion to a cancer antigen-binding nanobody. The resulting conjugate selectively degraded mucins on cancer cells, promoted cell death in culture models of mucin-driven growth and survival, and reduced tumor growth in mouse models of breast cancer progression. This work establishes a blueprint for the development of biologics that degrade specific protein glycoforms on target cells.

Glycoproteomics has accelerated in recent decades owing to numerous innovations in the analytical workflow. In particular, new mass spectrometry strategies have contributed to inroads in O-glycoproteomics, a field that lags behind N-glycoproteomics due to several unique challenges associated with the complexity of O-glycosylation. This review will focus on progress in sample preparation, enrichment strategies, and MS/MS techniques for the identification and characterization of O-glycoproteins.

Proteoglycans are a small but diverse family of proteins that play a wide variety of roles at the cell surface and in the extracellular matrix. In addition to their glycosaminoglycan (GAG) chains, they are N- and O-glycosylated. All of these types of glycosylation are crucial to their function but present a considerable analytical challenge. We describe the combination of serial proteolysis followed by the application of higher-energy collisional dissociation (HCD) and electron transfer/higher-energy collisional dissociation (EThcD) to optimize protein sequence coverage and glycopeptide identification from proteoglycans. In many cases, the use of HCD alone allows the identification of more glycopeptides. However, the localization of glycoforms on multiply glycosylated peptides has remained elusive. We demonstrate the use of EThcD for the confident assignment of glycan compositions on multiply glycosylated peptides. Dense glycosylation on proteoglycans is key to their biological function; thus, developing tools to identify and quantify doubly glycosylated peptides is of interest. Additionally, glycoproteomics searches identify glycopeptides in otherwise poorly covered regions of proteoglycans. The development of these and other analytical tools may permit glycoproteomic similarity comparisons in biological samples.

Proteins and lipids decorated with glycans are found throughout biological entities, playing roles in biological functions and dysfunctions. Current analytical strategies for these glycan-decorated biomolecules, termed glycoconjugates, rely on ensemble-averaged methods that do not provide a full view of positions and structures of glycans attached at individual sites in a given molecule, especially for glycoproteins. We show single-molecule analysis of glycoconjugates by direct imaging of individual glycoconjugate molecules using low-temperature scanning tunneling microscopy. Intact glycoconjugate ions from electrospray are soft-landed on a surface for their direct single-molecule imaging. The submolecular imaging resolution corroborated by quantum mechanical modeling unveils whole structures and attachment sites of glycans in glycopeptides, glycolipids, N-glycoproteins, and O-glycoproteins densely decorated with glycans.

Repeat proteins are common in all domains of life and exhibit a wide range of functions. One class of repeat protein contains solenoid folds where the repeating unit consists of β-strands separated by tight turns. β-solenoids have distinguishing structural features such as handedness, twist, oligomerisation state, coil shape and size which give rise to their diversity. Characterised β-solenoid repeat proteins are known to form regions in bacterial and viral virulence factors, antifreeze proteins and functional amyloids. For many of these proteins, the experimental structure has not been solved, as they are difficult to crystallise or model. Here we use various deep learning-based structure-modelling methods to discover novel predicted β-solenoids, perform structural database searches to mine further structural neighbours and relate their predicted structure to possible functions. We find both eukaryotic and prokaryotic adhesins, confirming a known functional linkage between adhesin function and the β-solenoid fold. We further identify exceptionally long, flat β-solenoid folds as possible structures of mucin tandem repeat regions and unprecedentedly small β-solenoid structures. Additionally, we characterise a novel β-solenoid coil shape, the FapC Greek key β-solenoid as well as plausible complexes between it and other proteins involved in Pseudomonas functional amyloid fibres.

