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

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 beta-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.     

Effect of lipids and proteins on the viscosity of gastric mucus glycoprotein

The effect of associated lipids and covalently bound fatty acids, and the contribution of serum albumin and secretory IgA to the viscosity of dog gastric mucus glycoprotein was investigated. Using a cone/plate viscometer at shear rates between 1.15 - 230s -1, it was found that extraction of associated lipids from the glycoprotein lead to 80-85% decrease in the viscosity. Further loss (39%) in viscosity of the delipidated glycoprotein occurred following removal of covalently bound fatty acids. Reassociation of the delipidated glycoprotein with its neutral lipids increased the viscosity 3-fold, a 2.5-fold increase was obtained with glycolipids, and 2-fold with phospholipids. Preincubation of purified mucus glycoprotein with albumin or IgA resulted in the increase in viscosity. This increase in viscosity was proportional to albumin concentration up to 10%, and to IgA concentration up to 5%. The results show that interaction of lipids and proteins with mucus glycoprotein contributes significantly to the viscosity of gastric mucus.     

Hydrogen ion diffusion in dog gastric mucus glycoprotein: effect of associated lipids and covalently bound fatty acids

The effect of neutral lipids, glycolipids and phospholipids associated with dog gastric mucus glycoprotein, and that of covalently bound fatty acids on the ability of glycoprotein to retard the diffusion of hydrogen ion was investigated. Purified mucus glycoprotein in its native form, placed between equimolar (0.155M) solutions of HCl and NaCl in a specially designed two-compartment chamber, caused a 90% reduction in permeability to hydrogen ion when compared with a layer of NaCl. Extraction of associated lipids lead to a 68% increase in permeability of the glycoprotein to hydrogen ion, while removal of the covalently bound fatty acids increased further the diffusion rate by 6%. Reassociation of the delipidated glycoprotein with its neutral lipids reduced the permeability to hydrogen ion by 34%, an 11% reduction was obtained with glycolipids, and 23% with phospholipids. Since neutral lipids account for 47% of the glycoprotein lipids, glycolipids 41.1% and phospholipids 11.9%, the quantitative decrease in permeability of the delipidated glycoprotein following its reassociation with phospholipids is 2.7 times greater than that of neutral lipids and 7.3 times greater than that of glycolipids.     

Enhancement of the physicochemical qualities of gastric mucus by sofalcone.

The effect of prolonged administration of an antiulcer drug, sofalcone, on the physicochemical properties of gastric mucus was investigated. The experiments were conducted with groups of rats receiving twice daily for three consecutive days a dose of 100 mg/kg sofalcone, while the control group received daily doses of vehicle. The rats were sacrificed 16 h after the last dose and gastric mucosa subjected to physicochemical measurements. The results revealed that sofalcone evoked a 23% increase in mucus gel dimension, while sulfo- and sialomucins content of the gel increased by 54 and 25%, respectively. These changes were accompanied by a 16% increase in mucus H+ retardation capacity, 2-fold increase in viscosity, and a 39% increase in the gel hydrophobicity. The mucus elaborated in the presence of sofalcone contained 67% more covalently bound fatty acids, exhibited 10% lower content of protein, 30% higher content of carbohydrate, and 18% higher content of lipids. The mucus of the sofalcone group also showed an increase in the proportion of the high molecular weight mucus glycoprotein form, which in the control group accounted for about 30% of gel mucin, while its content in mucus gel of animals receiving sofalcone reached the value of 50%. The results indicate that sofalcone enhances the protective qualities of mucus component of gastric mucosal barrier.     

