Analysis of Mucin-Domain Glycoproteins Using Mass Spectrometry.

Mucin-domain glycoproteins are characterized by their high density of glycosylated serine and threonine residues, which complicates their analysis by mass spectrometry. The dense glycosylation renders the protein backbone inaccessible to workhorse proteases like trypsin, the vast heterogeneity of glycosylation often results in ion suppression from unmodified peptides, and search algorithms struggle to confidently analyze and site-localize O-glycosites. We have made a number of advances to address these challenges, rendering mucinomics possible for the first time. Here, we summarize these contributions and provide a detailed protocol for mass spectrometric analysis of mucin-domain glycoproteins. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Enrichment of mucin-domain glycoproteins Basic Protocol 2: Enzymatic digestion of mucin-domain glycoprotein(s) Basic Protocol 3: Mass spectrometry data collection for O-glycopeptides Basic Protocol 4: Mass spectrometry data analysis of O-glycopeptides.

Comprehensive Characterization of the Viscoelastic Properties of Bovine Submaxillary Mucin (BSM) Hydrogels and the Effect of Additives.

This study presents a comprehensive characterization of the viscoelastic and structural properties of bovine submaxillary mucin (BSM), which is widely used as a commercial source to conduct mucus-related research. We conducted concentration studies of BSM and examined the effects of various additives, NaCl, CaCl2, MgCl2, lysozyme, and DNA, on its rheological behavior. A notable connection between BSM concentration and viscoelastic properties was observed, particularly under varying ionic conditions. The rheological spectra could be well described by a fractional Kelvin-Voigt model with a minimum of model parameters. A detailed proteomics analysis provided insight into the protein, especially mucin composition within BSM, showing MUC19 as the main component. Cryo-scanning electron microscopy enabled the visualization of the porous BSM network structure. These investigations give us a more profound comprehension of the BSM properties, especially those pertaining to viscoelasticity, and how they are influenced by concentration and environmental conditions, aspects relevant to the field of mucus research.

Insights into the Chemistry, Structure, and Biological Activity of Human Salivary MUC7 Fragments and Their Cu(II) and Zn(II) Complexes.

Mucin 7 (MUC7) is one of the salivary proteins whose role in the innate immune system is widely known, but still, neither its mechanism of action nor the impact of its metal coordination is fully understood. MUC7 and its fragments demonstrate potent antimicrobial activity, serving as a natural defense mechanism for organisms against pathogens. This study delves into the bioinorganic chemistry of MUC7 fragments (L1─EGRERDHELRHRRHHHQSPK; L2─EGRERDHELRHRR; L3─HHHQSPK) and their complexes with Cu(II) and Zn(II) ions. The antimicrobial characteristics of the investigated peptides and their complexes were systematically assessed against bacterial and fungal strains at pH 5.40 and pH 7.40. Our findings highlight the efficacy of these systems against Streptococcus sanguinis, a common oral cavity pathogen. Most interestingly, Zn(II) coordination increased (or triggered) the MUC7 antimicrobial activity, which underscores the pivotal role of metal ion coordination in governing the antimicrobial activity of human salivary MUC7 fragments against S. sanguinis.

The forecasting power of the mucin-microbiome interplay in livestock respiratory diseases.

Complex respiratory diseases are a significant challenge for the livestock industry worldwide. These diseases considerably impact animal health and welfare and cause severe economic losses. One of the first lines of pathogen defense combines the respiratory tract mucus, a highly viscous material primarily composed of mucins, and a thriving multi-kingdom microbial ecosystem. The microbiome-mucin interplay protects from unwanted substances and organisms, but its dysfunction may enable pathogenic infections and the onset of respiratory disease. Emerging evidence also shows that noncoding regulatory RNAs might modulate the structure and function of the microbiome-mucin relationship. This opinion paper unearths the current understanding of the triangular relationship between mucins, the microbiome, and noncoding RNAs in the context of respiratory infections in animals of veterinary interest. There is a need to look at these molecular underpinnings that dictate distinct health and disease outcomes to implement effective prevention, surveillance, and timely intervention strategies tailored to the different epidemiological contexts.

Collagen/gelatin and polysaccharide complexes enhance gastric retention and mucoadhesive properties.

