The sugar code: letters and vocabulary, writers, editors and readers and biosignificance of functional glycan-lectin pairing.

Ubiquitous occurrence in Nature, abundant presence at strategically important places such as the cell surface and dynamic shifts in their profile by diverse molecular switches qualifies the glycans to serve as versatile biochemical signals. However, their exceptional structural complexity often prevents one noting how simple the rules of objective-driven assembly of glycan-encoded messages are. This review is intended to provide a tutorial for a broad readership. The principles of why carbohydrates meet all demands to be the coding section of an information transfer system, and this at unsurpassed high density, are explained. Despite appearing to be a random assortment of sugars and their substitutions, seemingly subtle structural variations in glycan chains by a sophisticated enzymatic machinery have emerged to account for their specific biological meaning. Acting as 'readers' of glycan-encoded information, carbohydrate-specific receptors (lectins) are a means to turn the glycans' potential to serve as signals into a multitude of (patho)physiologically relevant responses. Once the far-reaching significance of this type of functional pairing has become clear, the various modes of spatial presentation of glycans and of carbohydrate recognition domains in lectins can be explored and rationalized. These discoveries are continuously revealing the intricacies of mutually adaptable routes to achieve essential selectivity and specificity. Equipped with these insights, readers will gain a fundamental understanding why carbohydrates form the third alphabet of life, joining the ranks of nucleotides and amino acids, and will also become aware of the importance of cellular communication via glycan-lectin recognition.

Glycan variation and evolution in the eukaryotes.

In this review, we document the evolution of common glycan structures in the eukaryotes, and illustrate the considerable variety of oligosaccharides existing in these organisms. We focus on the families of N- and O-glycans, glycosphingolipids, glycosaminoglycans, glycosylphosphatidylinositol (GPI) anchors, sialic acids (Sias), and cytoplasmic and nuclear glycans. We also outline similar and divergent aspects of the glycans during evolution within the groups, which include inter- and intraspecies differences, molecular mimicry, viral glycosylation adaptations, glycosyltransferase specificity relating to function, and the natural dynamism powering these events. Finally, we present an overview of the patterns of glycosylation found within the groups comprising the Eukaryota, namely the Deuterostomia, Fungi, Viridiplantae, Nematoda, and Arthropoda.

Eukaryotic protein glycosylation: a primer for histochemists and cell biologists.

Proteins undergo co- and posttranslational modifications, and their glycosylation is the most frequent and structurally variegated type. Histochemically, the detection of glycan presence has first been performed by stains. The availability of carbohydrate-specific tools (lectins, monoclonal antibodies) has revolutionized glycophenotyping, allowing monitoring of distinct structures. The different types of protein glycosylation in Eukaryotes are described. Following this educational survey, examples where known biological function is related to the glycan structures carried by proteins are given. In particular, mucins and their glycosylation patterns are considered as instructive proof-of-principle case. The tissue and cellular location of glycoprotein biosynthesis and metabolism is reviewed, with attention to new findings in goblet cells. Finally, protein glycosylation in disease is documented, with selected examples, where aberrant glycan expression impacts on normal function to let disease pathology become manifest. The histological applications adopted in these studies are emphasized throughout the text.

Mucins: a biologically relevant glycan barrier in mucosal protection.

BACKGROUND: The mucins found as components of mucus gel layers at mucosal surfaces throughout the body play roles in protection as part of the defensive barrier on an organ and tissue specific basis. SCOPE OF THE REVIEW: The human MUC gene family codes up to 20 known proteins, which can be divided into secreted and membrane-associated forms each with a typical protein domain structure. The secreted mucins are adapted to cross link in order to allow formation of the extended mucin networks found in the secreted mucus gels. The membrane-associated mucins possess membrane specific domains which enable their various biological functions as part of the glycocalyx. All mucins are highly O-glycosylated and this is tissue specific and linked with specific biological functions at these locations. Mucin biology is dynamic and the processes of degradation and turnover are well integrated with biosynthesis to maintain a continuous mucosal protection against all external aggressive forces. Interaction of mucins with microflora plays an important role in normal function. Mucins are modified in a variety of diseases and this may be due to abberant mucin peptide or glycosylation. MAJOR CONCLUSIONS: Mucins represent a family of glycoprotein having fundamental roles in mucosal protection and communication with external environment. GENERAL SIGNIFICANCE: The review emphasises the nature of mucins as glycoproteins and their role in presenting an array of glycan structures at the mucosal cell surface.

