Sulfation in vitro of mucus glycoprotein by submandibular salivary gland: effects of prostaglandin and acetylsalicylic acid.

Enzymatic sulfation of mucus glycoprotein by rat submandibular salivary gland and the effect of prostaglandin and acetylsalicylic acid on this process were investigated in vitro. The sulfotransferase enzyme which catalyzes the transfer of sulfate ester group from 3'-phosphoadenosine-5'-phosphosulfate to submandibular gland mucus glycoprotein has been located in the detergent extracts of Golgi-rich membrane fraction of the gland. Optimum enzyme activity was obtained at pH 6.8 with 0.5% Triton X-100, 25 mM NaF and 4 mM MgCl2, using the desulfated glycoprotein. The enzyme was also capable of sulfation of the intact mucus glycoprotein, but the acceptor capacity of such glycoprotein was 68% lower. The apparent Km of the submandibular gland sulfotransferase for salivary mucus glycoprotein was 11.1 microM. The 35S-labeled glycoprotein product of the enzyme reaction gave in CsCl density gradient a 35S-labeled peak which coincided with that of the glycoprotein. This glycoprotein upon reductive beta-elimination yielded several acidic 35S-labeled oligosaccharide alditols which accounted for 75% of the 35S-labeled glycoprotein label. Based on the analytical data, the two most abundant oligosaccharides were identified as sulfated tri- and pentasaccharides. The submandibular gland sulfotransferase activity was stimulated by 16,16-dimethyl prostaglandin E2 and inhibited by acetylsalicylic acid. The rate of enhancement of the glycoprotein sulfation was proportional to the concentration of prostaglandin up to 2.10(-5) M, at which point a 31% increase in sulfation was attained. The inhibition of the glycoprotein sulfation by acetylsalicylic acid was proportional to the drug concentration up to 2.5.10(-4) M at which concentration a 48% reduction in the sulfotransferase activity occurred. The apparent Ki value for sulfation of salivary mucus glycoprotein in presence of acetylsalicylic acid was 58.9 microM. The results suggest that prostaglandins may play a role in salivary mucin sulfation and that this process is sensitive to such nonsteroidal anti-inflammatory agents as acetylsalicylic acid.

Lipid composition of the secretion from human bronchial explant culture.

In this study, we identified and quantitated the lipid components in the secretions of human bronchial explants cultured in a serum-free medium over a period of 50 days. Total lipids represented 6% of the dry material. This amount is considerably lower than that reported for 'normal' human sputum and suggests that the latter is contaminated by serum transudates, alveolar secretions, and microorganisms. Such serum-free culture systems are highly suitable to study cell physiology as it relates to human disease.

Glycosphingolipids of guinea pig gastric mucosa.

Glycosphingolipids have beenn isolated from guinea pig gastric mucosa and their composition and content determined. The neutral glycospingolipids were found to consist of mono-, di-, tri- and pentaglycosylceramide. The acidic glycosphingolipids wee represented by galactosyl and lactosyl sulfatides, and GM4, GM3 and GD3 gangliosides. None of the analyzed glycolipids contained N-acetylglucosamine and fucose.

Mucus glycoprotein of human saliva: differences in the associated and covalently bound lipids with caries.