This article has focused on collagen-gelatin, the gelation process, as well as blend interaction between collagen/gelatin with various polysaccharides to boost mucoadhesion and gastric retention. The interaction between mucoadhesive materials and mucin layers is of significant interest in the development of drug delivery systems and biomedical applications for effective targeting and prolonged time in the gastrointestinal tract. This paper reviews the current advancement and mucoadhesive properties of collagen/gelatin and different polysaccharide complexes concerning the mucin layer and interactions are briefly highlighted. Collagen/gelatin and polysaccharide blends biocompatible and biodegradable, the complex biomolecules have shown encouraging mucoadhesive properties due to their cationic nature and ability to form hydrogen bonds with mucin glycoproteins. The mucoadhesion mechanism was attributed to the electrostatic interactions between the positively charged amino (NH2) groups of blend biopolymers and the negatively charged sialic acid residues present in mucin glycoprotein. At the end of this article, the encouraging prospect of collagen/polysaccharide complex and mucin glycoprotein is highlighted.

Overcoming antibiotic resistance caused by genetic mutations of Helicobacter pylori with mucin adhesive polymer-based therapeutics.

Herein, we describe the 3'-sialyllactose-polyethyleneimine-chlorine e6 conjugate (3PC), meticulously engineered to effectively target Helicobacter bacteria (H. pylori) within the gastric environment. The composition of 3PC comprises polyethyleneimine, a cationic polymer, 3'-sialyllactose, which exhibits a specific binding affinity for H. pylori surface proteins, and a photosensitizer capable of generating oxygen radicals in response to specific wavelengths. The distinctive feature of 3PC lies in its capacity to enhance interaction with the anionic mucus layer facilitated by electrostatic forces. This interaction results in prolonged residence within the intestinal environment. The extended vacation in the intestinal milieu overcomes inherent limitations that have historically impeded conventional antibiotics from efficiently reaching and targeting H. pylori. 3PC can be harnessed as a potent tool for antibacterial photodynamic therapy, and its versatility extends to addressing the challenges posed by various antibiotic-resistant strains. The exceptional efficacy of 3PC in enhancing intestinal residence time and eradicating H. pylori has been robustly substantiated in animal models, particularly in mice. In summary, 3PC is a formidable agent capable of eradicating H. pylori, irrespective of its antibiotic resistance status, by efficiently penetrating and selectively targeting the mucus layer within the gastric environment.

Mucus-Mimicking Mucin-Based Hydrogels by Tandem Chemical and Physical Crosslinking.

Mucosal tissues represent a major interface between the body and the external environment and are covered by a highly hydrated mucins gel called mucus. Mucus lubricates, protects and modulates the moisture levels of the tissue and is capitalized in transmucosal drug delivery. Pharmaceutical researchers often use freshly excised animal mucosal membranes to assess mucoadhesion and muco-penetration of pharmaceutical formulations which may struggle with limited accessibility, reproducibility, and ethical questions. Aiming to develop a platform for the rationale study of the interaction of drugs and delivery systems with mucosal tissues, in this work mucus-mimicking mucin-based hydrogels are synthesized by the tandem chemical and physical crosslinking of mucin aqueous solutions. Chemical crosslinking is achieved with glutaraldehyde (0.3% and 0.75% w/v), while physical crosslinking by one or two freeze-thawing cycles. Hydrogels after one freeze-thawing cycle show water content of 97.6-98.1%, density of 0.0529-0.0648 g cm⁻3, and storage and loss moduli of ≈40-60 and ≈3-5 Pa, respectively, that resemble the properties of native gastrointestinal mucus. The mechanical stability of the hydrogels increases over the number of freeze-thawing cycles. Overall results highlight the potential of this simple, reproducible, and scalable method to produce artificial mucus-mimicking hydrogels for different applications in pharmaceutical research.

Mass Spectrometry-Compatible Elution Technique Enables an Improved Mucin-Selective Enrichment Strategy to Probe the Mucinome.

Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of healthy and disease-driven biological functions. Previously, we developed a mucin-selective enrichment strategy by employing a catalytically inactive mucinase (StcE) conjugated to a solid support. While this method was effective, it suffered from low throughput and high sample requirements. Further, the elution step required boiling in SDS, thus necessitating an in-gel digest with trypsin. Here, we introduce innovative elution conditions amenable to mucinase digestion and downstream analysis using mass spectrometry. This increased throughput and lowered sample input while maintaining mucin selectivity and enhancing the glycopeptide signal. We then benchmarked this technique against different O-glycan binding moieties for their ability to enrich mucins from various cell lines and human serum. Overall, the new method outperformed our previous procedure and all of the other enrichment techniques tested. This allowed for the effective isolation of more mucin-domain glycoproteins, resulting in a high number of O-glycopeptides, thus enhancing our ability to analyze the mucinome.

