Role of associated and covalently bound lipids in salivary mucin hydrophobicity: Effect of proteolysis and disulfide bridge reduction

doi:10.1016/S0006-291X(88)80471-6

Biochemical and Biophysical Research Communications

Volume 151, Issue 3, 30 March 1988, Pages 1046-1053

https://doi.org/10.1016/S0006-291X(88)80471-6 Get rights and content

Summary

The hydrophobic properties of salivary mucus glycoprotein were investigated by fluorescence spectroscopy using bis(8-anilino-1-naphthalene-sulfonate). The mucin, purified from rat submandibular salivary gland, was subjected to removal of associated and covalently bound lipids, degradation with pronase, and reduction with β-mercaptoethanol, and titrated with the probe. Analyses of fluorescence data revealed the presence of 49 ± 5 hydrophobic binding sites in the intact mucin molecule, a 69% increase in the number of binding sites occurred following extraction of associated lipids, while the removal of covalently bound fatty acids caused a 25% decrease in the binding sites. Proteolytic destruction of the nonglycosylated regions of the glycoprotein essentially abolished the probe binding, whereas reduction produced glycoprotein subunits whose combined number of hydrophobic binding sites was 2.4 times greater than that of mucus glycoprotein polymer. The results suggest that associated and covalently bound lipids contribute to hydrophobic characteristics of salivary mucin and that the hydrophobic binding sites reside on the nonglycosylated regions of this glycoprotein buried within its core.

