Two types of rat gastric mucus glycoprotein subunits

Gastric mucus glycoproteins were extracted with 2% Triton X-100 from rat gastric corpus and antrum and purified by CsCl equilibrium centrifugation. Corpus mucus glycoproteins were degraded into what appeared to be two "subunits" (Mw 4.4 x 10(5) and 6 x 10(6)) by the reduction of disulfide bonds. Papain digestion of the latter produced glycopeptides with a molecular weight of approximately 4.4 x 10(5). This type of subunit had carbohydrate chains with about 9 sugars attached to every 2 amino acid residues. Papain digestion of the former type of subunit revealed no change in the elution profile on Bio-Gel A-15m. This type of subunit had carbohydrate chains with 17-19 sugars attached to every 3 amino acid residues. The subunit of antral mucus glycoproteins was essentially the same as the former type of corpus subunits in molecular weight (Mw 4.4 x 10(5)) and average oligosaccharide chain length. These results suggest that there are two distinct types of mucus glycoprotein subunits in rat stomach.     

Biosynthesis, processing and secretion of mucus glycoprotein in the rat stomach

For the study of the biosynthesis, processing and secretion of mucus glycoproteins in rat gastric mucous cells, antibodies were raised against purified gastric mucus glycoproteins and against deglycosylated gastric mucus glycoproteins. Indirect immunofluorescence analysis of gastric mucosa sections revealed that both antibodies specifically labelled the mucus glycoprotein-synthesizing cells in the gastric mucosa. Stomach segments were pulse-labelled with [35S]cysteine and chased for various times. The radioactively labelled (glyco)proteins were quantitatively immunoprecipitated and analyzed by SDS-polyacrylamide gel electrophoresis. Less than 3% of the total radioactivity incorporated in protein was found to be present in mucus glycoproteins. Antibodies raised against native mucus glycoproteins recognized only high-molecular-weight mucus glycoproteins, while the antibodies against deglycosylated glycoproteins also bound to probable precursor forms. The synthesis of mature mucus glycoproteins (Mr greater than 300 000) required about 90 min. After 3 h of chase, only a small portion of the pulse-labelled mucus glycoproteins had been secreted; the majority of the radioactive glycoproteins at that time was still associated with the tissue. Immature (glyco)proteins were not secreted into the medium.     

Functional interactions of gastric mucus glycoprotein

The concentration-dependence of viscosity in solutions of purified glycoprotein from pig gastric mucus is of the form expected for simple polymer entanglement. At higher concentrations, however, a weak viscoelastic gel is formed, whose mechanical spectrum (over the frequency range 10⁻²---10² rad s⁻¹) indicates a more stable mechanism of interchain association, and is closely similar to that of native mucus. On prolonged exposure to solvent, reconstituted gels redissolve, while native mucus retains its structural integrity (as characterized by the storage modulus, G′) but releases a significant, variable amount of glycoprotein. On proteolytic digestion or disulphide reduction of the glycoprotein to its component subunits, network structure is lost, but the mechanical spectra of the resulting solutions show interactions beyond simple entanglement. From this evidence we suggest that in the sub-micrometre-sized ‘domains’ in which native mucus is secreted, the carbohydrate side chains of component glycoprotein molecules are interdigitated in a comparatively stable arrangement, with the polymeric subunit structure of the glycoprotein conferring the branching required for development of a three-dimensional network, and with a substantial, variable sol-fraction of free glycoprotein within the interstices of the gel. On solubilization of native mucus, the ‘domain’ structure is destroyed irreversibly. Interaction between domains, and between individual molecules in gels reconstituted from the component glycoprotein after extraction and purification, is by more transient, non-specific interdigitation and entanglement, to confer the overall flow and spreading characteristics of the gel.     

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.     

Identification of gastric mucosal mucus glycoprotein sulfotransferase.

