In this chapter, we are presenting two assay formats for glycomic discovery and validation: Fluorescent Immune-Lectin Assay (FILA) and Fluorescent Lectin Assay (FLA). Both methods rely on conjugation of lectins to highly sensitive Europium nanoparticles (Eu-NPs) that provides specific detection of glycans. The FILA method is based on antibody immobilization, where biotinylated antibodies capture target mucin onto microplate wells before lectin-Eu-NP binding. In contrast, the FLA method is based on hydrophobic interactions that allow direct attachment of mucins or mucin-type glycoproteins to the plate surface without requiring a capturing antibody. The FLA method also use the same lectin-Eu-NP detection strategy. Each method is described in a detailed workflow from buffer and sample preparation, detection and obtaining signals, to concentration measurement. Evaluation protocols for both methods are provided at the end of this chapter. These assays provide effective and flexible strategies for screening of glycoproteins and mucin-type molecules and their glycosylation in complex biological samples such as plasma (e.g., lubricin in plasma) or mucosal surfaces (e.g., mucins in tear fluid). Orthogonal tests using mass spectrometry (MS) and western blot analysis are recommended to enhance certainty in glycosylation profiles obtained from either the FILA or FLA method. Ultimately, these methods support researchers to select and implement practical approaches for clinical and diagnostic applications in glycomics.

Mammalian mucosal surfaces are coated with excreted mucus composed primarily of large, heavily O-glycosylated proteins known as mucins. Such extensive O-glycosylation often makes mucin recalcitrant to conventional proteases, making mucins challenging to study by techniques requiring proteolysis. Many mucosa-associated microbes, however, encode mucolytic proteases (O-glycopeptidases or mucinases) that are O-glycan dependent. O-glycopeptidases display substantial diversity in both glycan and peptide sequence recognition, constituting a versatile toolkit for selective cleavage of glycoproteins bearing distinct glycan structures. Exploiting this diversity requires efficient methods for O-glycopeptidase discovery and characterization of substrate selectivity. Here, we describe a high-throughput FÖrster resonance energy transfer (FRET)-based assay for screening O-glycopeptidase activity and glycan selectivity.

Mucins are heavily glycosylated proteins that form protective mucus barriers at host-environment interfaces. Mucin genes frequently contain exonic variable number tandem repeat (exVNTR) domains that encode peptides enriched in proline, threonine, and serine. These repeat domains create substantial challenges for comparative and population genetic analyses because short-read sequencing often collapses repeat arrays and obscures haplotype structure. Recent advances in long-read sequencing, pangenome resources, and specialized VNTR analysis tools now enable systematic investigation of mucin genetics and evolution. In this chapter, we present practical protocols for identifying candidate mucin genes across species, annotating mucin exVNTRs from long-read genome assemblies, and genotyping exVNTR alleles in large short-read sequencing cohorts. We further outline analytical strategies for evaluating natural selection acting on mucin repeat domains. Together, these protocols enable systematic identification, structural resolution, and evolutionary analysis of mucin variation across species and human populations.

The intestinal mucus layer is a highly hydrated hydrogel that forms a critical barrier separating the gut microbiota from the epithelial surface while permitting nutrient exchange and luminal transport. Despite its central role in intestinal homeostasis, native mucus remains difficult to study due to its transparency, fragility, and poor preservation by conventional fixation methods, which often distort its structure or detach it from the epithelium. These limitations have restricted investigation of mucus organization, barrier properties, and enzymatic processes under physiological conditions. Here, we describe experimental approaches for the visualization and functional analysis of intestinal mucus using live ex vivo explant preparations combined with confocal microscopy. This system preserves the native architecture, cellular heterogeneity, and mucus attachment of intact epithelial tissue while allowing controlled experimental manipulation. Fluorescent lectin-based labeling enables resolution of spatially distinct mucus domains, including crypt-associated plumes and intercrypt mucus, which differ in glycosylation patterns and penetrability. Functional barrier properties are assessed using bead penetration assays, providing quantitative measures of mucus thickness and resistance to particle infiltration. In addition, the use of fluorescent probes and peptide substrates permits detection and spatial mapping of endogenous and microbial enzymatic activity within the mucus layer. These methods enable investigation of how enzymatic remodeling influences mucus structure and barrier integrity. Together, these protocols provide a versatile platform for studying mucus dynamics, organization, and function in a physiologically relevant context, bridging the gap between in vitro systems and in vivo studies.