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Citation Excerpt :
However, mucins are not the only lubricious component of salivary pellicles [21]. Moreover, mucins have a tendency to interact with other biological molecules [22,23], and it is known that the lubrication performance of mucin films can be improved, becoming closer to that of salivary pellicles, if mixed with other salivary fractions [17]. Thus, while a number of works indicate that an outer layer of anchored oral mucins mixed with other salivary components mediates the highly efficient boundary lubrication exhibited by reconstituted salivary pellicles, the nature of these additional pellicle components and the mechanisms by which they influence the structure and lubrication properties of mucins remains unknown.
Salivary pellicles i.e., thin films formed upon selective adsorption of saliva, protect oral surfaces against chemical and mechanical insults. Pellicles are also excellent aqueous lubricants. It is generally accepted that reconstituted pellicles have a two-layer structure, where the outer layer is mainly composed of MUC5B mucins. We hypothesized that by comparing the effect of ionic strength on reconstituted pellicles and MUC5B films we could gain further insight into the pellicle structure.
Salivary pellicles and MUC5B films reconstituted on solid surfaces were investigated at different ionic strengths by Force Spectroscopy, Quartz Crystal Microbalance with Dissipation, Null Ellipsometry and Neutron Reflectometry.
Our results support the two-layer structure for reconstituted salivary pellicles. The outer layer swelled when ionic strength decreased, indicating a weak polyelectrolyte behavior. While initially the MUC5B films exhibited a similar tendency, this was followed by a drastic collapse indicating an interaction between exposed hydrophobic domains. This suggests that mucins in the pellicle outer layer form complexes with other salivary components that prevent this interaction. Lowering ionic strength below physiological values also led to a partial removal of the pellicle inner layer. Overall, our results highlight the importance that the interactions of mucins with other pellicle components play on their structure.
Sulfate-based lipids: Analysis of healthy human fluids and cell extracts
2019, Chemistry and Physics of Lipids
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In fact, targeted MS-based approaches have been widely used for the SL detection but also due to their ability for structural characterization by tandem mass spectrometry (MS/MS) experiments. Despite the many studies conducted on the identification of SL in plasma (Lee et al., 2016; Mirzaian et al., 2015), saliva (Slomiany et al., 1988, 1983, 1978), urine (Yao et al., 2016), tear fluid (Lam et al., 2014), gastric fluid (Iwamori et al., 2000), seminal plasma (Sion et al., 2001), lipoproteins (Reis et al., 2013; Serna et al., 2015), red blood cells (Muskiet et al., 1983; Przybylska et al., 1995), blood platelets (Blache et al., 1995), and tissues brain (Moyano et al., 2013), kidney (Tadano-Aritomi et al., 2001), eye lenses (Seng et al., 2014), sperm cells (Fujimoto et al., 2000; Kongmanas et al., 2010; Zhang et al., 2005), hair (Cho et al., 2010; Drosche et al., 1994; Serizawa et al., 1990) and nails (Serizawa et al., 1990) and quantification by multi-reaction monitoring (MRM) (Fong et al., 2013; Kongmanas et al., 2010; Moyano et al., 2013; Sánchez-Guijo et al., 2015; Yao et al., 2016), these were mostly focused on specific steroid sulfates in individual fluids and cells (Esquivel et al., 2018; Fong et al., 2013; Lam et al., 2014; Przybylska et al., 1995; Sánchez-Guijo et al., 2015; Sion et al., 2001; Wang et al., 2018) leaving a whole panel of high Mw SL unaccounted for. In this study, the optimisation of targeted PIS 97 detection conditions to include low and high Mw SL (Figs. 1 and 2) revealed the presence of sulfate steroids, sulfoglycolipids, sulfated hypurate lipids, N-acyl taurine lipids and other unidentified sulfate lipid-based compounds identified in the same mass spectra (Fig. 3).
Sulfate-based lipids (SL) have been proposed as players in inflammation, immunity and infection. In spite of the many biochemical processes linked to SL, analysis on this class of lipids has only focused on specific SL sub-classes in individual fluids or cells leaving a range of additional SL in other biological samples unaccounted for.
This study describes the mass spectrometry screening of SL in lipid extracts of human fluids (saliva, plasma, urine, seminal fluid) and primary human cells (RBC, neutrophils, fibroblasts and skin epidermal) using targeted precursor ion scanning (PIS) approach. The PIS 97 mass spectra reveal a wide diversity of SL including steroid sulfates, sulfoglycolipids and other unidentified SL, as well as metabolites such as taurines, sulfated polyphenols and hypurate conjugates. Semi-quantification of SL revealed that plasma exhibited the highest content of SL whereas seminal fluid and epithelial cells contained the highest sulphur to phosphorous (S/P) ratio.
The complexity of biofluids and cells sulfateome presented in this study highlight the importance of expanding the panel of synthetic sulfate-based lipid standards. Also, the heterogenous distribution of SL provides evidence for the interplay of sulfotransferases/sulfatases, opening new avenues for biomarker discovery in oral health, cardiovascular, fertility and dermatology research areas.
Gel-forming mucin interactome drives mucus viscoelasticity
2018, Advances in Colloid and Interface Science
Citation Excerpt :
By contrast, other reports have noted relatively abundant lipids in mucus gels (1–2% of the total weight). Several classes of lipids have been described, including neutral lipids, the major class with more than half of the lipid fraction, and to a lesser extent glycolipids and phospholipids [114–116]. Their role is presumably to create a hydrophobic barrier at the mucus layer surface.
Mucus is a hydrogel that constitutes the first innate defense in all mammals. The main organic component of mucus, gel-forming mucins, forms a complex network through both reversible and irreversible interactions that drive mucus gel formation. Significant advances in the understanding of irreversible gel-forming mucins assembly have been made using recombinant protein approaches. However, little is known about the reversible interactions that may finely modulate mucus viscoelasticity, which can be characterized using rheology. This approach can be used to investigate both the nature of gel-forming mucins interactions and factors that influence hydrogel formation. This knowledge is directly relevant to the development of new drugs to modulate mucus viscoelasticity and to restore normal mucus functions in diseases such as in cystic fibrosis. The aim of the present review is to summarize the current knowledge about the relationship between the mucus protein matrix and its functions, with emphasis on mucus viscoelasticity.
