Mucin Biosynthesis Research Articles

Effect of roxatidine bismuth citrate (MX1) against acetylsalicylic acid- and indomethacin-induced gastric mucosal damage in rats.

We have studied the effect of the newly synthesized agent, roxatidine bismuth citrate (N-[3-(3-(1-piperidinyl-methyl)phenoxy)propyl]-hydroxyacetamide-2- hydroxypropane-1,2,3-tricarboxylate-bismuth(3+) complex), code name MX1, against acetylsalicylic acid (ASA)- and indomethacin-induced gastric mucosal damage in rats. Effects of MX1 (12.5, 50, 125, 184, 250 mg/kg) were compared to the effects of equimolar doses of roxatidine and bismuth subcitrate. Effect of MX1 (10(-6) M) on mucin biosynthesis measured by [3H] glucosamine incorporation in rat gastric corpus has been determined. MX1-pretreatment dose-dependently decreased the mean ulcer number and length in all doses used in an extent similar to that of roxatidine and more pronounced in comparison with bismuth subcitrate. The morphometrical results have been confirmed histomorphologically. The biosynthesis of mucin was found to be significantly enhanced after MX1 addition. The results of the present study suggest that MX1 has a gastroprotective effect against ASA- and indomethacin-induced ulcers which might be due both to its H2-blocking and mucus-stimulating activity.

Changes to mucins in uninvolved mucosa and at the tumour site in gastric adenocarcinoma of intestinal type.

1. Mucin histochemistry is markedly altered in the stomach in intestinal-type adenocarcinoma. To increase understanding of these changes we have examined the content and distribution of carbohydrate in mucus glycopolypeptides isolated from non-malignant antrum, and from the uninvolved gastric mucosa and tumour site of patients with this disease. 2. The content of carbohydrate declined by 12.6% (P = 0.02) in mucus glycopolypeptides from uninvolved gastric mucosa when compared with those from non-malignant antrum, and by a further 25.4% (P < 0.001) in mucus glycopolypeptides from the tumour site. The first of these changes was accompanied by a significant decrease in the number of carbohydrate chains/1000 amino acid residues, and a significant increase in the number of monosaccharide units in each carbohydrate chain. The second of these changes was accompanied by significant decreases in both the number of carbohydrate chains/1000 amino acid residues, and in the number of monosaccharide units in each carbohydrate chain. 3. The number of sulphated monosaccharide units/100 carbohydrate chains increased from a mean of 7.2 in mucus glycopolypeptides from non-malignant antrum to a mean of 27.2 (P < 0.001) in preparations from uninvolved gastric mucosa and 22.7 (P < 0.001) in preparations from the tumour site. 4. Evidence is presented that these structural changes to mucus glycopolypeptides from the malignant stomach are due to an abnormal mucin biosynthesis by metaplastic goblet cells and/or immature gastric-type mucous cells within the uninvolved mucosa, and immature mucous cells at the tumour site.

Structural requirements for roxatidine in the stimulant effect of rat gastric mucin synthesis and the participation of nitric oxide in this mechanism.