Sulfation of mucus glycoproteins, reaction catalyzed by Golgi resident sulfotransferase, is an important event in posttranslational processing of gastric mucins. Here we report the purification of mucus glycoprotein sulfotransferase enzyme from the microsomal fraction of rat gastric mucosa. The enzyme was released from the membrane with 0.5% Triton X-100 and precipitated from the 100,000xg supernatant with 90% ice-cold acetone. The enzyme activity (44.7 pmol/mg/45 min) in the precipitate was enriched nearly 10-fold compared to Triton X-100 extract of microsomal membrane (4.2 pmol/mg/45 min). On SDS-PAGE, the enzyme gave a single 43 kDa protein band, which was active towards mucin, but did not catalyze the sulfation of galactosylceramide. The study is the first to report the characteristics of a sulfotransferase enzyme specific for gastric mucin.     

Gastric mucus glycoprotein in different rat strains

The extent of gastric damage induced by aspirin was found to differ according to rat strain. The occurrence of ulcers varied, from high to low, in the following strain order: Donryu, Sprague-Dawley (SD) and Wistar. The content of corpus mucus glycoprotein was essentially the same in all the strains: about 6 mg as hexose of dry tissue. Antral mucus glycoprotein content increased in the order Wistar, SD and Donryu: 7.1, 8.3 and 9.1 mg, respectively. Gastric mucus glycoprotein carbohydrate composition was essentially the same in all three strains. The relatively low proportions of N-acetylglucosamine, galactose and sialic acid from the antrum was a characteristic feature in contrast to mucus glycoprotein from the corpus which contained a high proportion of these sugars.     

Characterization of mucus glycoprotein fatty acyltransferase from gastric mucosa

A fatty acyltransferase activity which catalyzes the transfer of palmitic acid from palmitoyl coenzyme A to gastric mucus glycoprotein has been demonstrated in the rat gastric mucosa. Subcellular fractionation studies revealed that the enzyme activity was present in a Golgi-rich membrane fraction. Optimum enzymatic activity for acylation of mucus glycoprotein was obtained with 0.5% Triton X-100, 25 mM NaF, and 2 mM dithiothreitol at a pH of 7.4. The enzymatic activity increased proportionally, over a given range, with increased concentrations of both substrates and of enzyme. The apparent Km of the enzymes for the undegraded mucus glycoprotein was 4.5 X 10(-7) M and for palmitoyl-CoA, 3.8 X 10(-5) M. The 14C-labeled product of the reaction cochromatographed on Bio-Gel A-50 column and migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with gastric mucus glycoprotein. Treatment of this 14C-labeled glycoprotein with mild alkali released hexane-extractable product which was identified as [14C]palmitate. The enzyme was also capable of fatty acylation of the deglycosylated glycoprotein, but did not catalyze the transfer of palmitic acid to the proteolytically degraded mucus glycoprotein. This indicates that the acceptor site for fatty acyltransferase is situated in the protease-susceptible nonglycosylated region of the mucus glycoprotein polymer.     

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.     

Isolation and structural analysis of rat gastric mucus glycoprotein suggests a homogeneous protein backbone.

We isolated monomeric gastric mucus glycoprotein from the rat stomach by applying three successive CsCl-density-gradient steps in the continuous presence of guanidinium chloride. The rat gastric mucin was pure as compared with mucin isolated without the chaotropic reagent. In addition, the presence of guanidinium chloride resulted in a better preservation of the protein moiety. The purified mucin was fractionated according to buoyant density and chemically radiolabelled on tyrosine or cysteine residues and digested with specific proteinases. Analysis of mucin fractions of various densities gave identical peptide patterns, suggesting that the fractions contain a common protein backbone. Electron-microscopic images of the individual mucin molecules were recorded using rotary shadowing. They showed large filamentous molecules with a mean length of 208 nm that, after proteolytic digestion, yielded glycopeptides with a mean length of 149 nm. Heterogeneity in buoyant density and electrophoretic mobility is located in this large glycopeptide which remains after proteolytic digestion. Metabolic labelling of the mucin with [35 S]sulphate and [3H]galactose, followed by purification and proteolytic digestion, revealed that this glycopeptide accounts for most of the mass and contains relatively little protein, but probably all the oligosaccharides and sulphate. As this protein part is masked by the oligosaccharides, detailed study by the methods described was not possible. The results indicate that rat gastric mucin is homogeneous in a major part of the protein backbone and that the heterogeneity of the molecule originates most likely from differences in sulphate and/or sugar composition.     