A high molecular weight mucus glycoprotein has been isolated from submandibular saliva of caries-resistant and caries-susceptible individuals by a procedure involving fractionation on Bio-Gel P-100 and A-50 columns followed by equilibrium density-gradient centrifugation in CsCl. The purified caries-resistant mucus glycoprotein displayed a buoyant density of 1.50 and accounted for 9.5% of the dry weight of caries-resistant saliva. The caries-susceptible mucus glycoprotein represented 14.1% of the dry weight of caries-susceptible saliva and gave a buoyant density of 1.43. Both glycoproteins exhibited similar protein and carbohydrate content, but the caries-resistant mucus glycoprotein contained 28.7% less associated lipids and 3-times less covalently bound fatty acids than the caries-susceptible mucus glycoprotein. The associated lipids were represented by neutral lipids, glycolipids and phospholipids, whereas the covalently bound fatty acids consisted mainly of hexadecanoate, octadecanoate and docosanoate. Extraction of associated lipids caused the caries-resistant glycoprotein to band in CsCl gradient at the density of 1.54 and caused the caries-susceptible glycoprotein to band at the density of 1.52. A further shift in the buoyant densities occurred following removal of the covalently bound fatty acids, and both glycoproteins banded at the density of 1.57. While the intact caries-resistant and caries-susceptible glycoproteins were susceptible to proteolysis by pronase, the lipid-rich caries-susceptible glycoprotein was degraded to a lesser extent. Extraction of associated lipids increased the degradation of both glycoproteins, but the caries-susceptible glycoprotein still remained 25% less susceptible. However, the susceptibility to pronase of the delipidated and deacylated caries-resistant and caries-susceptible glycoproteins was essentially identical. The caries-resistant and caries-susceptible mucus glycoproteins also differed in susceptibility to peptic degradation. The apparent Km values for intact caries-resistant and caries-susceptible glycoproteins were 10.5 X 10(-7) M and 8.1 X 10(-7) M, while the values for the delipidated and deacylated caries-resistant and caries-susceptible glycoproteins were 13.0 X 10(-7) M and 12.4 X 10(-7) M. The results suggest that the differences in the content of associated lipids and covalently bound fatty acids are responsible for the different physiochemical characteristics of caries-resistant and caries-susceptible salivary mucus glycoproteins, which may be determining factors in the resistance to caries.

In vitro fatty acid acylation of mucus glycoprotein from sublingual salivary glands.

The enzyme activity which catalyzes the transfer of palmitic acid from palmitoyl-coenzyme A to sublingual gland mucus glycoprotein has been demonstrated in the detergent extracts of the microsomal fraction of rat sublingual and parotid salivary glands. The acyltransferase activity of this fraction was similar in both types of glands. Further subcellular fractionation performed on sublingual glands revealed that the enzyme is associated with the Golgi-rich membrane fraction. Optimum enzymatic activity for fatty acylation of mucus glycoprotein was obtained using 0.5% Triton X-100, 2 mM dithiothreitol, 25 mM NaF, and 10 mM MgCl2 at a pH of 7.4. Higher concentrations of NaF, MgCl2 and dithiothreitol, however, were inhibitory. The apparent Km of the sublingual glands microsomal enzyme for mucus glycoprotein was 0.55 mg/ml and for palmitoyl-CoA, 3.5 X 10(-5) M. A 15% decrease in the acyltransferase activity was obtained with the reduced and alkylated mucus glycoprotein and it showed no activity towards the proteolytically degraded glycoprotein. The 14C-labeled product of the enzyme reaction gave in CsCl density gradient a band at the density of 1.49 in which the 14C label coincided with the glycoprotein. The 14C label in this glycoprotein was susceptible to deacylation with hydroxylamine, and the released labeled material was identified as palmitate.

Lipid composition of tracheobronchial secretions from normal individuals and patients with cystic fibrosis.

The lipid composition of tracheobronchial secretions from normal individuals and patients with cystic fibrosis was investigated. Lipids were extracted from he dialyzed and lyophilized samples, and fractionated on silicic acid columns into neutral lipids, glycolipids and phospholipids. The lipids contained each fraction were separated into individual components by thin-layer chromatography and quantified. The secretions of patients with cystic fibrosis and were found to contain about 30% more lipids than that of normal individuals and exhibited elevated levels of cholesterol, phospholipids and glycosphingolipids. The level of free fatty acids and glyceroglucolipids was higher in the normal secretions. The phospholipids of cystic fibrosis secretions exhibited higher content of sphingomyelin and phosphatidylserine, while the normal samples contained more lysophosphatidylcholine. The glycosphingolipids of both types of samples consisted mainly of glucosyl- and lactosylceramides. The major glyceroglucolipid of the normal tracheobronchial secretions was tetraglucosyl glyceroglucolipid, whereas hexa-and octaglucosyl glyceroglucolipids were the predominant compounds of the cystic fibrosis secretions.

Prostaglandin effect on the enzymatic sulfation of mucus glycoprotein in gastric mucosa.