What interactions drive the salivary mucosal pellicle formation?
2014, Colloids and Surfaces B: Biointerfaces
Citation Excerpt :
This heavily glycosylated protein is thought to be an essential part of the enamel and mucosal pellicle [7] and with its gel forming properties is thought to be essential for lubrication in the oral cavity [15]. MUC5B has both hydrophilic domains and hydrophobic domain patches [12,30], however its hydrophobic domains are within the non-glycosylated region and it is possibly covalently bound lipids from saliva that contribute to its hydrophobic nature [34]. Its low level of binding may also be due to the lack of membrane bound MUC1 on the particle surfaces [35,36] which could be essential in the binding of MUC5B to the mucosa in the oral cavity and development of the pellicle.
The bound salivary pellicle is essential for protection of both the enamel and mucosa in the oral cavity. The enamel pellicle formation is well characterised, however the mucosal pellicle proteins have only recently been clarified and what drives their formation is still unclear. The aim of this study was to examine the salivary pellicle on particles with different surface properties (hydrophobic or hydrophilic with a positive or negative charge), to determine a suitable model to mimic the mucosal pellicle. A secondary aim was to use the model to test how transglutaminase may alter pellicle formation. Particles were incubated with resting whole mouth saliva, parotid saliva and submandibular/sublingual saliva. Following incubation and two PBS and water washes bound salivary proteins were eluted with two concentrations of SDS, which were later analysed using SDS-PAGE and Western blotting. Experiments were repeated with purified transglutaminase to determine how this epithelial-derived enzyme may alter the bound pellicle. Protein pellicles varied according to the starting salivary composition and the particle chemistry. Amylase, the single most abundant protein in saliva, did not bind to any particle indicating specific protein binding. Most proteins bound through hydrophobic interactions and a few according to their charges. The hydrophobic surface most closely matched the known salivary mucosal pellicle by containing mucins, cystatin and statherin but an absence of amylase and proline-rich proteins. This surface was further used to examine the effect of added transglutaminase. At the concentrations used only statherin showed any evidence of crosslinking with itself or another saliva protein.
In conclusion, the formation of the salivary mucosal pellicle is probably mediated, at least in part, by hydrophobic interactions to the epithelial cell surface.
Salivary phospholipid secretion in response to β-adrenergic stimulation is mediated by Src kinase-dependent epidermal growth factor receptor transactivation
2004, Biochemical and Biophysical Research Communications
Communication between receptor tyrosine kinase and G protein-coupled receptor (GPCR)-mediated signaling is recognized as a common integrator linking diverse aspects of intracellular signaling systems. Here, we report that G protein-coupled β-adrenergic receptor activation leading to stimulation of salivary phospholipid release occurs with the involvement of epidermal growth factor receptor (EGFR). Using sublingual gland acinar cells, we show that prosecretory effect of isoproterenol on phospholipid release was subjected to suppression by EGFR kinase inhibitor, PD153035, and wortmannin, an inhibitor of PI3K, but not by PD98059, an inhibitor of extracellular signal regulated kinase (ERK). Furthermore, wortmannin, but not the ERK inhibitor, caused the reduction in the acinar cell secretory responses to β-adrenergic agonist-generated cAMP as well as adenyl cyclase activator, forskolin. The acinar cell phospholipid secretory responses to isoproterenol, moreover, were inhibited by PP2, a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR phosphorylation. Taken together, our data are the first to demonstrate the requirement for Src kinase-dependent EGFR transactivation in regulation of salivary phospholipid secretion in response to β-adrenergic GPCR activation.
Mechanisms of oesophageal mucosal defence
2000, Bailliere's Best Practice and Research in Clinical Gastroenterology
The integrity of the oesophageal mucosa depends upon an equilibrium between aggressive factors, predominantly acid and pepsin, and protective mechanisms. Protective mechanisms operate within the oesophageal mucosa as pre-epithelial, epithelial and post-epithelial defences. Only the protective components of the oesophageal pre-epithelial defence can be tested in vivo in humans. It has been recently demonstrated that human oesophageal submucosal glands elaborate mucous secretion rich in bicarbonate and non-bicarbonate buffers, mucin, prostaglandin E₂, epidermal growth factor (EGF) and transforming growth factor α (TGFα). This oesophageal secretion, accompanied by similarly protective factors within the swallowed saliva, defines the protective potential of the oesophageal pre-epithelial defence that exists in the form of a mucus-buffer layer covering the oesophageal mucosa and which retards the back-diffusion of hydrogen ions. It has also been demonstrated that patients with severe erosive reflux oesophagitis exhibit qualitative impairment in both the salivary and oesophageal components of the oesophageal pre-epithelial defence. Furthermore, patients with endoscopically negative gastro-oesophageal reflux disease have a significantly stronger oesophageal pre-epithelial defence than patients with erosive reflux oesophagitis. On the other hand, African–Americans, who are less likely to develop erosive reflux oesophagitis, have a stronger oesophageal pre-epithelial defence than do Caucasians. The salivary component of the oesophageal pre-epithelial defence can be enhanced by mastication and the administration of cisapride, whereas oesophageal secretion can only be significantly augmented by cisapride.

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Role of associated and covalently bound lipids in salivary mucin hydrophobicity: Effect of proteolysis and disulfide bridge reduction

Summary

Arch. Biochem. Biophys.

Arch. Oral Biol.

Biochim. Biophys. Acta

Arch. Oral Biol.

Biochim. Biophys. Acta

Prog. Lipid Res.

Arch. Oral Biol.

Arch. Biochem. Biophys.

J. Biol. Chem.