Site directed processing: Role of amino acid sequences and glycosylation of acceptor glycopeptides in the assembly of extended mucin type O-glycan core 2

Background

The assembly of Ser/Thr-linked O-glycans of mucins with core 2 structures is initiated by polypeptide GalNAc-transferase (ppGalNAc-T), followed by the action of core 1 β3-Gal-transferase (C1GalT) and core 2 β6-GlcNAc-transferase (C2GnT). β4-Gal-transferase (β4GalT) extends core 2 and forms the backbone structure for biologically important epitopes. O-glycan structures are often abnormal in chronic diseases. The goal of this work is to determine if the activity and specificity of these enzymes are directed by the sequences and glycosylation of substrates.

Methods

We studied the specificities of four enzymes that synthesize extended O-glycan core 2 using as acceptor substrates synthetic mucin derived peptides and glycopeptides, substituted with GalNAc or O-glycan core structures 1, 2, 3, 4 and 6.

Results

Specific Thr residues were found to be preferred sites for the addition of GalNAc, and Pro in the + 3 position was found to especially enhance primary glycosylation. An inverse relationship was found between the size of adjacent glycans and the rate of GalNAc addition. All four enzymes could distinguish between substrates having different amino acid sequences and O-glycosylated sites. A short glycopeptide Galβ1–3GalNAcα-TAGV was identified as an efficient C2GnT substrate.

Conclusions

The activities of four enzymes assembling the extended core 2 structure are affected by the amino acid sequence and presence of carbohydrates on nearby residues in acceptor glycopeptides. In particular, the sequences and O-glycosylation patterns direct the addition of the first and second sugar residues by ppGalNAc-T and C1GalT which act in a site directed fashion.

General significance

Knowledge of site directed processing enhances our understanding of the control of O-glycosylation in normal cells and in disease.     

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.     

Association of Cell Surface Mucins with Galectin-3 Contributes to the Ocular Surface Epithelial Barrier

Maintenance of an intact mucosal barrier is critical to preventing damage to and infection of wet-surfaced epithelia. The mechanism of defense has been the subject of much investigation, and there is evidence now implicating O-glycosylated mucins on the epithelial cell surface. Here we investigate a new role for the carbohydrate-binding protein galectin-3 in stabilizing mucosal barriers through its interaction with mucins on the apical glycocalyx. Using the surface of the eye as a model system, we found that galectin-3 colocalized with two distinct membrane-associated mucins, MUC1 and MUC16, on the apical surface of epithelial cells and that both mucins bound to galectin-3 affinity columns in a galactose-dependent manner. Abrogation of the mucin-galectin interaction in four different mucosal epithelial cell types using competitive carbohydrate inhibitors of galectin binding, β-lactose and modified citrus pectin, resulted in decreased levels of galectin-3 on the cell surface with concomitant loss of barrier function, as indicated by increased permeability to rose bengal diagnostic dye. Similarly, down-regulation of mucin O-glycosylation using a stable tetracycline-inducible RNA interfering system to knockdown c1galt1 (T-synthase), a critical galactosyltransferase required for the synthesis of core 1 O-glycans, resulted in decreased cell surface O-glycosylation, reduced cell surface galectin-3, and increased epithelial permeability. Taken together, these results suggest that galectin-3 plays a key role in maintaining mucosal barrier function through carbohydrate-dependent interactions with cell surface mucins.Mucosal surfaces comprise more than 400 m² of the total surface area in humans (compared with 1.8 m² for skin) and are, thus, by far the largest area of contact with the environment (1). Epithelial cells in mucosal surfaces are continuously faced with the critical function of forming a protective apical barrier that prevents cellular damage and infection while allowing the exchange of molecules with the extracellular milieu. Loss of barrier function is ascribed to numerous mucosal pathologies, such as dry eye (a disease affecting more than 5 million people in the United States), severe asthma, and inflammatory bowel disease (2–4). Integral to the apical surface of mucosal epithelia are cell surface-associated mucins, a group of high molecular weight glycoproteins defined by the presence of long amino-terminal, extracellular domains containing extensive sites for O-glycan attachment. O-Glycosylation is the most abundant post-translational modification of mucins and constitutes up to 80% of mucin''s mass. It is thought that specific cell surface mucins and their O-glycans provide protection to the mucosal surface (5). Data from knock-out mice deficient in cell surface mucin MUC1 and core 3 β-1,3-N-acetylglucosaminyltransferase, an enzyme involved in the synthesis of mucin-type O-glycans in human colon, indicate the requirement for mucins and their O-glycans in maintaining barrier integrity in the gastrointestinal tract and the eye (6–9). However, the mechanism by which cell surface-associated mucins and their O-glycans contribute to forming the mucosal barrier on the epithelial glycocalyx remains poorly characterized.Galectins are a family of animal β-galactoside-binding lectins, defined by their evolutionarily conserved carbohydrate recognition domain (10, 11). As many as 15 galectins have been identified in mammals, and they are widely distributed among different types of cells and tissues (12). Galectins have been implicated in numerous biological processes, including tumor cell adhesion and progression, immunity, inflammation, wound healing, and development (11, 13, 14). Galectin-3 is a 35-kDa protein originally identified as Mac-2, a cell surface antigen expressed on murine thioglycollate-elicited peritoneal macrophages (15). It is now established that galectin-3, like other galectins, can interact in a multivalent fashion and cross-link glycan ligands on cell surface receptors, such as with epidermal growth factor receptors and α5β1 integrin, to generate molecular lattices (16, 17). In this study we investigate whether galectin-3 participates in mucosal barrier function through its interaction with cell surface-associated mucins. We demonstrate here that two distinct cell surface mucins, MUC1 and MUC16, interact with galectin-3 on the apical surface of epithelial cells and that carbohydrate-mediated mucin-galectin-3 interactions play an important role in maintaining mucosal barrier function.     