The role of covalently bound fatty acids in the degradation of human gastric mucus glycoprotein

The undegraded high-molecular-weight glycoprotein of human gastric mucus has been isolated free of noncovalently bound proteins and lipids, as judged by gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, cesium chloride density gradient centrifugation, and lipid analysis. Mild alkaline methanolysis of the thoroughly delipidated glycoprotein revealed that, on the average, the native undegraded glycoprotein contains 2.9 mol of acyl linked fatty acids/mg glycoprotein. The low-molecular-weight glycoprotein subunits, obtained after pepsin digestion, contain 2 nmol of acyl linked fatty acids/mg glycopeptide. The highest content of covalently bound fatty acids was found in the fraction of glycoprotein which remained undegraded after pepsin digestion. On the average, 10.2 mol of fatty acids/mg was substituted on this pepsin-resistant glycoprotein. After deacylation with hydroxylamine, the undegraded pepsin-resistant glycoprotein became susceptible to proteolytic cleavage. The obtained results suggest that fatty acids covalently bound to gastric mucus glycoprotein are involved in the regulation of proteolytic digestion of mucus glycoprotein in the stomach.     

Isolation of fatty acids covalently bound to the gastric mucus glycoprotein of normal and cystic fibrosis patients

Covalently bound fatty acids were found in strictly purified and delipidated gastric mucus glycoprotein of normal and cystic fibrosis individuals. The susceptibility of this linkage to methanolic KOH and hydroxylamine treatment indicated the ester bond between fatty acids and glycoprotein. On the average, 2.9 nmol fatty acid/mg glycoprotein were found in normal samples, and 12.2 nmol/mg glycoprotein in samples derived from cystic fibrosis. In normal gastric mucus glycoprotein the covalently linked fatty acids consisted of hexadecanoate (47.0%), octadecanoate (22.0%), tetracosanoate (5.9%), octadecenoate (14.5%) and tetracosenoate (6.0%). In cystic fibrosis mucus glycoprotein the covalently bound fatty acids were comprised mainly of hexadecanoate (36.5%), octadecanoate (48.7%) and octadecenoate (8.6%). These data indicate that cystic fibrosis gastric mucus glycoprotein is highly acylated and perhaps this is the major defect of glycoproteins in this disease.     

Gastric mucin hydrophobicity: effects of associated and covalently bound lipids, proteolysis, and reduction

The hydrophobic properties of gastric mucus glycoprotein were investigated using the fluorescent probe, bis(8-anilino-1-naphthalenesulfonate). The glycoprotein was subjected to removal of associated and covalently bound lipids, peptic degradation, and disulfide bridge reduction. Fluorescence titration data revealed the presence of 55 hydrophobic binding sites in the intact mucin molecule, 71 binding sites in the glycoprotein devoid of associated lipids, and 53 binding sites in the glycoprotein devoid of associated lipids and covalently bound fatty acids. Proteolytic digestion of the glycoprotein with pepsin essentially abolished the probe binding, while reduction of disulfide bridges resulted in glycoprotein subunits whose combined number of binding sites was about 3 times greater than that of the mucin polymer. The binding of the probe to mucus glycoprotein varied with the pH of the medium, being highest at pH 2.0 and lowest at pH 9.0. The results indicate that lipids contribute to the hydrophobic character of gastric mucin and that hydrophobic binding sites reside on the nonglycosylated regions of the glycoprotein polymer buried within its core.     

Only trace amounts of fatty acids are found in pure mucus glycoproteins

The presence of noncovalently associated lipids and covalently bound fatty acids was investigated in preparations of mucus glycoproteins obtained by using density-gradient centrifugation in CsCl/guanidinium chloride. No phospholipids, glycolipids, cholesterol, or triglycerides could be detected. However, small amounts of extractable fatty acids were consistently found, the sum of which ranged from 0.3 to 0.9 micrograms/mg of glycoprotein. The amount of fatty acid released after subsequent treatment with KOH ranged from 0 to 27 ng/mg of glycoprotein. We conclude that density-gradient centrifugation in CsCl/guanidinium chloride is very efficient in removing noncovalently associated lipids from mucus glycoproteins and that covalently bound fatty acids are probably not present in the macromolecules.     

Covalently bound fatty acids in the gastrointestinal epithelial cell membrane.