A glycocalyx permeability assay for in situ assessment of transmembrane mucin barrier function ABSTRACT: The small intestine lacks a dense, attached mucus layer and instead relies on an apical cell surface glycocalyx as an alternative barrier mechanism to regulate host-microbiota interactions. However, quantitatively assessing the integrity of the glycocalyx barrier in a physiologically relevant setting has remained challenging. Here, we introduce the Glyco-TRAPP assay, a live imaging-based approach that enables direct, quantitative measurement of glycocalyx permeability and bacteria-epithelium interactions in intact intestinal tissue. By enabling direct functional assessment of barrier integrity in both human biopsies and experimental models, Glyco-TRAPP provides a powerful tool to uncover how molecular defects at the cell surface, compromising for example transmembrane mucin expression, localization, and glycosylation, impact glycocalyx barrier function. Glyco-TRAPP provides a functional readout that complements conventional structural or molecular analyses.

Mucins are highly glycosylated proteins, a subset of which form the structural basis of mucus barriers at mucosal surfaces. In the gastrointestinal tract, the secreted gel-forming mucin MUC2 is the principal component of the intestinal mucus layer and plays a critical role in maintaining host-microbiota homeostasis. MUC2 undergoes extensive post-translational glycosylation mediated by numerous glycosyltransferases, producing a dense network of O- and N-linked glycans that regulate mucus structure, microbial interactions, and barrier function. Disruption of mucus integrity has been implicated in a range of diseases including infection, inflammation, and cancer. Accurate visualization and quantification of mucus architecture are therefore essential for understanding mucus biology and its role in disease pathogenesis. Histological analysis remains the most accessible approach for studying mucus structure in situ. Recent work has revealed that the inner (barrier) layer can be subdivided into two distinct sublayers (b1 and b2) with different cellular origins and glycan compositions. These structural features necessitate analytical approaches capable of quantifying mucus thickness, spatial organization, and microbial proximity with high reproducibility. Here we describe a set of complementary workflows for the visualization and quantitative analysis of intestinal mucus. These include brightfield histochemical staining using Alcian Blue, fluorescence lectin labeling to resolve mucus sublayers, and combined mucin-bacterial fluorescence in situ hybridization for confocal imaging. In addition, we present automated image analysis pipelines implemented in ImageJ/Fiji for reproducible quantification of mucus thickness and spatial structure. Together, these approaches provide a practical framework for studying mucus barrier biology and mucus-microbe interactions in health and disease.

Mucins, the fundamental structural components of mucus, constitute a diverse family of complex glycoproteins. They are classified into two main categories: secreted mucins and surface-associated mucins, both of which serve critical roles in protecting and lubricating epithelial surfaces. The extensive glycosylation of mucins is pivotal to their diverse functions, influencing hydration, lubrication, microbial interactions and structural integrity. Glycans on mucin facilitate water retention and lubrication by binding and trapping water molecules. They also act as decoys, preventing microbial adhesion and mediating interactions with commensal and pathogenic bacteria, as well as attenuating microbial virulence factors. Furthermore, glycans contribute to the physical structure of mucus, supporting its barrier functions. Dysregulation of mucin glycosylation has been implicated in various pathologies, including cancer, Inflammatory bowel disease (IBD), and cystic fibrosis. Notably, specific patterns of mucin glycosylation have been used as biomarkers, for example in IBD, underscoring the importance of distinguishing between glycosylated vs non-glycosylated mucins.

M13 phage makes it possible to produce DNA-encoded display of any molecules stable in water by prospective DNA-barcoding. For example, chemical conjugation of a glycan to an M13 virion that contains a prospectively introduced DNA barcode in the M13 genome creates a DNA-barcoded glycophage. In this glycophage, there is a 1:1 correspondence between the DNA sequence inside the phage and the structure of the glycan displayed on the surface of the phage. The same construct simultaneously illustrates the concept of DNA-encoded multivalency because there is a one-to-one correspondence between the DNA barcode and the displayed density. In this chapter, we show that a collection of these DNA-barcoded multivalent glycophages, known as Liquid Glycan Array (LiGA), can measure the glycan-recognition profile of mucin-binding proteins. Using DNA-encoded multivalency, we make an intriguing observation that a simple ultra-dense display of D-galactose on M13 "looks like mucin" because it binds to the proteolytically inactive E447D mutant of mucinase StcE (StcEE447D). The binding is specific because StcEE447D does not bind to the ultra-high-density display of the enantiomer control, L-galactose, nor to the diastereomer controls (D-glucose and D-mannose).