The effect of 16,16-dimethyl prostaglandin E2 (DMPGE2) on the sulfation of mucus glycoprotein in gastric mucosa was investigated. The enzymatic activity which catalyzes the transfer of the sulfate ester group from 3'-phosphoadenosine-5'-phosphosulfate to gastric mucus glycoprotein was located in the detergent extracts of Golgi-rich membrane fraction of antral and body mucosa of rat stomach. The sulfotransferase activity of this fraction from body mucosa, however, was 35% higher than that from the antrum. The enzyme exhibited optimum activity at pH 6.8 using 0.5% Triton X-100 and 30 mM NaF. The apparent Km of the enzyme for sulfation of mucus glycoprotein was 10.5 microM, and the sulfate ester was found incorporated into the carbohydrate chains of the glycoprotein. Introduction of DMPGE2 to the reaction mixtures led to an enhancement in the rate of mucus glycoprotein sulfation. The rate of enhancement was proportional to the concentration of DMPGE2 up to 1.0 x 10(-4) M and was of the competitive type, with an apparent Km value of 6.7 microM. Since sulfated mucins play an important role in gastric mucosal defense and the increase in their sulfation occurred at levels of prostaglandin present in gastric mucosa, the observed effect may be of significance to gastric mucosal defense in vivo.

Enhancement of the lipid content and physical properties of gastric mucus by geranylgeranylacetone.

The effects of intragastric administration of geranylgeranylacetone (GGA) on the content, composition and physical properties of the mucus component of the gastric mucosal barrier were investigated. One group of rats received twice daily for 3 consecutive days a dose of 100 mg/kg body weight of GGA, while the control group was subjected to daily doses of the vehicle. Sixteen hours following the last dose, the animals were killed, and their stomach was cut open and subjected to measurements of the adherent mucus gel content, analysis of its lipids and molecular forms of elaborated mucin, and evaluation of the viscosity and H+ retardation capacity. The results revealed that GGA elicited a 62% increase in the adherent mucus gel and caused a marked decrease in the proportion of the lower molecular weight mucin. Furthermore, the mucus of the GGA group exhibited a 67% higher content of covalently bound fatty acids and contained 46% more total lipids which were greatly (143%) enriched in phospholipids. The physical measurements demonstrated that mucus elaborated in the presence of GGA also exhibited 2.3 times higher viscosity and had a 32% greater ability to retard the diffusion of H+ than the mucus of the control group. The results suggest that GGA exerts a profound effect on the lipid content and the properties of gastric mucus associated with the maintenance of the mucosal integrity.

Lipid composition of submandibular saliva from normal and cystic fibrosis individuals.

The submandibular saliva of patients with cystic fibrosis was found to contain about 66% more lipids/100 ml of saliva than that of normal individuals and exhibited elevated levels of neutral lipids, phospholipids, and glycolipids. No significant differences were noted in the proportions of individual neutral lipid and phospholipid components present in both types of samples. The glycolipids of normal saliva consisted entirely of glyceroglucolipids, whereas those of cystic fibrosis saliva, in addition to glyceroglucolipids, also contained small amounts of glycosphingolipids. These quantitative and qualitative differences may affect the physicochemical properties of the secretion.

Association of lipids with proteins and glycoproteins in human saliva.

The distribution of lipids in the fractions of parotid and submandibular saliva following Bio-Gel A-50 column chromatography was measured. Over 50% of the total lipids of submandibular saliva was found in the fraction which contained mainly the high-molecular-weight glycoprotein. This fraction also contained most of the glycolipids, free fatty acids, phospholipids, and cholesterol. In the parotid saliva, the fraction containing the basic glycoprotein (the major glycoprotein fraction of parotid saliva) contained 35% of the total saliva lipids and was enriched in phospholipids ana cholesterol esters.

Lipids of supragingival calculus.

The matrix of supragingival calculus constitutes 15.7% of the calculus dry weight and contains 54.9% protein and 10.2% lipids. Of the total lipids, 61.8% are represented by neutral lipids, 28% by glycolipids, and 10.2% by phospholipids. The neutral lipids exhibit a high content of free fatty acids (63.9%) and triglycerides (15.8%). The glycolipids are comprised of simple glycosphingolipids (17.2%), mainly lactosyl- and glucosylceramides, and of neutral and sulfated glyceroglucolipids (82.8%). The phospholipids contain large quantities of phosphatidylethanolamine (34.2%) and diphosphatidylglycerol (25.5%). Comparison with salivary and submandibular stone lipids indicates that both saliva and bacteria contribute to the lipid content of supragingival calculus.