1. The structural requirements of the histamine H2-receptor antagonist, roxatidine (2-acetoxy-N-(3-[m-(1-piperidinylmethyl)phenoxy]-propyl)acetamide hydrochloride), for the stimulant effect on mucin biosynthesis and their relation to histamine H2-receptor antagonism were identified by considering the structural analogues of this drug using an organ culture system of the rat stomach and competition studies with [125I]iodoaminopotentidine ([125I]-APT) binding to membranes of the guinea pig striatum. 2. [3H]Glucosamine incorporation into mucin during 5 h incubation period was stimulated by roxatidine and its structural analogues A (2-hydroxy-N-(3-[m-(1-piperidinylmethyl)phenoxy]-propyl)acetamide) and B (N-(3-[m-(1-piperidinylmethyl)phenoxy]-propyl)acetamide). This effect was seen in mucosal cultures of the corpus, but not antrum, region. 3. Structural analogues, in which the length of the flexible chain between the benzene ring and the amide structure differs from that of roxatidine, failed to activate mucin synthesis. No significant change in mucus synthesis occurred with the addition of analogues in which the piperidine ring attached to the benzene ring via a methylene bridge was changed. 4. Specific [125I]-APT binding to the histamine H2 receptor of guinea pig brain membranes was inhibited by roxatidine and all structural analogues used in this study, except F (N-(3-[m-(N, N-dimethyl-aminomethyl)phenoxy]-propyl)acetamide). 5. Ranitidine at 10(-4) M did not suppress the roxatidine-induced increase in [3H]glucosamine incorporation into mucin. 6. Roxatidine-induced stimulation of [3H]glucosamine incorporation into mucin was completely blocked by the addition of either NG-nitro-L-arginine (10(-5) M) or 2-(4-carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide sodium salt (10(-5) M). The inhibitory action of NG-nitro-L-arginine was totally reversed by L-arginine (5 x 10(-3) M). 7. These results suggest that the cardinal chemical features of roxatidine for the activation of mucin biosynthesis in the corpus region of the rat stomach are the appropriate length of the flexible chain between the amide structure and the aromatic ring system bearing the methylpiperidinyl group at the meta position. The activity of roxatidine and its analogues to stimulate mucin synthesis is not related to their histamine H2 receptor antagonistic activity. Roxatidine-induced activation of mucin biosynthesis in the corpus tissue is mediated by nitric oxide.

Biosynthesis of mucins (MUC2-6) along the longitudinal axis of the human gastrointestinal tract

Little is known about the biosynthesis of mucin molecules in humans. Our aim was to examine the mucin biosynthesis (MUC2-6) along the longitudinal axis of the healthy human gastrointestinal tract. Biopsies of human stomach and small and large intestine were metabolically labeled with 35S-labeled amino acids, [35S]sulfate, or[3H]galactose, immunoprecipitated with antibodies against MUC2-6, and analyzed by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), MUC5AC [apparent molecular weight (M(r)) 500,000] and MUC6 (apparent M(r) 400,000) were detected in the stomach but not in the small or large intestine, MUC3 (apparent M(r) 550,000) was detected in duodenum and jejunum, MUC2 (apparent M(r)600,000) was detected throughout the small and large intestine, and MUC4 (apparent M(r) > 900,000) was detected predominantly in the large intestine. Interestingly, some individuals displayed double bands of MUC2 and MUC3 precursors, suggesting allelic variation within the respective genes. Between small and large intestine mature secreted MUC2 showed differences in mobility on SDS-PAGE, suggesting differences in glycosylation. Each of the MUC2, MUC3, MUC4, MUC5AC, and MUC6 precursors could be distinguished electrophoretically, and each showed region-specific expression along the gastrointestinal tract.

O-Glycosylation and Cellular Differentiation in a Subpopulation of Mucin-Secreting HT-29 Cell Line

Malignant transformation of epithelial cells is associated with abnormal glycosylation of mucins. The aim of this work was to evaluate the changes in the O-glycosylation processes during differentiation of tumor cells by performingin vitroreactions using crude microsomal preparations obtained from a subpopulation of HT-29 cells capable of differentiating into mucin-secreting cells (HT-29 MTX cells). The reactions of O-glycosylation were carried out at different times of culture: before confluence (Day 5), when cells are still undifferentiated, and after confluence (Day 21), when cells display a mucin-secreting phenotype. As acceptor for the UDP-N-acetylgalactosamine:polypeptideNacetylgalactosaminyltransferase (GalNAc transferase), the peptide motif TTSAPTTS (tandem repeat deduced from MUC5AC human gastric gene, expressed in HT-29 MTX cells) was used. A higher rate of enzyme activity was observed in preconfluent cells, and analysis by capillary electrophoresis and electrospray mass spectrometry showed a different pattern of galactosaminylation in pre- and postconfluent cells. Core 1 UDP-galactose:N-acetyl-α-galactosaminyl-R 3-β-galactosyltransferase (3-β-galactosyltransferase) activityalso decreased with the differentiation, whereas CMP-neuraminic acid:galactose–β-1, 3-N-acetyl-α-galac- tosaminyl-R 3-α-sialyltransferase activity increased. In comparison, the evolving process of mucin biosynthesis was tested by the analysis of purified mucins of HT-29 MTX cells, in amino acid and carbohydrate composition, and immunoreactivity assays using several antibodies and lectins. The results suggested that (i) no mucins were detected at Day 5, while the GalNAc transferase and 3-β-galactosyltransferase activities were already at high rates; (ii) the mucins purified from postconfluent cells showed a high content of sialic acid in an α-2,3-linkage to galactose residues; and (iii) cellular differentiation seemed to be accompanied by more regulated processes of glycosylation. This study of the O-glycosylation in HT-29 MTX cells is thus an interesting approach to analyzing the regulation of mucin biosynthesis during cellular differentiation.

