Salivary Mucins MUC5B Research Articles

Investigation of Hydrocolloid Plant Polysaccharides as Potential Candidates to Mimic the Functions of MUC5B in Saliva

The successful substitution of complex physiological fluids, such as human saliva, remains a major challenge in drug development. Although there are a large number of saliva substitutes on the market, their efficacy is often inadequate due to short residence time in the mouth, unpleasant mouthfeel, or insufficient protection of the teeth. Therefore, systems need to be identified that mimic the functions of saliva, in particular the salivary mucin MUC5B and the unique physiological properties of saliva. To this end, plant extracts known to contain hydrocolloid polysaccharides and to have mucus-forming properties were studied to evaluate their suitability as saliva substitutes. The aqueous plant extracts of Calendula officinalis, Fucus sp. thalli, and lichenan from Lichen islandicus were examined for composition using a range of techniques, including GC-MS, NMR, SEC, assessment of pH, osmolality, buffering capacity, viscoelasticity, viscoelastic interactions with human saliva, hydrocolloid network formation, and in vitro cell adhesion. For this purpose, a physiologically adapted adhesive test was developed using human buccal epithelial cells. The results show that lichenan is the most promising candidate to mimic the properties of MUC5B. By adjusting the pH, osmolality, and buffering capacity with K2HPO4, it was shown that lichenan exhibited high cell adhesion, with a maximum detachment force that was comparable to that of unstimulated whole mouth saliva.

Proteomic response in Streptococcus gordonii DL1 biofilm cells during attachment to salivary MUC5B

ABSTRACT Background Salivary mucin MUC5B seems to promote biodiversity in dental biofilms, and thereby oral health, for example, by inducing synergistic ‘mucolytic’ activities in a variety of microbial species that need to cooperate for the release of nutrients from the complex glycoprotein. Knowledge of how early colonizers interact with host salivary proteins is integral to better understand the maturation of putatively harmful oral biofilms and could provide key insights into biofilm physiology. Methods The early oral colonizer Streptococcus gordonii DL1 was grown planktonically and in biofilm flow cell systems with uncoated, MUC5B or low-density salivary protein (LDP) coated surfaces. Bacterial cell proteins were extracted and analyzed using a quantitative mass spectrometry-based workflow, and differentially expressed proteins were identified. Results and conclusions Overall, the proteomic profiles of S. gordonii DL1 were similar across conditions. Six novel biofilm cell proteins and three planktonic proteins absent in all biofilm cultures were identified. These differences may provide insights into mechanisms for adaptation to biofilm growth in this species. Salivary MUC5B also elicited specific responses in the biofilm cell proteome. These regulations may represent mechanisms by which this mucin could promote colonization of the commensal S. gordonii in oral biofilms.

Oral mucosal pellicle as an immune protection against micro-organisms in patients with recurrent aphthous stomatitis: A hypothesis

Recurrent Aphthous Stomatitis (RAS) is the most common ulcerative diseases of oral mucosa affecting an estimate of 20% of the world’s population. Majority of the people affected by RAS are under 30 years of age. RAS is located on the lining (non-keratinized) oral mucosa, i.e. buccal mucosa, lateral side of the tongue, soft palate, lip mucosa, or the floor of mouth. An aphthous ulcer develops when lymphocytic cells infiltrate into the epithelium and cause an edema due to transient inflammatory stimuli. Bacteria, viruses and fungi have been suggested to cause aphthous lesions, but findings regarding oral pathogens are conflicting. Prior consensus has been that RAS is a multifactorial condition, with microbes, allergies, nutritional deficiencies, genetic factors, certain illnesses, immunodeficiency, hormonal changes, trauma and stress among others, contributing to the condition. In spite of many suggestions and investigations, the etiology and pathophysiology of RAS remains uncertain.Our hypothesis focuses on mucin proteins that have been shown to play a role in the formation of protective mucosal pellicle, which serves as the first line of defense between oral epithelium and pathogens within the oral cavity. Mucins, including transmembrane mucin 1 (MUC1), and salivary mucins MUC5B and MUC7 form a protein network that is strongly retained to oral epithelium. The role of the mucosal pellicle in pathophysiology of RAS is unknown. Structural variations have been found in the salivary MUC7 terminal end oligosaccharides in RAS patients, rendering the protein unable to agglutinate pathogens. Furthermore, low levels of MUC1 fail to provide a scaffold for assembly of salivary mucins. We introduce a new hypothesis, the alterations in the structure of these glycoproteins could have a profound impact on the oral mucosal barrier function. On the other hand, micro-organisms secreting their mucolytic enzymes destroy the mucosal pellicle causing oral ulcers.

