Y.H. Liau

Glycosulfatase activity of helicobacter pylori toward gastric mucin

A glycosulfatase activity toward sulfated gastric mucus glycoprotein was identified in the extracellular material elaborated by H. pylori, a bacteria implicated in the etiology of gastric disease. Upon acetone precipitation, an active enzyme fraction at 64% acetone was obtained which on SDS-PAGE gave a major 30kDa protein band. The H. pylori glycosulfatase exhibited maximum activity (314.8 pmol/mg protein/h) at pH 5.7 in the presence of Triton X-100 and CaCl2, and was capable of removal of the sulfate ester groups situated at C-6 of N-acetylglucosamine, galactose and glucose. However, the enzyme was ineffective toward galactosylceramide and lactosylceramide sulfates which contain the sulfate ester group on C-3 of galactose. The results suggest that H. pylori is capable of overcoming the interference by sulfated mucus glycoprotein with its colonization of gastric mucosa.

Helicobacter pylori lipopolysaccharide effect on the synthesis and secretion of gastric sulfomucin

The effect of H. pylori lipopolysaccharide on the synthesis and secretion of sulfated mucin in gastric mucosa was investigated using mucosal segments incubated in the presence of [3H]proline, [3H]glucosamine and [35S]Na2SO4. The lipopolysaccharide, while showing no discernible effect on the apomucin synthesis was found to inhibit the process of mucin glycosylation and sulfation, which at 100 micrograms/ml lipopolysaccharide reached the optimal inhibition of 65%. The analysis of mucin secretory responses revealed that the lipopolysaccharide by first 15 min caused a 57% stimulation in sulfomucin secretion followed thereafter by inhibition, which reached maximum of 32% by 45 min. The results suggest that colonization of gastric mucosa by H. pylori may be detrimental to the process of gastric sulfomucin synthesis and secretion.

Role of sulfation in post-translational processing of gastric mucins.

1. Gastric mucosal segments were incubated in MEM supplemented with various sulfate concentrations in the presence of [3H]glucosamine, [3H]proline and [35S]Na2SO4, with and without chlorate, an inhibitor of 3-phosphoadenosine-5-phosphosulfate formation. 2. Incorporation of glucosamine and sulfate depended upon the sulfate content of the medium and reached a maximum at 300 microM sulfate. Introduction of chlorate into the medium, while having no effect on protein synthesis as evidenced by proline incorporation, caused, at its optimal concentration of 2 mM, a 90% decrease in mucin sulfation and a 40% drop in glycosylation. 3. At low sulfate content in the medium and in the presence of chlorate, the incorporation of sulfate and glucosamine was mainly into the low molecular-weight form of mucin. An increase in sulfate in the medium caused an increase in the high molecular-weight form of mucin and in the extent of sulfation in its carbohydrate chain. 4. The results suggest that the sulfation process is an early event taking place at the stage of mucin subunit assembly and that sulfate availability is essential for the formation of the high molecular-weight mucin polymer.

Role of sulphation in post-translational processing of rat salivary mucins

Segments of rat submandibular salivary gland were incubated in MEM supplemented with 10-800 microM sulphate in the presence of [3H]-glucosamine, [3H]-proline and [35S]-Na2SO4, with 0-8 mM chlorate, an inhibitor of 3-phosphoadenosine-5-phosphosulphate formation. Incorporation of glucosamine and sulphate depended upon the sulphate content of the medium and reached a maximum at 400 microM sulphate. The introduction of chlorate into the medium, while having no effect on the protein synthesis as shown by [3H]-proline incorporation, caused, at its optimal concentration of 4 mM, a 90% decrease in mucin sulphation and a 29% drop in mucin glycosylation. At low sulphate content in the medium and in the presence of chlorate the incorporation of sulphate and glucosamine was mainly into the low molecular-weight form of mucin. An increase in sulphate in the medium caused an increase in the high molecular-weight form of mucin and in the extent of sulphation in its carbohydrate chain. This effect of sulphate was, however, inhibited by chlorate. The results suggest that sulphation takes place at an early stage of mucin assembly and that sulphate availability is essential for the formation of the high molecular-weight mucin.

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.