Mucin Biopolymers Prevent Bacterial Aggregation by Retaining Cells in the Free-Swimming State

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Highlights? Mucin biopolymers reduce bacterial adhesion to underlying substrates ? Bacterial motility is maintained or increased in the presence of mucins ? Mucins block aggregate formation by motile bacteria ? Immotile Pseudomonas aeruginosa can form alginate and Psl-dependent flocs in mucus     

FAB-MS characterization of sialyl Lewis x determinants on polylactosamine chains of human airway mucins secreted by patients suffering from cystic fibrosis or chronic bronchitis

Although a large body of structural data exists for bronchial mucins from cystic fibrosis (CF) and chronic bronchitis (CB) patients, little is known about terminal structures carried on poly-N-acetyllactosamine antennae. Such structures are of interest because they are potential ligands for bacterial adhesins and other lectins. In this study, we have used fast atom bombardment mass spectrometry (FAB-MS) to examine terminal sequences released by endo--galactosidase from O-glycans obtained by reductive elimination of bronchial mucins purified from the sputum of 8 CF and 8 CB patients. Our data show that, although the polylactosamine antennae of CF and CB mucins have several terminal sequences in common, they differ significantly in their sialyl Lewis^x (NeuAc2-3Gal1-4[Fuc1-3]GlcNAc1-) content. Thus all examined mucins from CF patients carry sialyl Lewis^x on their polylactosamine antennae, whereas this type of epitope is present on only three out of the eight CB mucins examined, notably in the airways of one CB patient which were heavily infected by Pseudomonas aeruginosa as are the airways of all the CF patients. This suggests that, in airway mucins, the expression of sialyl Lewis^x on polylactosamine antennae is probably more related to inflammation and infection than to a direct effect of the CF defect.     

Influence of TNFalpha on the sialylation of mucins produced by a transformed cell line MM-39 derived from human tracheal gland cells

In order to investigate the influence of inflammation on the peripheral glycosylation of airway mucins, a human respiratory glandular cell line (MM-39) was treated by TNF. The expression and the activity of sialyl- and fucosyl-transferases, involved in the biosynthesis of peripheral carbohydrate determinants like sialyl-Lewis x, were investigated by RT-PCR and by HPAEC respectively. The mRNA steady-state level of sialyl- (ST3Gal III) and of fucosyl- (FUT3) transferases was moderately up-regulated by TNF; a 52% increase of 2,3-sialyltransferase activity was also observed in TNF-stimulated MM-39 cells. After metabolic radio-labelling with [³H]glucosamine and [³H]fucose, the mucins released inthe culture supernatant were purified by Sepharose CL-4B, density-gradient centrifugation and treatment with glycosaminoglycans-degrading enzymes. The mucins, released in the culture supernatant from control MM-39 cells, were constituted by two populations of molecules having the same 1.39–1.44 mg/ml density but carrying either high or low amounts of sialic acid residues at their periphery. TNF was able to increase the sialylation of the weakly sialylated mucins. This effect and the enhancement of the 2,3-sialyltransferase activity by TNF argue in favour of a regulation of the mucin sialylation by this pro-inflammatory cytokine. Despite the moderate overexpression of FUT3, no fucosylation of mucins produced by MM-39 cells was induced by TNF. In conclusion, the influence of TNF on the sialylation of mucins could explain why the mucins from infected patients suffering either from cystic fibrosis or from chronic bronchitis are more sialylated.     