Lipid composition of human parotid salivary gland stones.

The parotid gland stone matrix constitutes 20.2% of the stone dry weight and contains 8.5% of lipids. Of the total lipids, 74% are represented by neutral lipids, 17% by glycolipids, and 9% by phospholipids. The neutral lipids exhibit a high content of free fatty acids (52.7%) and cholesteryl esters (31.1%). The glycolipids are composed of simple glycosphingolipids (7.1%), and of neutral and sulfated glyceroglucolipids (92.9%). Phosphatidylethanolamine and diphosphatidylglycerol are the principal constituents of the phospholipid fraction.

Modulation of buccal mucosal calcium channel activity by salivary mucins.

The effect salivary mucus glycoproteins on the activity of calcium channel isolated from buccal mucosal cell membranes was investigated. The channel complex following reconstitution into phospholipid vesicles exhibited an active 45Ca2+ uptake and responded to calcium channel activator, BAY K8644, and the antagonist, PN200-110. The uptake of 45Ca2+, while only moderately (15%) affected by the intact mucus glycoprotein was significantly inhibited (60-64%) by the acidic mucus glycoprotein fractions. This effect was associated with the sialic acid and sulfate ester groups of the carbohydrate chains. The channel complex in the presence of epidermal growth factor (EGF) and ATP responded by an increase in protein tyrosine phosphorylation of 55 and 170kDa proteins, and the vesicles containing the phosphorylated channels showed a 46% increase in 45Ca2+ uptake. The phosphorylation and the calcium uptake were susceptible to inhibition by a specific tyrosine kinase inhibitor, genistein. The binding of EGF to buccal mucosal calcium channel receptor protein was also inhibited (36-41%) by acidic mucus glycoprotein. The reduction in binding was dependent upon the presence of sulfate ester and sialic acid groups, as evidenced by the loss of the glycoprotein inhibitory capacity following removal of these groups. The results for the first time demonstrate that salivary mucins actively participate in the modulation of the buccal mucosal calcium channel activity, a process of importance to the preservation of soft oral integrity.

Role of sulfation in the processing of gastric mucins.

The role of sulfation in the processing of mucus glycoprotein in gastric mucosa was investigated. Rat gastric mucosal segments were incubated in MEM at various medium sulfate concentrations in the presence of [35S]Na2SO4, [3H]glucosamine and [3H]proline, with and without chlorate an inhibitor of PAPS formation. The results revealed that the mucin sulfation attained maximum at 300 microM medium sulfate concentration. Introduction of chlorate into the incubation medium, while having no effect on the protein synthesis as evidenced by [3H]proline incorporation, caused at its optimal concentration of 2 mM a 90% decrease in mucin sulfation and a 40% drop in mucin glycosylation. Evaluation of mucin molecular forms distribution indicated the predominance of the high molecular mucin form in the intracellular fraction and the low molecular mucin from in the extracellular fraction. Increase in medium sulfate caused an increase in the high molecular weight mucin form in both fractions, and this effect was inhibited by chlorate. Also, higher medium sulfate concentrations led to a higher degree of sulfation in the high molecular weight mucin form, the effect of which was inhibited by chlorate. The results suggest that the sulfation process is an early event taking place at the stage of mucin subunit assembly and is required for mucin polymer formation. Hence, the disturbances in mucin sulfation process could be detrimental to the maintenance of gastric mucus coat integrity.

Glycosulfatase activity of Helicobacter pylori towards gastric sulfomucin: effect of nitecapone.