Mucins are high-molecular-weight glycoproteins essential for the hydration, lubrication, and protective barrier functions of epithelial surfaces. Beyond these physical properties, their dense O-glycan brushes serve as potent multivalent ligands that regulate immune communication through interactions with sialic acid-binding immunoglobulin-like lectins (Siglecs) and other glycan-binding receptors. The translation of native mucins into functional biomaterials is often hindered by the mechanical instability of noncovalent assemblies, and investigations of mucin biology can require well-defined model mucin materials with controlled properties. This chapter presents a detailed protocol for domain-specific covalent crosslinking, a strategy to engineer mucin-based hydrogels with controlled architecture and bioactivity. By selectively targeting either the protein backbone (via carbodiimide/NHS coupling of bioorthogonal handles) or the glycan side chains (via mild periodate oxidation and oxyamine or reductive amination conjugation), the spatial arrangement and mesh architecture of the resulting hydrogel network can be dictated. The two crosslinking approaches yield materials with distinct gelation kinetics, protease susceptibility, pore structure, and immune-modulatory properties, while preserving the bioactive sialic acid residues critical for receptor engagement. The protocol covers the full workflow from mucin dissolution and chemical modification through purification, gelation, and characterization by NMR, HPLC-PAD, and rheometry. By providing a detailed roadmap for manipulating mucin macromolecular architecture, this chapter enables both the design of instructive biomaterials for drug delivery and tissue engineering, and the construction of well-defined model glycoprotein systems for fundamental research on glycan-mediated biology.

Mucin glycoproteins are the major structural components of mucus, imparting its gel consistency. Not only do mucins form a protective barrier that traps debris and keeps pathogenic bacteria at a safe distance from host cells, but they display structurally complex glycans that feed beneficial microbes and directly interact with microbial pathogens to regulate gene expression and virulence. This chapter provides a conceptual framework for mucin-microbe interaction studies and integrated protocols spanning the purification of biologically relevant mucins, isolation of complex glycan pools for mechanistic studies, and key experimental approaches for microbiological investigations of mucin function. Together, we hope these protocols will enable the exploration of yet-undiscovered roles of glycans in bacterial physiology and across domains of life.

This chapter describes practical workflows for the characterization and application of O-glycoproteases and mucinases in mass spectrometry-based analysis of mucin-domain glycoproteins. Dense O-glycosylation limits the effectiveness of conventional proteases, making specialized enzymes essential for generating informative peptides. We outline a stepwise strategy that begins with rapid gel-based assays to determine enzyme activity, glycan dependence, and sensitivity to sialylation. These qualitative experiments identify suitable substrates and guide optimization of enzyme to substrate ratios and digestion conditions. We then detail preparation of mucin samples for LC-MS/MS, including optional glycosidase treatment, controlled proteolysis, cleanup, and acquisition parameters that enable confident identification of glycopeptides. Particular emphasis is placed on interpreting fragmentation data and combining collisional and electron-based dissociation to determine peptide sequence and glycosite localization. Because automated searches remain error prone for O-glycopeptides, we provide guidelines for manual validation and for constructing cleavage motifs that incorporate both amino acid and glycan preferences. Together, these approaches establish a general framework for defining enzyme specificity and selecting appropriate tools for complex biological samples. Comprehensive characterization of mucin-degrading enzymes expands the analytical toolbox for studying mucin biology and improves the ability to map site-specific O-glycosylation with molecular precision.

Mucins are central to cellular health and their dysregulation is implicated in many diseases, including cancers. Detailing biophysical implications of mucin dysregulation could therefore be crucial to diagnostic and/or therapeutic development. Investigating relationships between mucin O-glycan sequence and structural properties is challenged by the inherent heterogeneity and flexibility of these macromolecules. Herein, we review an ecosystem of computational tools for conducting and analyzing molecular dynamics (MD) simulations of mucins. We expressely detail MD simulation procedures and showcase two published examples: the simulation of the canonical mucin MUC5B and T-cell immunoglobulin mucin domain containing proteins 3 and 4. We aim to increase accessibility of simulations to glycobiologists, and to increase awareness of mucins to computational chemists, with the goal of facilitating future mucin simulation efforts.