Characterization of intestinal O-glycome in reactive oxygen species deficiency.

Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation resulting from an inappropriate inflammatory response to intestinal microbes in a genetically susceptible host. Reactive oxygen species (ROS) generated by NADPH oxidases (NOX) provide antimicrobial defense, redox signaling and gut barrier maintenance. NADPH oxidase mutations have been identified in IBD patients, and mucus layer disruption, a critical aspect in IBD pathogenesis, was connected to NOX inactivation. To gain insight into ROS-dependent modification of epithelial glycosylation the colonic and ileal mucin O-glycome of mice with genetic NOX inactivation (Cyba mutant) was analyzed. O-glycans were released from purified murine mucins and analyzed by hydrophilic interaction ultra-performance liquid chromatography in combination with exoglycosidase digestion and mass spectrometry. We identified five novel glycans in ileum and found minor changes in O-glycans in the colon and ileum of Cyba mutant mice. Changes included an increase in glycans with terminal HexNAc and in core 2 glycans with Fuc-Gal- on C3 branch, and a decrease in core 3 glycans in the colon, while the ileum showed increased sialylation and a decrease in sulfated glycans. Our data suggest that NADPH oxidase activity alters the intestinal mucin O-glycans that may contribute to intestinal dysbiosis and chronic inflammation.

An unusual dual sugar-binding lectin domain controls the substrate specificity of a mucin-type O-glycosyltransferase.

N-acetylgalactosaminyl-transferases (GalNAc-Ts) initiate mucin-type O-glycosylation, an abundant and complex posttranslational modification that regulates host-microbe interactions, tissue development, and metabolism. GalNAc-Ts contain a lectin domain consisting of three homologous repeats (α, ß, and γ), where α and ß can potentially interact with O-GalNAc on substrates to enhance activity toward a nearby acceptor Thr/Ser. The ubiquitous isoenzyme GalNAc-T1 modulates heart development, immunity, and SARS-CoV-2 infectivity, but its substrates are largely unknown. Here, we show that both α and ß in GalNAc-T1 uniquely orchestrate the O-glycosylation of various glycopeptide substrates. The α repeat directs O-glycosylation to acceptor sites carboxyl-terminal to an existing GalNAc, while the ß repeat directs O-glycosylation to amino-terminal sites. In addition, GalNAc-T1 incorporates α and ß into various substrate binding modes to cooperatively increase the specificity toward an acceptor site located between two existing O-glycans. Our studies highlight a unique mechanism by which dual lectin repeats expand substrate specificity and provide crucial information for identifying the biological substrates of GalNAc-T1.

Water Sorption and Structural Properties of Human Airway Mucus in Health and Muco-Obstructive Diseases.

Muco-obstructive diseases change airway mucus properties, impairing mucociliary transport and increasing the likelihood of infections. To investigate the sorption properties and nanostructures of mucus in health and disease, we investigated mucus samples from patients and cell cultures (cc) from healthy, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) airways. Atomic force microscopy (AFM) revealed mucin monomers with typical barbell structures, where the globule to spacer volume ratio was the highest for CF mucin. Accordingly, synchrotron small-angle X-ray scattering (SAXS) revealed more pronounced scattering from CF mucin globules and suggested shorter carbohydrate side chains in CF mucin and longer side chains in COPD mucin. Quartz crystal microbalance with dissipation (QCM-D) analysis presented water sorption isotherms of the three types of human airway mucus, where, at high relative humidity, COPD mucus had the highest water content compared to cc-CF and healthy airway mucus (HAM). The higher hydration of the COPD mucus is consistent with the observation of longer side chains of the COPD mucins. At low humidity, no dehydration-induced glass transition was observed in healthy and diseased mucus, suggesting mucus remained in a rubbery state. However, in dialyzed cc-HAM, a sorption-desorption hysteresis (typically observed in the glassy state) appeared, suggesting that small molecules present in mucus suppress the glass transition.

Extraction of Mucins from the Mammalian Intestinal Tract.