Myristic, palmitic, stearic, oleic and linoleic acids have been identified as the covalently bound fatty acids in the monkey gastrointestinal mucosal membrane proteins and among them palmitolation was predominant. Distribution studies in various regions of the gastrointestinal mucosa showed no significant difference in the content and composition of covalently bound fatty acids in these membrane and most of the fatty acids were found to be ester linked. Total membranes from isolated crypt and villus enterocytes and colonocytes had similar composition of these fatty acids. Covalently bound fatty acid levels were higher in the small intestinal brush border membrane. As suggested for the mucus glycoproteins, covalently bound fatty acids in the intestinal epithelial cell membrane may protect these membranes from proteolytic damage from the luminal proteases.     

The effect of sialoadenectomy on gastric mucosal integrity in the rat: roles of epidermal growth factor and prostaglandin E2

Removal of the salivary glands (SALX) in rats has been shown to increase the susceptibility of gastric mucosa to ulcerogens. In the present study, we have investigated the role of specific salivary glands in this response. In addition, we have examined whether a functional link exists between the salivary glands, epidermal growth factor (EGF), and prostaglandin E2 (PGE2) by determining whether SALX decreases the responsiveness of the mucosa to the protective actions of either of both of these agents. Removal of the parotid salivary glands did not significantly increase ulceration in response to intragastric administration of 100% w/v ethanol. Animals were examined 60 min after ethanol administration. Removal of the submandibular-sublingual gland complexes was associated with a significant increase in the area of mucosal damage and a decrease in gastric pit depth in ethanol-treated animals when compared with sham-operated control rats. Furthermore, in both SALX and control animals, exogenous PGE2 and EGF resulted in a dose-dependent reduction in both groups of animals, although the protective effects of PGE2 and EGF were attenuated in SALX rats. PGE2 and EGF administered in combination resulted in the same degree of protection in both SALX and control rats. Sialoadenectomy resulted in a reduction in mucosal PGE2 synthesis. EGF administration did not consistently increase mucosal PGE2 synthesis. Conversely, sialoadenectomy did not reduce mucosal levels of EGF nor did exogenous PGE2 consistently increase salivary or mucosal content of EGF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of EGF is associated with the delay of ulcer healing by cigarette smoking

Cigarette smoking is associated with peptic ulcer diseases. Smokers have lower levels of salivary epidermal growth factor (EGF) than nonsmokers. We investigated whether reduction of EGF is involved in the delay of gastric ulcer healing by cigarette smoking. Rats with acetic acid-induced ulcers were exposed to cigarette smoke (0, 2, or 4% vol/vol) 1 day after ulcer induction. EGF level was elevated 1 day after ulcer induction in salivary glands and serum, and 4 days after ulcer induction in the gastric mucosa. However, cigarette smoke depressed these beneficial effects and EGF mRNA expression in salivary glands and gastric mucosa. Cigarette smoke delayed gastric ulcer healing and reduced cell proliferation, angiogenesis, and mucus synthesis. Exogenous EGF (10 and 20 microg/kg i.v.) before smoke exposure reversed the adverse effects of cigarette smoke, whereas vascular endothelial growth factor level and nitric oxide synthase activity were unaffected. It is concluded that the detrimental effect of cigarette smoke on ulcer healing is a consequence of reduction of angiogenesis, cell proliferation, and mucus secretion through the depressive action on EGF biosynthesis and its mRNA expression in salivary glands and gastric mucosa.     