Self-association of the glycopeptide antibiotic teicoplanin A2 in aqueous solution studied by molecular hydrodynamics.

The natural glycopeptide antibiotic teicoplanin is used for the treatment of serious Gram-positive related bacterial infections and can be administered intravenously, intramuscularly, topically (ocular infections), or orally. It has also been considered for targeting viral infection by SARS-CoV-2. The hydrodynamic properties of teicoplanin A2 (M1 = 1880 g/mol) were examined in phosphate chloride buffer (pH 6.8, I = 0.10 M) using sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge together with capillary (rolling ball) viscometry. In the concentration range, 0-10 mg/mL teicoplanin A2 was found to self-associate plateauing > 1 mg/mL to give a molar mass of (35,400 ± 1000) g/mol corresponding to ~ (19 ± 1) mers, with a sedimentation coefficient s20, w = ~ 4.65 S. The intrinsic viscosity [[Formula: see text]] was found to be (3.2 ± 0.1) mL/g: both this, the value for s20,w and the hydrodynamic radius from dynamic light scattering are consistent with a globular macromolecular assembly, with a swelling ratio through dynamic hydration processes of ~ 2.

Comparative hydrodynamic and nanoscale imaging study on the interactions of teicoplanin-A2 and bovine submaxillary mucin as a model ocular mucin.

Glycopeptide antibiotics are regularly used in ophthalmology to treat infections of Gram-positive bacteria. Aggregative interactions of antibiotics with mucins however can lead to long exposure and increases the risk of resistant species. This study focuses on the evaluation of potential interactions of the last line of defence glycopeptide antibiotic teicoplanin with an ocular mucin model using precision matrix free hydrodynamic and microscopic techniques: sedimentation velocity in the analytical ultracentrifuge (SV-AUC), dynamic light scattering (DLS) and atomic force microscopy (AFM). For the mixtures of teicoplanin at higher doses (1.25 mg/mL and 12.5 mg/mL), it was shown to interact and aggregate with bovine submaxillary mucin (BSM) in the distributions of both sedimentation coefficients by SV-AUC and hydrodynamic radii by DLS. The presence of aggregates was confirmed by AFM for higher concentrations. We suggest that teicoplanin eye drop formulations should be delivered at concentrations of < 1.25 mg/mL to avoid potentially harmful aggregations.

Molecular aspects of mucin biosynthesis and mucus formation in the bovine cervix during the periestrous period.

Mucus within the cervical canal represents a hormonally regulated barrier that reconciles the need to exclude the vaginal microflora from the uterus during progesterone dominance, while permitting sperm transport at estrus. Its characteristics change during the estrous cycle to facilitate these competing functional requirements. Hydrated mucin glycoproteins synthesized by the endocervical epithelium form the molecular scaffold of this mucus. This study uses the bovine cervix as a model to examine functional groups of genes related to mucin biosynthesis and mucus production over the periestrous period when functional changes in cervical barrier function are most prominent. Cervical tissue samples were collected from 30 estrus synchronized beef heifers. Animals were slaughtered in groups starting 12 h after the withdrawal of intravaginal progesterone releasing devices (controlled internal drug releases) until 7 days postonset of estrus (luteal phase). Subsequent groupings represented proestrus, early estrus, late estrus, metestrus, and finally the early luteal phase. Tissues were submitted to next generation RNA-seq transcriptome analysis. We identified 114 genes associated with biosynthesis and intracellular transport of mucins, and postsecretory modifications of cervical; 53 of these genes showed at least a twofold change in one or more experimental group in relation to onset of estrus, and the differences between groups were significant (P < 0.05). The majority of these genes showed the greatest alteration in their expression in the 48 h postestrus and luteal phase groups.

The molecular biology of inflammatory bowel diseases.