Glycosaminoglycan patterns in gingival proteoglycans of rat with age

Among the potential biochemical indices that are closely associated with craniofacial development are the proteoglycans. Gingival segments from the palate of 4-, 6-, 8-, 12- and 18-week-old rats were incubated for 4 h in medium containing [3H]-glucosamine and [35S]-Na2SO4, and subjected to proteoglycan isolation and glycosaminoglycan analysis. Two distinct proteoglycan fractions differing in the degree of sulphation were obtained by ion-exchange chromatography. The incorporation of both labels in the undersulphated fraction increased with age; there was a pronounced decrease with age in the sulphated proteoglycan fraction. The undersulphated proteoglycans showed an age-dependent decrease in hyaluronic acid, and increase in dermatan sulphate and chondroitin 4- and 6-sulphates. Gel filtration of the sulphated proteoglycan fraction yielded high and low molecular-weight proteoglycans, the glycosaminoglycans of which were particularly rich (61-76%) in dermatan sulphate. Smaller quantities of chondroitin 4- and 6-sulphates, and heparan sulphate were also present. All glycosaminoglycans showed a decrease in content with age. The findings suggest a possible correlation between gingival proteoglycan/glycosaminoglycan patterns and development.

Effect of ebrotidine on the synthesis and secretion of gastric sulfomucin

1. The effect of a new antiulcer agent, ebrotidine, on the synthesis and secretion of sulfomucin in gastric mucosa was investigated. Rat gastric mucosal segments were incubated in DMEM containing [3H]proline, [3H]glucosamine and [35S]Na2SO4 as markers for mucin synthesis, glycosylation and sulfation, in the presence of 0-150 microM ebrotidine. 2. The drug, while showing no discernible effect on the apomucin synthesis, evoked a dose-dependent increase in mucin glycosylation and sulfation, which at 100 microM ebrotidine, attained its maximum of 2.4 and 2.7-fold stimulation, respectively. 3. The analysis of mucin secretory responses revealed that ebrotidine caused a concentration-dependent enhancement in sulfomucin secretion which attained its maximum increase of 3.3-fold at 100-120 microM ebrotidine. Furthermore, the sulfomucin elaborated in the presence of ebrotidine exhibited a higher content of a large molecular-weight mucus glycoprotein form, the assembly of which is intimately associated with the sulfation event. 4. The results suggest that the ability of ebrotidine to enhance gastric sulfomucin synthesis and secretion may play an important role in the gastroprotective mechanism of action of this agent.

Nitecapone effect on the synthesis and secretion of gastric sulfomucin

1. The effect of a new antiulcer agent, nitecapone, on the synthesis and secretion of sulfomucin in gastric mucosa was investigated using mucosal segments incubated in the presence of [3H]proline, [3H]glucosamine and [35S]sulfate. 2. The drug, while showing no discernible effect on the apomucin synthesis, evoked a dose-dependent increase in mucin glycosylation and sulfation, which at 225 microM nitecapone, attained its maximum of 1.8 and 2.2-fold stimulation, respectively. 3. The analysis of mucin secretory responses revealed that nitecapone caused a concentration-dependent enhancement in sulfomucin secretion attaining maximum increase of 1.5-fold at 150 microM nitecapone. 4. The stimulatory effect of nitecapone on sulfomucin secretion was accompanied by 1.4-fold increase in mucosal cAMP level, and showed sensitivity to protein kinase A inhibitors, thus pointing towards the involvement of protein kinase A in mediation of gastric sulfomucin secretory responses to nitecapone. 5. The ability of nitecapone to enhance sulfomucin synthesis and secretion could be of importance to the gastroprotective action of this agent.

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.

Enhancement of gastric mucus phospholipid secretion by an antiulcer agent, ebrotidine.

1. Rat gastric mucosal cells, subjected to phospholipid labeling by incubating the cell suspension in DMEM with [3H]choline, were exposed to different concentrations (0-150 microM) of H2-receptor antagonists, ebrotidine and ranitidine, and the phospholipid secretory responses were evaluated. 2. In the absence of the drugs, the secretion of choline-containing phospholipids over a 1 hr period averaged 3.97% of the total cellular labeled phospholipids. Ebrotidine caused a dose-dependent increase in the rate of phospholipid secretion which was most pronounced at 1 hr and persisted for at least 2 hr. The maximal effect was attained at 120 microM ebrotidine giving a 36% increase in phospholipid secretion. 3. The phospholipid secretory response to ebrotidine was accompanied by an increase in gastric mucosal cell cAMP level which reached a maximum value of 2.1-fold over that of controls at 1 hr. Ranitidine, in contrast, neither evoked increase in cAMP level nor caused any stimulation in phospholipid secretion. 4. The results indicate that the gastroprotective properties of ebrotidine are associated with the ability of the drug to elicit a rapid stimulation in gastric mucus phospholipid secretion, and that ranitidine does not possess such property.