Changes in mucus glycoprotein synthesized in rat gastric mucosa exposed to ethanol

The resistance to proteolysis by pepsin of gastric mucus glycoprotein synthesized by tissue culture in the presence and absence of 0.1 M ethanol was investigated. The glycoprotein product of ethanol-supplemented culture was found to contain 68% less associated lipids and 81% less covalently bound fatty acids, but exhibited unaltered content of carbohydrate and protein. The lipid and fatty acyl deficient glycoprotein was 5-times more rapidly and 2-3-times more extensively degraded by pepsin than the glycoprotein synthesized in the absence of ethanol. Following delipidation with organic solvents and deacylation with hydroxylamine both glycoproteins were digested at the same rate and degraded to the same extent. The lower content of fatty acyl residues markedly affected the overall pattern of the proteolytic fragments identified by SDS gel electrophoresis. The peptides corresponding to the acylated fragments of control were degraded and an increase in the amount of smaller peptides was observed. The in vitro assays of the fatty acyltransferase activity towards the substrates obtained from control and alcohol-containing cultures revealed that the enzyme activity was similar and increased proportionally with increased concentration of both glycoprotein substrates and enzyme. However, addition of 0.1 M ethanol to the assay tubes containing complete incubation mixture decreased the acylation of either glycoprotein by 40%. Based on the results presented here, and on previous studies of mucus glycoprotein synthesis in the presence of ethanol, we conclude that ethanol interferes with the process of acylation of mucus glycoprotein with fatty acids.     

Interactions of EGF and ornithine decarboxylase activity in the regulation of gastric mucus synthesis in cigarette smoke exposed rats

Cigarette smoking has been shown to aggravate ulceration and delay ulcer healing. Smokers had a lower level of mucus in their stomachs. In the present study, we examined whether cigarette smoke or its extract reduced mucus production through the suppression of epidermal growth factor (EGF) associated with the reduction of polyamine biosynthesis both in vivo and in vitro. Ornithine decarboxylase (ODC) activities and mucus synthesis were determined in rat gastric mucosa and in human MKN-28 cells. Incubation of MKN-28 cells with EGF (0.01-1.00 ng/mL) significantly increased mucus synthesis in vitro, which was accompanied by an increase of ODC activity. Removal of salivary glands decreased the circulated EGF level and induced a significant reduction of mucus-secreting layer thickness in the gastric mucosa. Cigarette smoke or its extract markedly decreased mucus synthesis in vivo and in vitro, both of which could be completely reversed by intravenous administration of EGF (20 microg/kg) in rats or co-incubation with EGF (1 and 2 ng/mL) in MKN-28 cells. However, ODC activities, which were suppressed by cigarette smoke or its extract, were unaffected by intravenous administration of EGF in rats, or only partially reversed by co-incubation with EGF in MKN-28 cells. These findings indicate that both EGF and ODC activity represent two different entities in the modulation of cigarette smoking on gastric mucus synthesis. The action of EGF on mucus synthesis may only be partially if not dependent on ODC activity in the stomach.     

In vitro acylation of rat gastric mucus glycoprotein with [3H]palmitic acid

The incorporation of fatty acids into gastric mucus glycoproteins was studied by incubating rat gastric mucosal cell suspensions with [9,10-3H]palmitic acid and [3H]proline. The mucus glycoprotein polymer, secreted into the growth medium (extracellular) and that contained within the cells (intracellular), was purified from the other components of the secretion, thoroughly delipidated, and then analyzed for the radiolabeled tracers. Both pools of mucus glycoprotein, incubated in the presence of [3H]palmitic acid, contained radioactive label which could not be removed by gel filtration, CsCl density gradient centrifugation, sodium dodecyl sulfate-gel electrophoresis, or lipid extraction. Treatment of the purified mucus glycoprotein with 1 M hydroxylamine or 0.3 M methanolic KOH released the radioactivity, thus indicating that [3H]palmitic acid was covalently bound by ester linkage to the glycoprotein. The released radioactivity was associated mainly (87%) with palmitic acid. The incorporation ratio of [3H]proline to [3H]palmitic acid was 0.12:1.0 in the extracellular glycoprotein and 1.38:1.0 in the intracellular glycoprotein, which suggested that acylation of mucus glycoprotein occurs in the intracellular compartment after completion of its polypeptide core. The fact that incorporation of [3H]palmitic acid was greater in the glycoprotein subunits than in the glycoprotein polymer indicates that acylation takes place near the end of subunit processing but before their assembly into the high molecular weight mucus glycoprotein polymer.     