IBDs (inflammatory bowel diseases) are a group of diseases affecting the gastrointestinal tract. The diseases are multifactorial and cover genetic aspects: susceptibility genes, innate and adaptive responses to inflammation, and structure and efficacy of the mucosal protective barrier. Animal models of IBD have been developed to gain further knowledge of the disease mechanisms. These topics form an overlapping background to enable an improved understanding of the molecular features of these diseases. A series of articles is presented based on the topics covered at the Biochemical Society Focused Meeting The Molecular Biology of Inflammatory Bowel Diseases.

Trefoil factor family peptides are increased in the saliva of children with mucositis.

BACKGROUND: Mucositis is a painful ulcerative condition of the oral cavity and gastrointestinal tract, occurring in association with chemotherapy and radiotherapy regimes. Trefoil factor family peptides (TFF, trefoil peptides), present in saliva, contribute to epithelial restitution and repair and are therefore potentially important in the healing phase of mucositis. This study aimed to assess any changes in the levels of trefoil peptides in oncology patients with and without mucositis. METHODS: Saliva was collected from healthy children, pretreatment oncology patients, neutropenic patients on treatment with no oral disease and mucositic patients. TFF1, 2 and 3 were quantified using ELISA. RESULTS: In healthy children TFF2 and 3 were positively correlated with age (r = 0.454, p = 0.01 for TFF2; r = 0.410, p = 0.05 for TFF3 Spearman rank correlation). TFF3 was higher in mucositis compared to all other groups. A linear regression prediction model indicated that TFF3, but not TFF1 and TFF2, was significantly different in mucositic and healthy controls, suggesting an altered pattern of trefoil peptide secretion (p = 0.021). CONCLUSIONS: This study is the first to focus on trefoil peptides in paediatric saliva. It shows the correlation between TFF2, TFF3 and age in healthy children. Paediatric mucositis disease occurs in the presence of increased concentrations and an altered pattern of trefoil peptides.

What role do mucins have in the development of laryngeal squamous cell carcinoma? A systematic review.

Mucins are the dominant component in the protective mucus layer on mucosal surfaces including the larynx. Hence, they are part of the first line of defence against external stimuli including effect of smoking in the larynx. We asked whether existing published evidence supported the hypothesis that alteration in mucins expression/production is related to the laryngeal neoplastic process. The objective of this study is to review published evidence for mucins having an important role in normal laryngeal physiology and the development of laryngeal squamous cell carcinoma (SCC). We aimed to review all available literature on mucins in the larynx in order to develop hypotheses to be tested by future research. Thereby, new potential means of prevention and treatment of laryngeal cancer may be developed. A systematic search of all published literature was conducted. Systematic searches were done in the following databases: AMED, BNI, EMBASE, HMIC, MEDLINE, PsycINFO, CINAHL and HEALTH BUSINESS ELITE from their respective inception up to 11 February 2011. The following keywords were used in combination: mucin, larynx and squamous cell carcinoma. Altogether, 53 studies were identified; 43 studies were excluded following screening of the titles and abstracts. Full text manuscripts for ten studies were obtained for detailed evaluation and five studies were included in this review. No single study fulfilled all relevant criteria. Based on the included studies, we now know that MUC1 is definitely expressed in SCC larynx. However, there is no definitive evidence to suggest that MUC1 and MUC2 are aberrantly expressed in SCC larynx as compared to normal larynx. Further studies using the best available detection technique to detect MUC1, MUC2 and other possible relevant mucins i.e., MUC4 on adequate numbers of normal and SCC specimens are needed to confirm the findings of this review.

Differential expression proteomics of human colorectal cancer based on a syngeneic cellular model for the progression of adenoma to carcinoma.