Effects of histamine on mucin biosynthesis in rat gastric mucosa.

Mucin biosynthesis is stimulated by gastrin during the process of glycosylation in the corpus mucosa of the rat stomach. The purpose of this study was to clarify, using an organ culture technique, whether biosynthetic responses to histamine in the rat gastric mucin are the same as that to gastrin. Radiolabeled mucin was obtained from the corpus and antral mucosa of the rat stomach after in vitro incubation for 5 h with [3H]glucosamine (GlcN), [14C]threonine (Thr), and [35S]sulfate. Addition of histamine (10(-7)-10(-5) M) to the culture medium increased [3H]GlcN-labeled mucin in the corpus tissue in a concentration-dependent manner. In the antrum, there was no significant change in the biosynthetic activity of mucin in response to histamine. Histamine at 10(-5) M also increased the incorporation of both [35S]sulfate and [14C]Thr into the corpus mucin. These results indicate that histamine stimulates the biosynthesis of the mucin peptide, as well as the glycosylation step in the corpus, and suggest that the effect of histamine on mucin synthesis is distinct from that of gastrin.

The fucosylation and secretion of mucins synthesized in human bronchial cells vary according to growth conditions.

In order to evaluate the importance of an extracellular matrix on mucin secretion, human bronchial epithelial cells were cultured for 3 weeks on either plastic or type I collagen. By metabolically labeling cells with tritiated glucosamine, we found that cells grown on collagen secreted more radiolabeled mucins than cells grown on plastic, and their amount increased from week 1 to week 3 in both conditions. Secreted mucins were also assayed by ELISA using an anti-Lewis b monoclonal antibody. The amount of immunoreactive mucins secreted by cells grown on plastic during the first 2 weeks of culture was much lower than that of cells grown on collagen. This indicated that the expression of the Lewis b antigen on mucins secreted by cells grown on plastic was very low during the first 2 weeks of radiolabeling and increased during the third week to reach levels that were comparable to the levels of expression by cells grown on collagen. The expression of the Lewis b epitope and the mRNA level of fucosyltransferases FUT2 and FUT3, which are involved in Lewis b synthesis, varied in a similar way. The two fucosyltransferases were expressed later in cells cultured on plastic than in cells grown on collagen. These results suggest that growth conditions influence both the biosynthesis and secretion of respiratory mucins.

Stimulation of mucin metabolism in rat gastric mucosa by histamine.

We examined the effects of histamine on mucin localized in the different regions and layers of rat gastric mucosa by determining the changes in the content as well as the biosynthetic activity of the mucin. In vivo administration of 0.8 mg/kg of histamine, which could not induce a concomitant gastric acid secretion, caused a significant increase in the mucin content in the corpus mucosa, but not in the antral mucosa. This increase was due to a significant accumulation of the mucin in the mucus gel and surface mucosa of the corpus region, whereas that in the deep mucosa did not significantly change. In the in vitro incubation system of rat gastric mucosa, histamine and dibutyryl cyclic AMP significantly increased [3H]-labeled mucin in the corpus. The histamine-induced acceleration of mucin biosynthesis was suppressed by ranitidine, but not pyrilamine. In the antrum, the biosynthetic activity showed no significant change by histamine. These results suggest that histamine promotes mucin metabolism via histamine H2 receptors in the surface mucosal layer of the corpus.