Mucin-domain glycoproteins expressed on cancer cell surfaces play central roles in cell adhesion, cancer progression, stem cell renewal, and immune evasion. Despite abundant evidence that mucin-domain glycoproteins are critical to the pathobiology of head and neck squamous cell carcinoma (HNSCC), our knowledge of the composition of that mucinome is grossly incomplete. Here, we utilized a catalytically inactive point mutant of the enzyme StcE (StcE^E447D) to capture mucin-domain glycoproteins in head and neck cancer cell line lysates followed by their characterization using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), in-gel digestion, nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS), and enrichment analyses. We demonstrate the feasibility of this workflow for the study of mucin-domain glycoproteins in HNSCC, identify a set of mucin-domain glycoproteins common to multiple HNSCC cell lines, and report a subset of mucin-domain glycoproteins that are uniquely expressed in HSC-3 cells, a cell line derived from a highly aggressive metastatic tongue squamous cell carcinoma. This effort represents the first attempt to identify mucin-domain glycoproteins in HNSCC in an untargeted, unbiased analysis, paving the way for a more comprehensive characterization of the mucinome components that mediate aggressive tumor cell phenotypes. Data associated with this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD029420.

ConspectusN-Acetylgalactosamine (GalNAc)-type O-glycosylation is an essential posttranslational modification (PTM) that plays fundamental roles in biology. Malfunction of this PTM is exemplified by the presence of truncated O-glycans in cancer. For instance, the glycoprotein MUC1 is overexpressed in many tumor tissues and tends to carry simple oligosaccharides that allow for the presentation of different tumor-associated antigens, such as the Tn or sTn antigens (GalNAc-α-1-O-Thr/Ser and Neu5Acα2-6GalNAcα1-O-Ser/Thr, respectively). In other cases, such as tumoral calcinosis associated with O-glycosylation of the fibroblast growth factor 23, O-glycans are absent or less abundant. Significant progress has been made in determining the three-dimensional structures of biomolecules that recognize GalNAc, such as antibodies, lectins, mucinases, GalNAc-transferases, and other glycosyltransferases. Analysis of the complexes between these entities and GalNAc-containing glycopeptides, in most cases derived from crystallographic or NMR analysis, provides an understanding of the key structural elements that control molecular recognition of these glycopeptides. Here, we describe and compare the binding sites of these proteins in detail, focusing on how the GalNAc moieties interact selectively with them. We also summarize the differences and similarities in GalNAc recognition. In general, the recognition of GalNAc-containing glycopeptides is determined by hydrogen bonds between hydroxyl groups and the N-acetyl group of GalNAc with proteins, as well as CH-π contacts in which the hydrophobic α-face of the sugar and the methyl group of NHAc can be involved. The latter interaction usually provides the basis for selectivity. It is worth noting that binding of these glycopeptides depends primarily on recognition of the sugar moiety, with some exceptions such as a few anti-MUC1 antibodies that primarily recognize the peptide backbone and use the sugar to facilitate shape complementarity or to establish a limited number of interactions with the protein. Focusing specifically on the GalNAc moiety, we can observe that there is some degeneracy of interactions within the same protein families, likely due to substrate flexibility. However, when all studied proteins are considered together, despite the commonalities within each protein family, no pattern can be discerned between the different families, apart from the presence of common residues such as Tyr, His, or Asp, which are responsible for hydrogen bonds. The lack of a pattern can be anticipated, given the diverse functions of mucinases, glycosyltransferases, antibodies, and lectins. Finally, it is important to point out that the conformational differences observed in solution in glycopeptides bearing GalNAc-α-1-O-Ser or GalNAc-α-1-O-Thr also can be found in the bound state. This unique characteristic is exploited, for instance, by the enzyme C1GalT1 to broadly glycosylate both acceptor substrates. The findings summarized in this review may contribute to the rational structure-guided development of therapeutic vaccines, novel diagnostic tools for early cancer detection, and new cancer treatments for cancer with tailored anti-Tn or anti-STn antibodies or new drugs to inhibit GalNAc-T isoenzymes.