This is the first differential expression proteomics study on a human syngeneic cellular in vitro progression model of the colorectal adenoma-to-carcinoma sequence, the anchorage-dependent non-tumorigenic adenoma derived cell line AA/C1 and the derived anchorage-independent and tumorigenic carcinoma cell line AA/C1/SB10C. The study is based on quantitative 2-DE and is complemented by Western blot validation. Excluding redundancies due to proteolysis and post-translational modified isoforms of over 2000 protein spots, 13 proteins were revealed as regulated with statistical variance being within the 95th confidence level and were identified by peptide mass fingerprinting in MALDI MS. Progression-associated proteins belong to the functional complexes of anaerobic glycolysis/gluconeogenesis, steroid biosynthesis, prostaglandin biosynthesis, the regulation and maintenance of the cytoskeleton, protein biosynthesis and degradation, the regulation of apoptosis or other functions. Partial but significant overlap was revealed with previous proteomics and transcriptomics studies in colorectal carcinoma. Among upregulated proteins we identified 3-HMG-CoA synthase, protein phosphatase 1, prostaglandin E synthase 2, villin 1, annexin A1, triosephosphate isomerase, phosphoserine aminotransferase 1, fumarylacetoacetate hydrolase and pyrroline-5-carboxylate reductase 1 (PYCR1), while glucose-regulated protein 78, cathepsin D, lamin A/C and quinolate phosphoribosyltransferase were downregulated.

Structural glycobiology and human health.

The glycans (carbohydrates) form a diverse group of biomolecules which play active parts in most physiological processes. The field of structural glycobiology concerns the structures of the glycans themselves, the proteins which interact with them and the nature of the interactions between the two. The resulting information is important for our understanding of human health and disease, and for development of new therapeutic strategies. A series of articles is introduced based on the topics covered at the Structural Glycobiology and Human Health Biochemical Society Focused Meeting. Their subjects range from in-depth determinations of three-dimensional protein structure to broad screening techniques for glycan-protein interactions relevant to disease processes, including bacterial, parasitic and viral infections, inflammatory processes, cancer and diabetes.

Ionic liquids in oligosaccharide synthesis: towards mucin-type glycan probes.

The present article provides an overview on mucins and their role in biological processes, while aiming to familiarize readers with the current tools available for the synthesis of structurally defined mucin-type glycan probes including the advantages and potential applications of using ionic liquids in the synthesis of this important class of oligosaccharides. Furthermore, we also highlight recent developments in glycoarray technology that can enable high-sensitivity and high-throughput analysis of this important class of protein-carbohydrate interactions.

O-glycan inhibitors generate aryl-glycans, induce apoptosis and lead to growth inhibition in colorectal cancer cell lines.

Our studies provide direct evidence that O-glycosylation pathways play a role in the regulation of cell growth through apoptosis and proliferation pathways. A series of small molecular weight analogs of the GalNAc-alpha-1-O-serine/threonine structure based on 1-benzyl-2-acetamido-2-deoxy-alpha-O-d-galactopyranoside have been synthesized and tested in the human colorectal cancer cell lines PC/AA/C1/SB10C and HCA7/C29. Three inhibitors, 1-benzyl-2-acetamido-2-deoxy-alpha-O-D-galactopyranoside, and the corresponding 2-azido- and C-glycoside analogs were screened in these colorectal cancer cell lines at 0.5 mM and showed induction of apoptosis and downregulation of proliferation. Treatment of both cell lines with inhibitors led to changes in glycosylation detected with peanut lectin. The inhibition of glycosyltransferase activity in cell homogenates from human colorectal mucosal cells and cultured cell lines could be shown. The competitive action of the inhibitors resulted in the intracellular formation of 28 aryl-glycan products which were identified by MALDI and electrospray mass spectroscopy. The structures showed a differential pattern for each of the inhibitors in both cell lines. Gene array analysis of the glycogenes illustrated a pattern of glycosyltransferases that matched the glycan structures found in glycoproteins and aryl-glycans formed in the PC/AA/C1/SB10C cells; however, there was no action of the three inhibitors on glycogene transcript levels. The inhibitors act at both intermediary metabolic and genomic levels, resulting in altered protein glycosylation and aryl-glycan formation. These events may play a part in growth arrest.

The Interaction of the Gut Microbiota with the Mucus Barrier in Health and Disease in Human.