Role of capsaicin sensory nerves and EGF in the healing of gastric ulcer in rats

Accumulating evidence indicates that capsaicin sensitive afferent fibers play a pivotal role not only in gastroprotection but also in ulcer healing. Denervation of capsaicin sensitive afferent fibers exerts an adverse action on these effects. However, whether such an action is mediated through a depression on epidermal growth factor (EGF) is undefined. In this study, the effects of denervation of sensory neurons with capsaicin (100 mg/kg, s.c.) on acetic acid-induced chronic gastric ulcers and their relationship with the EGF expression in salivary glands, serum and gastric mucosa were investigated. Capsaicin significantly increased ulcer size, decreased gastric mucosal cell proliferation at the ulcer margin, angiogenesis in the granulation tissue and also gastric mucus content. Ulcer induction by itself dramatically elevated EGF levels in salivary glands and serum on day 1 and 4, and also in the gastric mucosa on day 4. However, capsaicin completely abolished these effects. It is concluded that stimulation of EGF expression in salivary glands and serum may be one of the mechanisms by which capsaicin sensitive nerves contribute to the gastroprotective and ulcer healing actions in the stomach.     

Endogenous fatty acids are covalently and noncovalently bound to interphotoreceptor retinoid-binding protein in the monkey retina

Interphotoreceptor retinoid-binding protein (IRBP) purified from monkey interphotoreceptor matrix contains relatively high concentrations of endogenous fatty acids, 6.51 mol/mol of protein. Sixty-five percent of the total fatty acid bound to IRBP was found to be noncovalently attached, with the remainder covalently bound. The fatty acids are not residual components of phospholipids or neutral lipids, as judged by microchemical methods. The major fatty acids bound to IRBP are: palmitic (35%), stearic (21%), palmitoleic (7%), oleic (29%), linoleic (6%) and docosahexaenoic acids (2%). These fatty acids account for about 90% of the total fatty acid bound to interphotoreceptor matrix proteins extracted with organic solvents. Thus, IRBP may function as an intercellular fatty acid carrier and may depend on the covalently bound fatty acids for anchoring in the outer leaflet of cell membranes.     

The synthesis and secretion of gastric mucus glycoprotein by mucosal cells cultured in the presence of ethanol

The effect of ethanol on the synthesis and secretion of mucus glycoprotein in gastric mucosal cells was investigated. The mucosal cell suspensions were subjected to a short-term (4 h) culture in the presence of 0-1.5 M ethanol, with [3H]proline and [3H]palmitic acid as markers for glycoprotein synthesis and acylation. The synthesized labeled mucus glycoprotein was isolated from the incubation medium (extracellular glycoprotein) and from the mucosal cells (intracellular glycoprotein), and analyzed. Depending upon the ethanol concentration in the cell culture medium, two distinct effects on the synthesis and secretion of mucus glycoprotein were observed. The cells cultured in the presence of 0.02-0.1 M ethanol showed increased ability for the incorporation of [3H]proline and [3H]palmitic acid, and for the secretion of the newly assembled mucus glycoprotein. The synthesis of the glycoprotein increased 18-fold, acylation 5-fold, and secretion 10-fold. The synthesized glycoprotein, however, contained four to five times less of acyl-bound fatty acids. Ethanol at 0.1-1.5 M caused a marked reduction (62-64%) in the mucus glycoprotein synthesis, but the amount of glycoprotein released to the medium remained constant. This indicated that higher concentrations of ethanol caused the release of the preformed intracellular mucus glycoprotein reserves. The results demonstrate that gastric mucosal cells incubated in the presence of ethanol exhibit impaired synthesis and secretion of mucus glycoprotein, and that the severity of impairment depends upon the ethanol concentration.     