In the intestine, mucus covering the mucosa plays a critical role in maintaining gut homeostasis by protecting the mucosa from invasion by commensal bacteria. The gut mucus is composed primarily of MUC2 mucin secreted by goblet cells. MUC2 is highly O-glycosylated, and O-glycans are necessary for the function and polymer structure of MUC2. In addition, recent evidence revealed that several glycan modifications, such as sialylation and sulfation, confer resistance of mucins to proteolysis and affect the viscosity and lubricity of mucus. Therefore, characterizing glycan structures of mucins is required to understand their functions fully. In this chapter, we describe how to purify secreted mucins from the mammalian intestine for analysis of their glycan structures. This description includes the extraction of MUC2 mucin from the mucosal surface of the mouse colon and colon explants.

Preparation of Jellyfish Mucin.

A mucin-type glycoprotein extracted from various species of jellyfish (JF) is named qniumucin (Q-mucin). Compared with general mucins, most of which are from mammals including humans, Q-mucin can be collected on a relatively large scale with high yield. Owing to its simple structure with low heterogeneity, Q-mucin has a potential to be developed into material mucins which opens various applications valuable to humans. On the basis of our present knowledge, here, we describe our protocol for the extraction of Q-mucin, which can be extracted from any JF species worldwide. Experimental protocols to identify the structure of Q-mucin are also introduced.

Supported Molecular Matrix Electrophoresis.

Distinct bands of mucins cannot be banded using a gel electrophoresis based on a molecular sieving effect due to their very large molecular weight and remarkable diversity in glycosylation. In contrast, membrane electrophoresis can separate mucins as round bands. Here, we present an analysis of mucin separation via membrane electrophoresis using a porous polyvinylidene difluoride membrane, which is highly stable against chemical modifications and various organic solvents. The separated mucins can not only be stained with dyes but also with antibodies and lectins, and glycans can be released from the excised bands and analyzed.

9-Fluorenylmethyl Chloroformate Labeling for O-Glycan Analysis.

Structural analysis of O-glycans from mucins and characterization of the interaction of these glycans with other biomolecules are essential for a full understanding of mucins. Various techniques have been developed for the structural and functional analysis of glycans. While 9-fluorenylmethyl chloroformate (Fmoc-Cl) is generally used to protect amino groups in peptide synthesis, it can also be used as a glycan-labeling reagent for structural analysis. Fmoc-labeled glycans are strongly fluorescent and can be analyzed with high sensitivity using liquid chromatography-fluorescence detection (LC-FD) analysis as well as being analyzed with high sensitivity by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Fmoc-labeled glycans can be easily delabeled and converted to glycosylamine-form or free (hemiacetal or aldehyde)-form glycans that can be used to fabricate glycan arrays or synthesize glycosyl dendrimers. This derivatization allows for the isolation from biological samples of glycans that are difficult to synthesize chemically, as well as the fabrication of immobilized-glycan devices. The Fmoc labeling method promises to be a tool for accelerating O-glycan structural analysis and an understanding of molecular interactions. In this chapter, we introduce the Fmoc labeling method for analysis of O-glycans and fabrication of O-glycan arrays.

Preparation of Mucin Glycopeptides by Organic Synthesis.

Mucins are sugar-rich glycoproteins. Glycoprotein sugar moieties are structurally diverse, making it difficult to obtain naturally pure glycoproteins. Chemical synthesis is a powerful tool for obtaining target or designed compounds. Automated peptide synthesizers are commercially available, and many use the solid-phase peptide synthesis (SPPS) method. In addition, some of these synthesizers apply microwave irradiation to obtain higher reaction yields, thereby enabling the synthesis of 40 to 50 amino acid residual glycopeptides. Theoretically, glycopeptides can be synthesized using methods similar to those used for peptide synthesis, but glycosylated amino acid synthons are less stable than amino acid synthons and are also very expensive. Therefore, they are not suitable for use in large excess amounts. Many of oligosaccharide-linked amino acid synthons are not commercially available, so they must be specially prepared, and they also require careful handling that demands specific organic synthesis experience and techniques. However, monosaccharide-linked amino acid synthons are commercially available and are relatively easy to handle. Here, as an entry into glycopeptide synthesis, we describe a typical glycopeptide synthesis procedure for a 27 amino acid residual MUC1 repeating unit with monosaccharides.

MALDI-TOF MS/MS Analysis of Permethylated O-Glycan Alditols Released from Mucins.

Mucin glycans are associated with the function of mucin in maintaining mucosal homeostasis. Therefore, the glycomic analysis of mucins is crucial. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most suitable methods for the glycomic analysis of mucin O-glycans. In this chapter, we describe methods for analyzing permethylated O-glycan alditols released from mucins by MALDI-TOF MS and MALDI-TOF tandem mass spectrometry (MALDI-TOF MS/MS).