Glycoproteins are major players in the mucus protective barrier in the gastrointestinal and other mucosal surfaces. In particular the mucus glycoproteins, or mucins, are responsible for the protective gel barrier. They are characterized by their high carbohydrate content, present in their variable number, tandem repeat domains. Throughout evolution the mucins have been maintained as integral components of the mucosal barrier, emphasizing their essential biological status. The glycosylation of the mucins is achieved through a series of biosynthetic pathways processes, which generate the wide range of glycans found in these molecules. Thus mucins are decorated with molecules having information in the form of a glycocode. The enteric microbiota interacts with the mucosal mucus barrier in a variety of ways in order to fulfill its many normal processes. How bacteria read the glycocode and link to normal and pathological processes is outlined in the review.

Alterations in the composition of the supramucosal defense barrier in relation to disease severity of ulcerative colitis.

Mucin glycoproteins and trefoil peptides play an important role in protection and repair of the gastrointestinal epithelium. This study investigates alterations in mucin and trefoil peptide gene expression and product localization in ulcerative colitis (UC). Product localization and message expression of mucin MUC1 to 6 and trefoil peptide TFF1 to 3 genes was analyzed in rectosigmoid tissue from a cohort of patients with active UC and compared with that of normal colorectal mucosa. MUC1 expression was upregulated in severe UC at the site of rupture of crypt abscesses. Reduction in MUC2 expression occurred in UC adjacent to ulceration. No alteration in MUC3 or MUC4 gene expression was detectable in UC compared with normal colorectal mucosa. No ectopic expression of MUC5AC, MUC5B, or MUC6 was identified in UC. Ectopic TFF1 expression was identified in tissues eliciting histological features of severe disease. Decreased TFF3 localization was demonstrated in UC tissues, but no TFF2 expression was detected in any colorectal specimens. Subtle alterations in composition of the supramucosal defense barrier exist in UC and vary in relation to clinical severity of disease. There is upregulation in mucin MUC1 at crypt abscesses and neo-expression of TFF1 trefoil peptide in severe disease.

Prognostic value of mucins in the classification of ampullary carcinomas.

The ampulla of Vater is of high clinical relevance with regard to influx of chyme, ascending inflammation, intubation during diagnostic and therapeutic endoscopic investigation, therapeutic papillotomy, and especially to malignant transformation. Little is known about the distribution of mucins in the ampulla. In this study, we have investigated the mucin distribution in the normal ampulla of Vater and compared it to duodenal mucosa and Brunner glands. Expression of mucins in the ampulla of Vater and duodenum was monitored by reverse transcription-polymerase chain reaction and localization of the products by immunohistochemistry. The samples investigated originated from 30 autopsy cases. Mucins MUC1, MUC3, MUC4, MUC5AC, MUC5B, MUC6, MUC7, and MUC8 were expressed in the ampulla of Vater. Immunohistochemistry revealed production of MUC4, MUC5AC, MUC5B, and MUC6. The mucin composition varied in comparison with the duodenum referring to MUC2, MUC7, and MUC8. Detected mucins contribute to innate immunity, epithelial restitution, and protection against the aggressive secretions of the liver, gall bladder, and pancreas. By cross-linking, they influence the rheological properties of the secretions in the ampulla and facilitate unidirectional flow into the duodenum. Knowledge of their pattern of expression has prognostic value with regard to the detection of malignancy. The observed differences in the mucin distribution between the duodenum and the ampulla of Vater support the use of MUC2, MUC7, and MUC8 as useful tool in the classification of ampullary carcinomas.

A high-density putative monomeric mucin is the major [35S]labelled macromolecular product of human colorectal mucins in organ culture.

We have studied the biosynthesis of mucins in organ cultures of human colon using isopycnic density-gradient centrifugation following pulse labelling with [(35)S]sulphate and [(3)H]-D-glucosamine. A high-density [(35)S]sulphate labelled component, of larger size than MUC2 monomers, appeared in the tissue and also in the medium. It was not degraded by reduction, trypsin digestion, digestion with chondroitin ABC lyase or heparan sulphate III lyase, but was cleaved into smaller fragments following alkaline borohydride treatment and appears to be a monomeric, mucin-like molecule containing a protease-resistant domain with a larger hydrodynamic volume than MUC2 monomers. Although this macromolecule incorporated much more radiolabel than MUC2, it was not detected using chemical analysis and thus appears to be a component with a high metabolic turnover present in a very small amount. Most of the [(3)H]-D-glucosamine label was associated with low-density material that was well separated from MUC2, which was poorly labelled. Most of MUC2 was associated with the tissue as an 'insoluble' complex. The amount of MUC2 remained constant and its associated radiolabel increased only slightly with time. Analysis of the MUC2 subunits from the reduced 'insoluble' complex showed the typical reduction-insensitive oligomers and confirmed that the radiolabel was associated with this mucin. The large size of the [(35)S]-labelled putative monomeric mucin makes it difficult to separate it from reduced insoluble complex MUC2. As a result, many studies of intestinal mucin synthesis and secretion in the past have most likely been performed on 'mixtures' of this mucin and MUC2 and are thus not possible to interpret as the metabolic behaviour of oligomeric mucins.