Biosynthesis of the MUC2 mucin: evidence for a slow assembly of fully glycosylated units

The human colonic cell line PC/AA was grown to near confluency over 24 days and labelled with [14C]proline and [3H]glucose over the last 48 h in culture. The cell layer was extracted with 6 M guanidinium chloride and the mature fully glycosylated mucins were isolated at a density of 1.45 g/ml by using density-gradient centrifugation in CsCl/4 M guanidinium chloride. These mucins were identified as MUC2 with an anti-peptide antibody. The macromolecules were fragmented by reduction into two distinct populations of MUC2 subunits as assessed by agarose electrophoresis. The MUC2 mucin was polydisperse in length, ranging from 500 nm to many microns and its molecular-mass distribution, assessed by rate-zonal centrifugation, ranged from 5 x 10(6) to 40 x 10(6) Da. However, the metabolically labelled MUC2 mucins, though found throughout the whole distribution, were of much smaller average size. Since the entire distribution is not uniformly radiolabelled over 48 h, the formation of the largest species must be preceded by glycosylation and occur slowly, over several days, via the assembly of fully glycosylated units which are likely to be at least dimers [Asker, Baeckstrom, Axelsson, Carlstedt, and Hansson (1995) Biochem. J. 308, 873-880].

Stimulation of mucin biosynthesis in rat gastric mucosa by FRG-8813 and its structural analogs

Certain chemical properties, which may determine the stimulatory actions of the new histamine H 2 receptor antagonist, FRG-8813 (2-(furfurylsulfinyl)- N-(4-[4-(piperidinomethyl)-2-pyridyl]oxy-( Z)-2-butenyl)acetamide), on mucin biosynthesis, were identified by considering the derivation of this drug using an organ culture system of the rat stomach. [ 3H]Glucosamine and [ 35S]sulfate incorporation was stimulated in the corpus region by FRG-8813 and its structural analog, compound A ( N-[4-[[4-(piperidinylmethyl)pyridyl]-2-oxy]-( Z)-2-butenyl]phthalimide). The chronotropic response to histamine in the guinea pig right atria was suppressed by FRG-8813 in a concentration-dependent fashion. In contrast, compound A did not suppress the histamine-induced response. Ranitidine at 10 −4 M did not suppress the FRG-8813-induced increase in [ 3H]glucosamine incorporation into mucin. These results suggest that the pyridine derivative and amide structure are chemically important in FRG-8813 as a stimulant on mucus metabolism. Also, this effect is not directly due to histamine H 2 receptor antagonism.

Early steps in the biosynthesis of MUC2 epithelial mucin in colon cancer cells.

Expression of the MUC2 mucin has been demonstrated in normal gastrointestinal and respiratory epithelium and in carcinomas of the gastrointestinal and respiratory tracts, breast, ovary, and bladder using RNA probes and (or) monoclonal antibodies reactive with peptide epitopes on the 23 amino acid tandem repeat. Mouse monoclonal antibodies 4F1 and 3A2 were previously obtained by immunization with mucin derived from the LS174T colon cancer cell line and a KLH conjugate of a synthetic MUC2 VNTR peptide. These antibodies react with distinct epitopes on synthetic VNTR peptides and with normal and malignant epithelial tissues. In the present study, we examined the biosynthesis of MUC2 in LS174T colon cancer cells, using these antibodies to immunoprecipitate labelled mucin. A very high molecular mass protein was immunoprecipitated following 1 min pulse labelling with [3H]threonine and [3H]proline. A slight increase in molecular mass was observed over the next 16 min; however, unlike the MUC1 mucin, there was no large difference in apparent molecular mass between the MUC2 protein precursor and fully processed mucin using separation by SDS-PAGE. O-Glycosylation began within 1 h of synthesis of the protein core. Mucin secretion into the culture medium was detected in the 2nd hour following synthesis and was largely completed within 4 h of synthesis. Secreted mucin was far less reactive with these monoclonal antibodies than the precursor protein.