A variety of glycan structures cover the surface of all cells and are involved in myriad biological processes, including but not limited to, cell adhesion and communication, protein quality control, signal transduction and metabolism, while also being intimately involved in innate and adaptive immune functions. Immune surveillance and responses to foreign carbohydrate antigens, such as capsular polysaccharides on bacteria and surface protein glycosylation of viruses, are the basis of microbial clearance, and most antimicrobial vaccines target these structures. In addition, aberrant glycans on tumors called Tumor-Associated Carbohydrate Antigens (TACAs) elicit immune responses to cancer, and TACAs have been used in the design of many antitumor vaccine constructs. A majority of mammalian TACAs are derived from what are referred to as mucin-type O-linked glycans on cell-surface proteins and are linked to the protein backbone through the hydroxyl group of either serine or threonine residues. A small group of structural studies that have compared mono- and oligosaccharides attached to each of these residues have shown that there are distinct differences in conformational preferences assumed by glycans attached to either "unmethylated" serine or ß-methylated threonine. This suggests that the linkage point of antigenic glycans will affect their presentation to the immune system as well as to various carbohydrate binding molecules (e.g., lectins). This short review, followed by our hypothesis, will examine this possibility and extend the concept to the presentation of glycans on surfaces and in assay systems where recognition of glycans by proteins and other binding partners can be defined by different attachment points that allow for a range of conformational presentations.

Mucins represent a largely untapped class of polymeric building block for biomaterials, therapeutics, and other biotechnology. Because the mucin polymer backbone is genetically encoded, sequence-specific mucins with defined physical and biochemical properties can be fabricated using recombinant technologies. The pendent O-glycans of mucins are increasingly implicated in immunomodulation, suppression of pathogen virulence, and other biochemical activities. Recent advances in engineered cell production systems are enabling the scalable synthesis of recombinant mucins with precisely tuned glycan side chains, offering exciting possibilities to tune the biological functionality of mucin-based products. New metabolic and chemoenzymatic strategies enable further tuning and functionalization of mucin O-glycans, opening new possibilities to expand the chemical diversity and functionality of mucin building blocks. In this review, we discuss these advances, and the opportunities for engineered mucins in biomedical applications ranging from in vitro models to therapeutics.

The large family of polypeptide GalNAc-transferases (GalNAc-Ts) controls with precision how GalNAc O-glycans are added in the tandem repeat regions of mucins (e.g., MUC1). However, the structural features behind the creation of well-defined and clustered patterns of O-glycans in mucins are poorly understood. In this context, herein, we disclose the full process of MUC1 O-glycosylation by GalNAc-T2/T3/T4 isoforms by NMR spectroscopy assisted by molecular modeling protocols. By using MUC1, with four tandem repeat domains as a substrate, we confirmed the glycosylation preferences of different GalNAc-Ts isoforms and highlighted the importance of the lectin domain in the glycosylation site selection after the addition of the first GalNAc residue. In a glycosylated substrate, with yet multiple acceptor sites, the lectin domain contributes to orientate acceptor sites to the catalytic domain. Our experiments suggest that during this process, neighboring tandem repeats are critical for further glycosylation of acceptor sites by GalNAc-T2/T4 in a lectin-assisted manner. Our studies also show local conformational changes in the peptide backbone during incorporation of GalNAc residues, which might explain GalNAc-T2/T3/T4 fine specificities toward the MUC1 substrate. Interestingly, we postulate that a specific salt-bridge and the inverse γ-turn conformation of the PDTRP sequence in MUC1 are the main structural motifs behind the GalNAc-T4 specificity toward this region. In addition, in-cell analysis shows that the GalNAc-T4 isoform is the only isoform glycosylating the Thr of the immunogenic epitope PDTRP in vivo, which highlights the relevance of GalNAc-T4 in the glycosylation of this epitope. Finally, the NMR methodology established herein can be extended to other glycosyltransferases, such as C1GalT1 and ST6GalNAc-I, to determine the specificity toward complex mucin acceptor substrates.