Current concepts of the structure and nature of mammalian salivary mucous glycoproteins

Summary The visco-elastic properties of salivary secretions are due to high molecular-weight glyco-proteins, known as mucins. Mucins are composed of numerous oligosaccharide side-chains O-glycosidically linked through 2-acetamido-2deoxy--d-galactose to the hydroxyl groups of seryl and threonyl residues of the protein core; on the average, every fourth amino acid residue is involved in such a bond. This work conveys their isolation and purification, compiles the compositional analysis of several mammalian submaxillary and sublingual mucins; defines the conditions of the alkaline -elimination reaction, its mechanism and importance in structural studies of glycoproteins, and briefly discusses the influence of stimuli on mucous secretions, as well as biosynthesis, structural diversity, and physiological role of salivary mucous glycoproteins.     

A Metabolic Basis for Endothelial-to-Mesenchymal Transition

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Structures of sulfated oligosaccharides isolated from the respiratory mucins of a non-secretor (O, Lea+b-) patient suffering from chronic bronchitis

Mucin glycopeptides were prepared from the respiratory mucus of a non-secretor, chronic bronchitic patient with blood group O, Lea+b-. Oligosaccharides were released by alkaline borohydride treatment and purified by anion-exchange chromatography, size-exclusion chromatography and high performance anion-exchange chromatography. Structural studies employed 400-MHz 1H-NMR spectroscopy and matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Nine monosulfated oligosaccharides ranging in size from tetra- to hexasaccharide, were fully characterized in this study. The sulfate group occurs either on the C-3 of a terminal galactose residue or on the C-6 of a N-acetylglucosamine residue. In keeping with the non-secretor status of the patient, no structure with an (1-2)-linked fucose residue was found. Five of the structures had fucose present in (1-3)-linkage in the X determinant, while only one oligosaccharide (compound 7b) was seen with fucose (1-4)-linked in the Lea determinant. Eight structures isolated from the mucins of the non-secretor patient had not been found previously in the respiratory mucins; they are listed below.     

Effects of Size and Geographical Origin on Atlantic salmon,Salmo salar,Mucin O-Glycan Repertoire

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Highlights

• •Atlantic salmon O-glycome expanded to 169 structures in three epithelia.
• •Low interindividual variation amongst all populations and geographical regions.
• •Small variations in glycosylation between geographical locations and fish size.
• •Prominent fucosylation in gastrointestinal mucins from Tasmanian fish.

     

Two glycosylation alterations of mouse intestinal mucins due to infection caused by the parasite Nippostrongylus brasiliensis

The glycosylation alterations of mouse small intestinal mucins during a 12-day infectious cycle caused by the parasite Nippostrongylus brasiliensis have been studied. The guanidinium chloride insoluble mucins were isolated at day 0 to 12 from the small intestine of infected and non-infected C57BL/6 mice. The O-linked oligosaccharides were released by reductive -elimination from the mucins and separated into neutral, sialylated and sulfated fractions. All fractions were analyzed by monosaccharide composition analysis and the neutral oligosaccharides were structurally characterized by gas chromatography/mass spectrometry. Two oligosaccharides containing blood group H-type epitopes (Fuc1-2Gal-) were transiently expressed with a maximum at day 6. Additional oligosaccharides with the common structure HexNAc-Gal-3GalNAcol were transiently induced with a maximum at day 10. Northern blot analysis on total RNA showed a transient expression at day 4–6 of the Fut2 gene encoding a Fuc1-2 fucosyltransferase, probably responsible for the detected blood group H-type epitopes. Comparisons with the corresponding infection in rat studied previously, revealed structurally different alterations, although occurring as transient events in both species. Both showed an induced blood group-type transferase halfway through the infection (a blood group A transferase in rat) and an induced transferase adding a terminal GalNAc (to a sialic acid- containing epitope in rat) towards the end of the infection. These differences between closely related species suggest rapid evolutionary alterations in glycosyltransferase expression.     

The elucidation of the structure of the core part of the LPS from Plesiomonas shigelloides serotype O17 expressing O-polysaccharide chain identical to the Shigella sonnei O-chain

Plesiomonas shigelloides O17 LPS contains the same O-antigenic polysaccharide chain as a causative agent of dysentery, Shigella sonnei. This polysaccharide can be used as a component of a vaccine against dysentery. Core part of the P. shigelloides O17 LPS was studied using NMR and mass spectrometry and the following structure was proposed: Significant similarity of the P. shigelloides O17 LPS core with the structure of the P. shigelloides O54 core was observed.     