MUC7 Level As A New Saliva Risk Factor For Dental Caries In Adult Patients.

Purpose: Data currently available indicate the significance of salivary mucins MUC5B and MUC7 in the protection of teeth against caries. Our study aimed to determine the relationship between dental caries in adults and levels of MUC5B and MUC7.Methods: The studies were conducted on 45 adult subjects selected on the basis of dental examination and calculation of the DMFT (Decayed, Missing, and Filled Teeth) index. Among these patients, two research groups were distinguished: group 1 included 19 caries-free subjects (DMFT = 0); and group 2 included 26 patients with severe caries (DMFT > 13.9). Samples of whole unstimulated saliva were collected and centrifuged. MUC5B and MUC7 content in saliva supernatant were estimated using an enzyme-linked immunosorbent sandwich assay (ELISA). Analysis of the obtained data receiver operating characteristic (ROC) curves was employed to define relationships between the contents of the studied mucins and the detected dental caries.Results: In subjects from group 1, the mean level of MUC5B amounted to 0.63 ± 0.35 ng/ml and this was significantly higher than the concentration of mucin in patients of group 2, which amounted to 0.38 ± 0.32 ng/ml (p = 0.023). The mean level of MUC7 amounted to 5.47 ± 1.18 ng/ml and this was significantly higher than the level of the mucin in group 2, which was 1.39 ± 0.86 ng/ml (p< 0.0001). In parallel, a relationship was detected between levels of the examined mucins and manifestation of dental caries. For MUC7, the optimal cut-off value was obtained (i.e. corresponding to 100% sensitivity and specificity), amounting to 2.5 ng/ml for the detection of dental caries risk.Conclusions: Development of dental caries is linked to reduced concentrations of MUC5B and MUC7. The level of MUC7 may represent a significant parameter clinically suitable for evaluation of disease risk.

Mechanisms of astringency: Structural alteration of the oral mucosal pellicle by dietary tannins and protective effect of bPRPs

The interaction of tannins with salivary proteins is involved in astringency. This paper focussed on saliva lining oral mucosae, the mucosal pellicle. Using a cell-based model, the impact of two dietary tannins (EgC and EgCG) on the mucosal pellicle structure and properties was investigated by microscopic techniques. The role of basic Proline-Rich-Proteins (bPRPs) in protecting the mucosal pellicle was also evaluated.At low (0.05 mM) tannin concentration, below the sensory detection threshold, the distribution of salivary mucins MUC5B on cells remained unaffected. At 0.5 and 1 mM, MUC5B-tannin aggregates were observed and their size increased with tannin concentration and with galloylation. In addition, 3 mM EgCG resulted in higher friction forces measured by AFM. In presence of bPRPs, the size distribution of aggregates was greatly modified and tended to resemble that of the “no tannin” condition, highlighting that bPRPs have a protective effect against the structural alteration induced by dietary tannins.

The Effect of Exercise on Salivary Viscosity.

A common experience after exercise is the presence of a thick and sticky saliva layer on the oral surfaces, which causes a feeling of a dry mouth. Since the salivary mucin MUC5B is responsible for the visco-elastic behavior of saliva, in the present study we explored the effect of exercise on both the salivary viscosity and the secretion of MUC5B in saliva. Twenty healthy dental students performed an aerobic exercise by cycling for 15 min on cycle-ergometers at a heart rate of 130–140 beats per minute. Saliva was collected at three time points: before exercise, immediately after exercise and after 30 min recovery. Salivary flow rate, viscosity, amylase activity, total protein, carbohydrate and MUC5B concentration were determined. Salivary flow rate, protein and amylase did not change significantly. Immediately after exercise, the salivary viscosity and carbohydrate concentration were significantly higher than at baseline and after 30 min recovery. Immediately after exercise, the MUC5B concentration was significantly higher than after 30 min recovery. It is concluded that the presence of thick saliva after exercise is at least partially due to an increased secretion of MUC5B.