A glycosulfatase activity towards human gastric sulfomucin was identified in the extracellular material elaborated by H. pylori, a pathogen implicated in the etiology of gastric disease. The purified enzyme exhibited maximum activity at pH 5.7 in the presence of 0.3% Triton X-100 and 100mM CaCl2, and displayed on SDS-PAGE an apparent molecular weight of 30kDa. The H. pylori glycosulfatase effectively caused desulfation of N-acetylglucosamine-6-sulfate and galactose-6-sulfate of the carbohydrate chains of mucins, as well as that of glucose-6-sulfate of glyceroglucolipids, but was ineffective towards galactosyl -and lactosylceramide sulfates which contain galactose-3-sulfate. The glycosulfatase activity towards human gastric sulfomucin was inhibited by an antiulcer agent, nitecapone, which at its optimal concentration (100 micrograms/ml) caused a 61% decrease in mucin desulfation. The results show that H. pylori through its glycosulfatase activity causes desulfation of sulfated mucins and glyceroglucolipids of the protective mucus layer, and that nitecapone is able to interfere with this detrimental action.

Rat-colonic, mucus glycoprotein.

A glycoprotein was isolated from rat-colonic mucosa. Analytical ultracentrifugation studies showed the glycoprotein to be homogeneous, having an apparent molecular weight of 9.0 X 10(5); no subunits could be detected in the presence of sodium dodecyl sulfate. It contained 14% of protein and 86% of carbohydrate. The principal sugars in the glycoprotein were galactose, fucose, sialic acid, 2-acetamido-2-deoxygalactose, and 2-acetamido-2-deoxyglucose. A small proportion of mannose was also present. The glycoprotein, apart from the usual carbohydrate constituents present in mucus glycoproteins, contained sulfate, but no uronic acid. High amounts of serine and threonine, and low contents of aromatic and traces of sulfur-containing amino acids, reflect a similarity of this glycoprotein to other mammalian mucus glycoproteins; it differs, however, by its high proportions of Asx + Glx (26 mol.%). Cleavage studies with alkaline borohydride indicated O-glycosidic linkages between N-acetylhexosamine and serine, and threonine, of the peptide core in the glycoprotein. Only about one third of the serine and threonine was linked to the carbohydrate side-chains, which averaged about 22 units in length and were apparently branched.

Lipids associated with rat small-intestinal mucus glycoprotein.

The lipid content and composition of rat small-intestinal mucus, and the purified mucus glycoprotein before and after Pronase digestion were investigated. The mucus, obtained by the instillation of intestine with 2M NaCl, was fractionated on Bio-Gel A-50 in the presence of 6M urea and the mucus glycoprotein free of noncovalently bound protein was isolated. A portion of the purified glycoprotein was subjected to Pronase digestion to yield glycopeptides. The native mucus, and the purified glycoprotein and glycopeptides were extracted with chloroform-methanol, and the lipids contained in the extracts were analyzed. The lipids accounted for 17.6 of the dry weight of mucus, 26.4 of the mucus glycoprotein, and 25.3% of the glycopeptides. In comparison to mucus, the lipids associated with mucus glycoprotein contained 1.9 times more phospholipids and 2.1 times more glycolipids, showed a 26% increase in neutral lipids, and were virtually free of glycosphingolipids. Treatment of the purified glycoprotein with Pronase led to a moderate (22.3%) loss in neutral lipids, 4.3-fold decrease in phospholipids, and 52.3% increase in glyceroglucolipids. The results indicate that while the interaction of mucus glycoprotein with phospholipids involves its Pronase-susceptible region, the interaction with glyceroglucolipids occurs in the glycosylated region of the glycoprotein that is resistant to proteolysis.

Physico-chemical characteristics of mucus glycoproteins and lipids of the human oral mucosal mucus coat in relation to caries susceptibility.