Patterns of mucin adherence to contact lenses.

PURPOSE: Contact lens wear alters the preocular fluid through factors that include tear deposits on the lens. In the current study, lens-adherent material was extracted to assess whether contact lenses sample mucins from the preocular fluid. METHODS: Discarded extended-wear contact lenses were collected from patients with no ocular surface disease. Mucins were extracted in guanidine hydrochloride (GuHCl) with protease inhibitors. After the supernatant was removed, the extraction was repeated twice with the addition of 10 mM dithiothreitol, making a total of three extractions. Mucins were isolated by cesium chloride (CsCl) gradient centrifugation and size fractionated on Sepharose CL2B. Charge distribution was analyzed on ion-exchange chromatography with a lithium perchlorate (LiClO(4)) gradient. RESULTS: Contact lens-adherent mucins comprised soluble mucins and mucins that required solubilization by (repeated) dithiothreitol treatment. MUC1, MUC4, MUC2, and MUC5AC mucins eluted mainly at low buoyant densities in extractions from lenses worn long term without disinfection and at successively higher buoyant densities from monthly disposable contact lenses. Mucins with little negative charge, which were observed in all extractions, and very highly negatively charged species, present in the second and third extractions from contact lenses, had no equivalents in tissue-extracted mucins. CONCLUSIONS: Mucins adhering to contact lenses are altered forms of intracellular mucins. Different degrees of adherence of mucins to contact lenses may occur, either because of mucin characteristics or after mucin complexation with adherent materials. In the context of good contact lens hygiene, their presence may offer some protection from toxicants in the tear film, because mucins could function as acceptors for charged moieties such as free radicals.

Characterization of mucins in human lacrimal sac and nasolacrimal duct.

PURPOSE: Mucins are polymers that may reduce drag and enhance tear outflow. Mucin expression and distribution in human efferent tear ducts were tested in the physiological state, and potential differences in the expression pattern were investigated in the presence of primary acquired dacryostenosis (PANDO). METHODS: Expression of mucins in human lacrimal sac and nasolacrimal ducts was monitored by reverse transcription-polymerase chain reaction analysis. The presence and distribution of MUC1, -2, -4, -5AC, -5B, -6, and -7 in epithelia of the efferent tear duct passage are assessed with antisera to mucin peptide cores. Twenty normal tissues from cadavers and surgical specimens from 20 patients with PANDO were tested. RESULTS: mRNAs for all mucins investigated were detected in healthy human lacrimal sacs and nasolacrimal ducts. MUC6 mRNA was detected in only about half of the investigated samples. A reduced level of MUC2, -5AC, and -5B mRNAs was observed in PANDO. Immunohistochemistry revealed MUC2 in goblet cells and single epithelial cells. Both MUC5AC and -5B were detected in goblet cells forming intraepithelial mucous glands. MUC7 was present only in columnar epithelial cells of the efferent tear duct system. No immunoreactivity was observed with antibodies against MUC1, -4, and -6 peptide cores. CONCLUSIONS: Human efferent tear ducts express and produce a broad spectrum of mucins that is partly comparable with that in the conjunctiva and the salivary glands. The mucin diversity of the efferent tear ducts could enhance tear transport and antimicrobial defense. Reduced levels of mucin mRNA in a nonfunctioning though patent segment of the lacrimal passage, which is associated with epiphora, suggests that mucins ease tear flow through the efferent tear ducts.