Immunocytological detection of salivary mucins (MUC5B) on the mucosal pellicle lining human epithelial buccal cells

The mucosal pellicle is defined as the protein film adsorbed onto oral mucosa. This study aimed at characterizing the ultrastructure of human epithelial buccal cells and localizing salivary mucins MUC5B, a major constituent of the mucosal pellicle. Cells were sampled from the buccal surface and prepared for Transmission Electron Microscopy using high-pressure freezing/cryosubstitution followed by immunogold labelling of MUC5B. Morphologically, cells were visualized as typical cells of the superficial layer of a squamous nonkeratinized epithelium with a partly degraded plasma membrane. The outer surface of the plasma membrane was lined with a biological material of medium electron density. MUC5B were detected in the extracellular space, and particularly in the vicinity of the plasma membrane, sometimes onto fibrils protruding from the membrane. This area was, therefore, considered as constituting the mucosal pellicle, which appeared as a mixed film of both salivary and epithelial components. The distribution of gold particles suggested that the surface of the pellicle was not uniform, and that the film thickness could reach up to 100 nm. This work showed the feasibility of visualizing and characterizing the mucosal pellicle directly on human epithelial buccal cells sampled in a noninvasive manner.

MUC5B levels in submandibular gland saliva of patients treated with radiotherapy for head-and-neck cancer: A pilot study

BackgroundThe salivary mucin MUC5B, present in (sero)mucous secretions including submandibular gland (SMG) saliva, plays an important role in the lubrication of the oral mucosa and is thought to be related to the feeling of dry mouth. We investigated if MUC5B levels in SMG saliva could distinguish between the presence or absence of severe dry mouth complaints 12 months after radiotherapy (RT) for head-and-neck cancer (HNC).FindingsTwenty-nine HNC patients with a residual stimulated SMG secretion rate of ≥0.2 ml/10 min at 12 months after RT were analyzed. MUC5B (in U; normalized to 1) and total protein levels (mg/ml) were measured in SMG saliva at baseline and 12 months after RT using ELISA and BCA protein assay, respectively. Overall, median MUC5B levels decreased after RT from 0.12 to 0.03 U (p = 0.47). Patients were dichotomized into none/mild xerostomia (n = 12) and severe xerostomia (n = 17) based on a questionnaire completed at 12 months. SMG and whole saliva flow rates decreased after RT but were comparable in both groups. The median MUC5B level was higher in patients with no or mild xerostomia compared to patients with severe xerostomia (0.14 vs 0.01 U, p = 0.22). Half of the patients with severe xerostomia had no detectable MUC5B at 12 months after RT. No differences in total protein levels were observed.ConclusionsQualitative saliva parameters like MUC5B need further investigation in RT-induced xerostomia. This pilot study showed a trend towards lower MUC5B levels in the SMG saliva of patients with severe xerostomia 12 months after RT for HNC.

Electron microscopic immunogold localization of salivary mucin MUC5B in human buccal and palatal glands

In recent years, minor salivary glands, due to their involvement in the health and homeostasis of the oral cavity, have been the focus of several research investigations. Despite the fact that a considerable amount of data has been collected, many aspects of their functional features, including the secretory components they produce, remain to be ascertained. In this study we have analyzed the ultrastructural distribution of the MUC5B mucin in human palatal and buccal glands by means of post-embedding immunoelectron microscopy. Thin sections of normal human buccal and palatal glands obtained at surgery, were treated with polyclonal antibodies to human salivary MUC5B. Intense MUC5B reactivity was observed in the secretory granules of mucous cells of all glands examined. The present results provide new data regarding the secretory pattern of MUC5B in human buccal and palatal glands, indicating their significant contribution to the maintenance of the mucous biofilm that protects buccal and palatal mucosal areas.