Mucus coat was isolated from oral epithelial surfaces of caries-resistant and caries-susceptible subjects, analysed for content and composition of lipids and mucus glycoproteins, and evaluated for physico-chemical characteristics. The mucus coat from caries-resistant subjects had a protein content similar to that of the caries-susceptible group but a higher content of carbohydrate and a lower content of lipids and covalently bound fatty acid. The carbohydrate component was mainly mucus glycoprotein, which accounted for 28.4% of the dry weight of caries-resistant mucus and 25.3% of caries-susceptible mucus. By chromatographic analysis on Bio-Gel A-50, both types of preparations had high (Mr approximately 2000 kdalton) and low (Mr approximately 300 kdalton) molecular-weight mucus glycoproteins. In the caries-susceptible mucus coat these two glycoproteins were in similar proportions, whereas the low molecular-weight glycoprotein predominated in caries-resistant mucus. In both preparations, the high molecular-weight glycoprotein was characterized by a high content of carbohydrates, associated lipids and covalently bound fatty acids, whereas the low molecular-weight glycoprotein was richer in protein and contained lesser amounts of associated and covalently bound lipids. Although the low molecular-weight glycoprotein showed only minor compositional differences with caries status, the high molecular-weight glycoprotein of the caries-resistant group had a 2.5 times lower content of covalently bound fatty acid, a 1.3 times lower content of associated lipids and contained 1.2 times more sulphate and sialic acid then that of the caries-susceptible group.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzymatic sulphation of mucus glycoprotein in rat sublingual salivary gland.

Sulphotransferase activity catalysing the transfer of sulphate ester group from 3'-phosphoadenosine-5'-phosphosulphate to salivary mucus glycoprotein was located in detergent extracts of the Golgi-rich membrane fraction of rat sublingual salivary glands. Optimum enzyme activity was obtained with 0.5 per cent Triton X-100, 20 mM NaF and 2 mM MgCl2, at pH 6.8, using desulphated sublingual salivary mucus glycoprotein. The enzyme was equally capable of sulphation of the proteolytically degraded and desulphated glycoprotein, whereas the acceptor capacity of intact salivary mucus glycoprotein was about four times lower. The Golgi enzyme preparation also catalysed the sulphation of galactosylceramide. However, the sulphation of mucus glycoprotein was not affected by the presence of this glycolipid, suggesting that the sulphotransferase involved in mucin sulphation is different from that responsible for the synthesis of galactosylceramide sulphate. The apparent Km of the sublingual-gland mucus glycoprotein sulphotransferase for salivary mucin was 7.7 microM. The 35S-labelled glycoprotein product of the enzyme reaction gave, in CsCl density gradient, a band in which the 35S label coincided with the glycoprotein. Alkaline borohydride reductive cleavage of this glycoprotein released the label into the reduced acidic oligosaccharide fraction. Upon thin-layer chromatography, two [35S]-oligosaccharides were detected. These were identified as penta- and heptasaccharides, each bearing a labelled sulphate ester group on the terminal N-acetylglucosamine residue. Based on the results of chemical and enzymatic analyses of the intact and desulphated compounds the following structures for these oligosaccharides are suggested: SO3----GlcNAc beta----Gal beta----GlcNAc beta----Gal beta----GlcNAc beta----(NeuAc----)GalNAc-ol and SO3----GlcNAc beta----Gal beta----GlcNAc beta----(NeuAc----)GalNAc-ol.

Effect of lipids on the lactic acid retardation capacity of tooth enamel and cementum pellicles formed in vitro from saliva of caries-resistant and caries-susceptible human adults.

The lipid content and composition of these pellicles and the effect of lipids on their ability to retard the diffusion of lactic acid were investigated. Lipids accounted for 22.4% of the dry weight of caries-resistant enamel pellicle and 19.2% of caries-resistant cementum pellicle; the pellicle of caries-susceptible enamel contained 24.6% lipids and that of caries-susceptible cementum, 23.4%. Enamel and cementum pellicles from caries-resistant saliva had a significantly lower content of neutral lipids and phospholipids, whereas the glycolipid content was lower, although not significantly, in caries-susceptible pellicles. Pellicles from caries-resistant saliva had a considerably greater capacity to retard lactic acid diffusion than those from caries-susceptible saliva. In all cases, the retardation capacity was clearly dependent upon the lipid constituents, removal of which caused a 50-52% drop in lactic acid impedance by caries-resistant enamel and cementum pellicles, and 32-35% drop by caries-susceptible pellicles. On reacting the delipidated pellicles with their lipids, it was found that, in all cases, the highest quantitative effect on the restoration of the retardation capacity occurred when phospholipids were added. The findings suggest that the events controlling the interaction of salivary phospholipids with enamel and cementum may determine the susceptibility of the tooth surface to demineralization by acids produced by cariogenic micro-organisms.