A cell line model of mucin biosynthesis in the human intestine

A cell line model of mucin biosynthesis in the human intestine

Morphometric analysis of intestinal mucins under different dietary conditions and gut flora in rats

Elucidation of the mechanisms that alter the biosynthesis, turnover, and degradation of intestinal mucins is relevant to the understanding of both the normal gut ecosystem and various intestinal diseases. In this study image analysis was used to quantify the effects of diet and microbial flora on the mucin composition of goblet and deep crypt cells, the number and volume density of mucin-containing cells, and the staining density of their stored mucins in the small and large intestine of germ-free and conventionally maintained rats fed two different diets. One was a coarsely ground commercial rodent diet containing crude fiber of cereal origin and the other a purified diet composed of finely powdered ingredients, including cellulose as a source of fiber. The changes in mucin production were also analyzed in germ-free rats colonized with a human flora. Feeding a commercial diet reduced the volume density of cells containing neutral and sulfomucins in the jejunum of conventional rats and the staining density of neutral and acidic mucins in the germ-free rats. Both rat and human floras reduced the number of cells containing acidic and sulfomucins and the staining density of neutral mucins in the small intestine of animals fed on a purified diet. However, inoculation of human flora increased the staining density of stored neutral and sulfated mucins in the cells of the large intestine. The results demonstrate that the dietary changes are influential in modifying the amount and proportion of mucins in the small intestine and the microbial flora in the large intestine.

Mucin biosynthesis and macromolecular assembly.

Mucin biosynthesis and macromolecular assembly.

Biosynthesis of intestinal mucins: MUC1, MUC2, MUC3 and more.

Biosynthesis of intestinal mucins: MUC1, MUC2, MUC3 and more.

Biosynthesis and molecular architecture of gel-forming mucins: implications from an amphibian model system

Biosynthesis and molecular architecture of gel-forming mucins: implications from an amphibian model system

In defense of the oral cavity: structure, biosynthesis, and function of salivary mucins.

The oral environment, like other mucosal surfaces of the body, is coated by a slimy, viscoelastic coat termed mucus. This adherent layer, consisting predom­ inately of salivary glycoproteins, proteins, and lipids, forms ajelly-like blanket that represents the perimeter defense of the tissue/environmental interface (77). The unique physicochemical and rheological properties of the mucus coat are contributed largely by the heavily O-glycosylated mucin-glycoproteins (mu­ cins). Salivary mucins are synthesized by the mucus acinar cells of the paired submandibular (SMG) and sublingual (SLG) glands, as well as minor salivary glands distributed throughout the palatal and buccal mucosa. Serous acinar cells, such as those found in the human or rat parotid glands, do not contribute to the production of salivary mucins. Much of our current understanding of salivary mucin function has been derived by inference. Various subjective and objective functional losses have been catalogued in persons who lack the ability to produce adequate levels of saliva (4). Relevant findings include (a) the sensation of oral dryness, partic­ ularly among those subjects with compromised mucus gland function (26); (b) difficulty with swallowing; and (c) an increased susceptibility to dental cavity (caries) formation and the appearance of opportunistic yeast infections (thrush mouth). These findings have led to a generalized view that saliva functions to (a) hydrate and lubricate the oral structures; (b) facilitate the oral phase of

Biosynthesis of human small intenstinal mucins

Biosynthesis of human small intenstinal mucins

Preparation of Anti-mucin Polypeptide Antisera to Study Mucin Biosynthesis

Mucins are very heavily O-glycosylated glycoproteins. For in depth studies on the cell biological aspects of mucins, anti-polypeptide antibodies are essential. We therefore developed a method for the preparation and screening of polyclonal antisera against mucin peptide epitopes. Mucins from five different tissues were isolated using CsCl/guanidinium.HCl density gradient centrifugation, and polyclonal antisera were prepared. Specificity for mucin peptide epitopes was determined by Western blotting, immunohistochemistry, and immunoprecipitation. The versatility of each anti-mucin antiserum for the study of mucin biosynthesis was tested in metabolic labeling experiments on tissue explants. All polyclonal antisera were directed primarily against peptide epitopes of mucin precursors as well as of fully glycosylated mucins. Each of the polyclonal antisera enabled us to study the mucin biosynthesis in the organ where the mucin was isolated from originally. Our mucin isolation method yields very pure mucins with sufficiently intact polypeptides to reproducibly elicit polyclonal anti-polypeptide antisera. As the sera recognized the polypeptides, primarily independent of the state of O-glycosylation, the intermediate steps in the biosynthesis of the mucins could be identified.

Biosynthesis of human small intestinal mucins

Biosynthesis of human small intestinal mucins