Ultrastructural localization of salivary mucins MUC5B and MUC7 in human labial glands

As a result of their presence throughout the mouth in the submucosa or between muscle fibers, minor salivary glands secrete directly and continuously into the oral cavity, providing mucosal surfaces with highly glycosylated proteins that are active in bacterial aggregation and in oral tissue lubrication. In this study, we investigated the ultrastructural localization of the MUC5B and MUC7 mucins in human labial glands by means of a postembedding immunogold technique. Thin sections of normal human labial glands, obtained during surgery, were incubated with polyclonal antibodies to human salivary mucins MUC5B and MUC7, and then with gold-labeled secondary antibodies. Specific MUC5B reactivity was found in the secretory granules of mucous cells of all glands examined, and was associated with the luminal membrane of duct cells. MUC7 labeling was observed in the granules of both mucous and seromucous secretory cells of the glandular parenchyma. Quantitative analyses demonstrated that seromucous granules have higher immunogold labeling densities for MUC7 than mucous granules. Our immunohistochemical data extend the results of previous light microscopic studies of MUC5B and MUC7 localizations, pointing out the significant contribution of human labial glands in the secretion process of these two mucins.

Studies on the exchange of early pellicle proteins by mucin and whole saliva

Adsorption of small pellicle proteins statherin or proline-rich protein 1 (PRP1), respectively, and subsequent adsorption of human whole saliva (HWS) or salivary mucin MUC5B, respectively, was studied using ellipsometry and total internal reflectance fluorescence. Differences in elution (using sodium dodecyl sulphate (SDS) solutions) between mixed and single protein films were also investigated. On both hydrophilic and hydrophobized surfaces HWS and MUC5B were found to adsorb to pre-adsorbed layers of statherin and PRP1, respectively. Statherin adsorption on both substrate types showed no or minor exchange by HWS or MUC5B and no change in SDS elution between mixed and single protein films. Small amounts of PRP1 were exchanged by HWS on both surface types and the SDS elutable fractions were similar or larger for mixed films compared to single protein films. PRP1 and MUC5B in sequence showed minor exchange of PRP1 on hydrophilic surfaces, while no exchange could be established on hydrophobized substrates. SDS elutable fractions decreased for PRP1 and MUC5B mixed films compared to single protein films. In conclusion, minor amounts of statherin and PRP1 are exchanged during the time course of the experiments, which indicates that these proteins may to a large extent remain incorporated in the pellicle.

The salivary mucin MUC5B and lactoperoxidase can be used for layer-by-layer film formation

In situ ellipsometry was used to study layer-by-layer film formation on hydrophilic and hydrophobized silica surfaces by alternating sequential adsorption of human mucin MUC5B and cationic proteins lysozyme, lactoferrin, lactoperoxidase or histatin 5, respectively. The stability of the multilayers was investigated by addition of sodium dodecyl sulfate solution (SDS). Atomic force microscopy was employed to investigate morphological structures on the surfaces during the layer-by-layer film build-up. It was clearly shown that, on both hydrophilic and hydrophobized silica, only MUC5B and lactoperoxidase showed the ability for multilayer formation, resulting in an approximately linear increase in adsorbed amount and film thickness with each deposition cycle. The net increase in amounts per cycle was larger on the hydrophilic silica. Further, MUC5B needs to be adsorbed first on the hydrophilic substrates to obtain this fast build-up behavior. Generally, addition of SDS solution showed that a large fraction of the adsorbed film could be desorbed. However, films on the hydrophobized silica were more resistant to surfactant elution. In conclusion, MUC5B–cationic protein multilayers can be formed on hydrophilic and hydrophobized silica, depending on the choice of the cationic protein as well as in which order the build-up is started on hydrophilic silica. Additionally, SDS disrupts the layer-by-layer film formed by MUC5B and lactoperoxidase.