A Localized Pseudomonas syringae Infection Triggers Systemic Clock Responses in Arabidopsis

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Supertertiary Structure of the MAGUK Core from PSD-95

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Highlights? Using NMR and SAXS, we depict the dynamic structure of the MAGUK core PSD-95 ? PDZ3 of the MAGUK core interacts with SH3 via its canonical peptide binding groove ? Binding of PDZ peptides, such as CRIPT, disrupts the interaction between PDZ3 and SH3 ? Rosetta modeling shows that linker residues L411/M412 dock into the PDZ3 binding site     

Increasing sequence diversity with flexible backbone protein design: the complete redesign of a protein hydrophobic core

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Highlights? Flexible backbone design has been used to mutate every position in a protein core ? The redesign is hyperthermostable (melting temperature >140°C) ? An NMR structure and an X-ray structure closely match the design model ? Designed backbone perturbations were accurately recapitulated     

Quantitation and structures of oligosaccharide chains in human trachea mucin glycoproteins

Human respiratory mucin glycoproteins from patients with cystic fibrosis were purified and oligosaccharide chains were released by treatment with alkaline borohydride. A neutral oligosaccharide alditol fraction was isolated from mucin obtained from a patient with A blood group determinant by chromatography on DEAF-cellulose and individual oligosaccharide chains were then isolated by gel filtration on BioGel P-6 columns and high performance liquid chromatography with gradient and isocratic solvent systems. The structures of the purified oligosaccharides were determined by methylation analysis, sequential glycosidase digestion and H-NMR spectroscopy. The amount of each chain was determined by compositional analysis. A wide array of discrete branched oligosaccharide structures that contain from 3 to 22 sugar residues were found. Many of the oligosaccharides are related and appear to be precursors of larger chains. The predominant branched oligosaccharides which accumulate contain terminal blood group H (Fuc2Ga14) or blood group A (Fuc2(Ga1NAc3) (Ga14) determinants which stop further branching and chain elongation. The elongation of oligosaccharide chains in respiratory mucins occurs on the 3-linked G1cNAc at branch points, whereas the 6-linked GlcNAc residue ultimately forms short side chains with a Fuc2 (Ga1NAc3) Gal4 G1cNAc6 structure in individuals with A blood group determinant.The results obtained in the current studies further suggest that even higher molecular weight oligosaccharide chains with analogous branched structures are present in some human respiratory mucin glycoproteins. Increasing numbers of the repeating sequence shown in the oligosaccharide below is present in the higher molecular weight chains. {ie75-1} This data in conjunction with our earlier observations on the extensive branching of these oligosaccharide chains helps to define and explain the enormous range of oligosaccharide structures found in human and swine respiratory mucin glycoproteins. Comparison of the relative concentrations of each oligosaccharide chain suggest that these oligosaccharides represent variations of a common branched core structure which may be terminated by the addition of a2-linked fucose to the 3/4 linked galactose residue at each branch point. These chains accumulate and are found in the highest concentrations in these respiratory mucins.     

Leucine Signals to mTORC1 via Its Metabolite Acetyl-Coenzyme A

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Colonic mucins in ulcerative colitis: evidence for loss of sulfation

Colonic tissue obtained at surgery from control individuals and patients with ulcerative colitis was used to isolate mucins and to prepare mucin glycopolypeptides by pronase digestion. These were compared with mucins labelled with [³⁵S] sulfate and [³H]-glucosamine after organ culture tissue samples from the same patients. A significant loss of mucin sulfation was detected in the colitis patients by both metabolic labelling and chemical analysis of the glycopolypeptides. A change in the size distribution of purified mucin oligosaccharides fractionated on BioGel P6 after release by -elimination was seen in both radiolabelled and non-labelled colitis mucins compared with controls. Amino acid analysis of the glycopolypeptides showed a close similarity to the expected ratio of serine:threonine:proline for MUC2 and did not vary between control and colitis groups. Analysis of the mucins confirmed >90% purity in the labelling experiments, characteristic behaviour on density gradient centrifugation and agarose gel electrophoresis in control and ulcerative colitis groups and differences in sulfation and turnover at various sites in the normal colon.Abbreviations WGA wheat germ agglutinin - UC ulcerative colitis - HRP horseradish peroxidase