Structural Elucidation of Sialylated O-Glycan Alditols Obtained from Mucins by Mass Spectrometry.

Acidic O-glycans having sialic acid and/or sulfate residue are abundantly expressed in intestinal mucins. However, structural elucidation of acidic O-glycans is a laborious and time-consuming task due to their large structural variations. Here, we describe a methodology of structural elucidation for sialylated O-glycan alditols from intestinal mucins using tandem mass spectroscopy. Methylesterification and mild periodate oxidation of sialylated O-glycan alditols assist mass analysis. This description includes the purification process of O-glycan alditols for structural analysis.

On the mucoadhesive properties of synthetic and natural polyampholytes.

HYPOTHESIS: The mucoadhesive characteristics of amphoteric polymers (also known as polyampholytes) can vary and are influenced by factors such as the solution's pH and its relative position against their isoelectric point (pHIEP). Whilst the literature contains numerous reports on mucoadhesive properties of either cationic or anionic polymers, very little is known about these characteristics for polyampholytes EXPERIMENTS: Here, two amphoteric polymers were synthesized by reaction of linear polyethylene imine (l-PEI) with succinic or phthalic anhydride and their mucoadhesive properties were compared to bovine serum albumin (BSA), selected as a natural polyampholyte. Interactions between these polymers and porcine gastric mucin were studied using turbidimetric titration and isothermal titration calorimetry across a wide range of pHs. Model tablets were designed, coated with these polymers and tested to evaluate their adhesion to porcine gastric mucosa at different pHs. Moreover, a retention study using fluorescein isothiocyanate (FITC)-labelled polyampholytes deposited onto mucosal surfaces was also conducted FINDINGS: All these studies indicated the importance of solution pH and its relative position against pHIEP in the mucoadhesive properties of polyampholytes. Both synthetic and natural polyampholytes exhibited strong interactions with mucin and good mucoadhesive properties at pH < pHIEP.

Altered O-linked glycosylation in benign and malignant meningiomas.

Background: Changes in protein glycosylation have been reported in various diseases, including cancer; however, the consequences of altered glycosylation in meningiomas remains undefined. We established two benign meningioma cell lines-SUT-MG12 and SUT-MG14, WHO grade I-and demonstrated the glycan and glycosyltransferase profiles of the mucin-type O-linked glycosylation in the primary benign meningioma cells compared with two malignant meningioma cell lines-HKBMM and IOMM-Lee, WHO grade III. Changes in O-linked glycosylation profiles in malignant meningiomas were proposed. Methods: Primary culture technique, morphological analysis, and immunocytochemistry were used to establish and characterize two benign meningioma cell lines. The glycan profiles of the primary benign and malignant meningiomas cell lines were then analyzed using lectin cytochemistry. The gene expression of O-linked glycosyltransferases, mucins, sialyltransferases, and fucosyltransferases were analyzed in benign and malignant meningioma using the GEO database (GEO series GSE16581) and quantitative-PCR (qPCR). Results: Lectin cytochemistry revealed that the terminal galactose (Gal) and N-acetyl galactosamine (GalNAc) were highly expressed in primary benign meningioma cells (WHO grade I) compared to malignant meningioma cell lines (WHO grade III). The expression profile of mucin types O-glycosyltransferases in meningiomas were observed through the GEO database and gene expression experiment in meningioma cell lines. In the GEO database, C1GALT1-specific chaperone (COSMC) and mucin 1 (MUC1) were significantly increased in malignant meningiomas (Grade II and III) compared with benign meningiomas (Grade I). Meanwhile, in the cell lines, Core 2 ß1,6-N-acetylglucosaminyltransferase-2 (C2GNT2) was highly expressed in malignant meningiomas. We then investigated the complex mucin-type O-glycans structures by determination of sialyltransferases and fucosyltransferases. We found ST3 ß-galactoside α-2,3-sialyltransferase 4 (ST3GAL4) was significantly decreased in the GEO database, while ST3GAL1, ST3GAL3, α1,3 fucosyltransferases 1 and 8 (FUT1 and FUT8) were highly expressed in malignant meningioma cell lines-(HKBMM)-compared to primary benign meningioma cells-(SUT-MG12 and SUT-MG14). Conclusion: Our findings are the first to demonstrate the potential glycosylation changes in the O-linked glycans of malignant meningiomas compared with benign meningiomas, which may play an essential role in the progression, tumorigenesis, and malignancy of meningiomas.