Identification and characterization of binding properties of Helicobacter pylori by glycoconjugate arrays

The microaerophilic bacterium Helicobacter pylori is well established for its role in development of different gastric diseases. Bacterial adhesins and corresponding binding sites on the epithelial surface allow H. pylori to colonize the gastric tissue. In this investigation, the adhesion of H. pylori to dot blot arrays of natural glycoproteins and neoglycoproteins was studied. Adhesion was detected by overlay with fluorescence-labeled bacteria on immobilized (neo)glycoproteins. The results confirmed the interaction between the adhesin BabA and the H-1-, Lewis b-, and related fucose-containing antigens. In addition, H. pylori bound to terminal alpha2-3-linked sialic acids as previously described. The use of a sabA mutant and sialidase treatment of glycoconjugate arrays showed that the adherence of H. pylori to laminin is mediated by the sialic acid-binding adhesin, SabA. The adhesion to salivary mucin MUC5B is mainly associated with the BabA adhesin and to a lesser extent with the SabA adhesin. This agrees with reports, that MUC5B carries both fucosylated blood group antigens and alpha2-3-linked sialic acids. The adhesion of H. pylori to fibronectin and lactoferrin persisted in the babA/sabA double mutant. Because binding to these molecules was abolished by denaturation rather than by deglycosylation, it was suggested to depend on the recognition of unknown receptor moieties by an additional unknown bacterial surface component. The results demonstrate that the bacterial overlay method on glycoconjugate arrays is a useful tool for exploration and the characterization of unknown adhesin specificities of H. pylori and other bacteria.

Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MS n

Neutral O-linked oligosaccharides released from the salivary mucin MUC5B were separated and detected by negative ion LC-MS and LC-MS 2. The resolution of the chromatography and the information obtained from collision induced dissociation of detected [M − H] − ions were usually sufficient to identify the sequence of individual oligosaccharides, illustrated by the fact that 50 different oligosaccharides ranging from disaccharides to nonasaccharides could be assigned from the sample. Fragmentation was shown to yield mostly reducing end sequence fragments (Z i and Y i ), enabling primary sequence assignment. Specific fragmentation pathways or patterns were also detected giving specific linkage information. The reducing end core (Gal/GlcNAcβ1-3GalNAcol or Gal/GlcNAcβ1-3(GlcNAcβ1-6)GalNAcol) could be deduced from the pronounced glycosidic C-3 cleavage and A i type cleavages of the reducing end GalNAcol, together with the non reducing end fragment from the loss of a single substituted GalNAcol. Substitution patterns on GlcNAc residues were also found, indicative for C-4 substitution ( 0,2A i − H 2O cleavage) and disubstitution of C-3 and C-4 (Z i /Z i cleavages). This kind of fragmentation can be used for assigning the mode of chain elongation (Galβ1-3/4GlcNAcβ1-) and identification of Lewis type antigens like Lewis a/x and Lewis b/y on O-linked oligosaccharides. In essence, negative ion LC-MS 2 was able to generate extensive data for understanding the overall glycosylation pattern of a sample, especially when only a limited amount of material is available.

Recovery of Intact 2-Aminobenzamide-Labeled O-Glycans Released from Glycoproteins by Hydrazinolysis

The initial step in quantitative analysis of O-linked glycans of glycoproteins is to release them in high yield, nonselectively, unmodified, and with a free reducing terminus. In contrast to other techniques, hydrazinolysis can meet these criteria. However, when analyzing pools of O-linked glycans as described in the accompanying article by L. Royle et al. (2002, Anal. Biochem. 304), some peeling of the glycans was observed. Critical steps in the sample preparation and glycan recovery were therefore evaluated by analyzing and identifying both intact O-glycans and degraded products. Synthetic O-glycopeptides were characterized by mass spectrometry. Released glycans were identical to those on the glycopeptide. O-Linked glycans from a range of glycoproteins of increasing complexity, namely, bovine serum fetuin, glycophorin A, and previously uncharacterized glycopeptides isolated from human salivary mucin Muc5B, were also analyzed. Quantitative analysis of the glycan profile confirmed that there was <2% peeling of O-glycans released by hydrazinolysis conditions of 60°C for 6 h, and recovered using the optimised procedure now described. This demonstrated that O-glycans can be prepared by hydrazinolysis without degradation and, as part of an analytical strategy, makes the analysis of O-glycans attached to low-microgram levels of naturally occurring glycoproteins feasible.