Role of mucus in gastric mucosal protection.

Even though there is no general agreement as to the mechanism of gastric mucosal protection, the consensus is that the initial brunt of luminal insults falls on the mucus layer which constitutes the only identifiable physical barrier between the gastric lumen and the mucosal surface. The continuous renewal and resilient nature of this layer efficiently counters peptic erosion of the gel, assures its viscoelastic and permselective properties, and provides a milieu for containment of the diffusing luminal acid by mucosal bicarbonate. Disturbances in this delicate balance lead to the impairment of the protective function of mucus resulting in gastric disease. Indeed, the weakening of gastric mucosal defense is intimately associated with the diminished viscoelastic qualities of mucus, decrease in hydrogen ion retardation capacity, and the extensive proteolysis of its mucin component. Although until recently the disintegration of the mucus coat was attributed exclusively to the enhanced activity of intragastric pepsin, our studies provided strong argument that a bacterial factor, namely infection by Helicobacter pylori, through the action of its protease and lipase enzymes also is highly detrimental to the integrity of gastric mucus. Hence, agents capable of interfering with the pathogenic activity of this bacteria are becoming the drugs of choice in peptic ulcer therapy.     

SIgA Binding to Mucosal Surfaces Is Mediated by Mucin-Mucin Interactions

The oral mucosal pellicle is a layer of absorbed salivary proteins, including secretory IgA (SIgA), bound onto the surface of oral epithelial cells and is a useful model for all mucosal surfaces. The mechanism by which SIgA concentrates on mucosal surfaces is examined here using a tissue culture model with real saliva. Salivary mucins may initiate the formation of the mucosal pellicle through interactions with membrane-bound mucins on cells. Further protein interactions with mucins may then trigger binding of other pellicle proteins. HT29 colon cell lines, which when treated with methotrexate (HT29-MTX) produce a gel-forming mucin, were used to determine the importance of these mucin-mucin interactions. Binding of SIgA to cells was then compared using whole mouth saliva, parotid (mucin-free) saliva and a source of purified SIgA. Greatest SIgA binding occurred when WMS was incubated with HT29-MTX expressing mucus. Since salivary MUC5B was only able to bind to cells which produced mucus and purified SIgA showed little binding to the same cells we conclude that most SIgA binding to mucosal cells occurs because SIgA forms complexes with salivary mucins which then bind to cells expressing membrane-bound mucins. This work highlights the importance of mucin interactions in the development of the mucosal pellicle.     

Correlation of gastric mucosal damage with sialic acid profile in rats: effect of hydrochloric acid, pepsin and hypertonic saline

Sialic acids occupy terminal positions on gastric mucus glycoprotein where they contribute to the high viscosity of mucin. Desialylation of mucus may lead to degradation of the mucus and eventually to the breakdown of the gastric mucus barrier. The effect of a variety of damaging agents (0.1 M HCl, 2 mg ml(-1) pepsin and 2 M NaCl) on sialic acid profile was determined in pylorus-ligated rats. The relationship between sialic acid, galactose, pyruvate and the extent of gastric mucosal damage were studied. Instillation of pepsin significantly increased total sialic acid, galactose and macroscopic mucosal lesions in the stomach. Instillation of 0.1 M HCl reduced the total sialic acid but this decrease was not significant. Acidity led to a significant increase in the amount of free sialic acid in the gastric instillates and the macroscopic lesions induced by acid was not significantly different from the control animals (0.15 M NaCl). 2 M NaCl induced the macroscopic lesions in the stomach and also free sialic acid in the instillates. Pepsin potentiates the action of 2 M NaCl. In all the agents examined with the exception of acid, it was observed that an increase in free sialic acid and galactose was accompanied by gastric mucosal erosion and elevation of pyruvate concentration. It is concluded that gastric acidity alone is not inherently damaging and that resistance of gastric mucosa to destructive agents may be dependent on the integrity of the sialic acids.