Cell surface glycans regulate key biological processes including immune signaling, cell communication, and pathogen recognition. Glycan-driven signaling is primarily mediated by glycan-binding proteins (lectins), whose functions depend on the identity and presentation of their glycoprotein ligands. However, identifying ligands for glycan-binding proteins remains challenging due to the structural complexity of carbohydrates and the importance of cellular context in determining binding specificity. Here, we describe a fluorescence-activated cell sorting (FACS)-based pooled CRISPR screening workflow for systematic identification of genetic factors that regulate lectin binding in living cells. The protocol covers lentiviral transduction of pooled sgRNA libraries and phenotypic selection of high- and low-lectin-binding populations by flow cytometry. Genomic DNA extraction, sequencing, and computational sgRNA enrichment analysis enable identification of genes influencing ligand biosynthesis and presentation. Subsequent analysis of these genetic factors can provide a comprehensive view of the structural determinants that govern lectin-glycan binding. The approach is compatible with CRISPR knockout, interference, and activation strategies, allowing broad interrogation of both loss- and gain-of-function effects. Key considerations for maintaining library coverage, optimizing sorting parameters, and performing robust statistical analysis are highlighted to maximize screening performance. Overall, this workflow offers a scalable framework for mapping glycan ligand landscapes in health and disease.

The cell-based mucin array is a versatile platform for expressing and interrogating recombinant mucin reporter proteins with representative patterning and customizable O-glycan structures. The platform is based on glycoengineered mammalian cell lines (HEK293/CHO), in which the glycosylation machinery is genetically rewritten to enable controlled display of specific O-glycan core structures and terminal epitopes with defined sialylation, fucosylation, and sulfation. Uniquely, the platform presents glycans in their native protein context, enabling investigation of how O-glycan density, clustering, and multivalency influence interactions with mucins. A conceptual framework for recognition of such glycan-context cues is provided by the patterned arrangement of O-glycans within mucin O-glycodomains, often composed of tandem repeat (TR) sequences with distinct O-glycosites resembling molecular barcodes. Mucin reporters mimic the serine-, threonine-, and proline-rich domains of natural mucins and mucin-like proteins or they can be designed as artificial Glycocarriers with model O-glycan cluster motifs. Reporters expressed as membrane-bound forms for cell display or as secreted fusion proteins for production can be applied in diverse bioassays. They have been used to probe glycan/mucin binding by viral and microbial adhesins, as well as human Siglec immune receptors. Moreover, the platform provides defined substrates for functional analyses of mucin and O-glycodomain degradation by microbial O-glycopeptidases or mucinases, revealing information of substrate specificities, cleavage points, and catalytic mechanisms. This chapter describes how the cell-based mucin array can be used to dissect interactions with mucins by glycan- and mucin-binding receptors as well as mucinases. The platform overcomes limitations of contemporary technologies by enabling studies with mimics of natural mucins and O-glycodomains that preserve clustered and patterned O-glycan contexts.

Mucins, the major polymeric glycoproteins constituting mucus hydrogels, are produced in a complex biosynthetic pathway during which numerous disulfide bonds and glycan modifications are introduced. High-resolution structures have been obtained for over-expressed mucin segments under conditions that mimic the Golgi apparatus, an important station for mucin modification in the secretory pathway. Golgi-like solution conditions promote the self-assembly of the mucin segments into compact, ordered states, which have the dual advantage of illuminating the mechanism for mucin polymerization and being amenable to analysis by single-particle cryo-electron microscopy (cryo-EM). While the structures of these ordered intermediate states may differ from the arrangement and properties of secreted, hydrated, full-length extracellular mucins in gels on epithelial surfaces, they provide insight into the organization of mucin globular domains, and in some cases "disordered" regions, and reveal fundamental aspects of mucin bioassembly. Extending cryo-EM studies to longer mucin fragments and additional mucins may expand our understanding of these remarkable macromolecules.