Modulation of gastric mucosal calcium channel activity by mucus glycoprotein

• 1.1. The effect of gastric mucus glycoprotein on the activity of calcium channel isolated from gastric epithelial cell membrane was investigated. The ⁴⁵Ca²⁺ uptake into the vesicle-reconstituted channels, while only moderately (14%) affected by the intact mucus glycoprotein, was found significantly inhibited (59%) by the acidic glycoprotein fraction. This effect was associated with the sialic acid and sulfate ester groups of the glycoprotein, as their removal caused a loss in the inhibition.
• 2.2. The channel complex in the presence of epidermal growth factor (EGF) and ATP responded by an increase in protein tyrosine phosphorylation of 55 and 170 kDa proteins, and the vesicles containing the phosphorylated channels showed a 50% increase in ⁴⁵Ca²⁺ uptake. The phosphorylation and the calcium uptake were susceptible to inhibition by a specific tyrosine kinase inhibitor, genistein.
• 3.3. The channel protein phosphorylation was inhibited by the acidic mucus glycoprotein, which also interfered with the binding of EGF to the channel protein. The inhibitory effect was dependent upon the presence of sulfate ester and sialic acid groups, as evidenced by the loss of the glycoprotein inhibitory capacity following their removal.
• 4.4. The results suggest that the acidic gastric mucus glycoproteins, by modulating the EGF-controlled calcium channel phosphorylation, play a major role in gastric mucosal calcium homeostasis.

     

Peptic erosion of gastric mucus in the rat

1. The effect of pepsin on the loss of mucus glycoprotein from the gastric epithelial mucus layer was studied in the rat. 2. Pepsin was instilled into the gastric lumen, and luminal contents were subsequently assayed. 3. Glycoprotein loss increased with luminal pepsin, up to a concentration of 1 mg pepsin/ml. 4. Luminal glycoprotein had a molecular size distribution intermediate between subunit, and native mucus glycoprotein of the epithelial mucus layer. 5. Incubation of gastric epithelial scrapings with pepsin demonstrated that insoluble, native mucus glycoprotein was rapidly degraded to soluble glycoprotein of similar molecular size distribution to that found in vivo in the lumen.     

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.     

Effect of ethanol on mucus glycoprotein fatty acyltransferase from gastric mucosa

The enzyme activity that catalyzes the transfer of palmitic acid from palmitoyl coenzyme A to the deacylated intact or deglycosylated gastric mucus glycoprotein was demonstrated in the detergent extracts of the microsomal fraction of antral and body mucosa of the rat stomach. Both types of mucosa exhibited similar acyltransferase activities and acceptor specificities. A 10-14% decrease in the fatty acyltransferase activity was observed with the reduced and S-carboxymethylated mucus glycoprotein, but the proteolytically degraded glycoprotein showed no acceptor capacity. This indicated that the acylation of mucus glycoprotein with fatty acids occurs at its nonglycosylated polypeptide regions and that some of the fatty acids may be linked via thiol esters. Optimum enzyme activity was obtained at pH 7.4 with the detergent Triton X-100, NaF, and dithiothreitol. The apparent Km values for the intact and deglycosylated mucus glycoproteins were 0.45 and 0.89 microM, respectively. The acyltransferase activity of the microsomal enzyme was inhibited by ethanol. With both intact and deglycosylated glycoprotein substrates, the rate of inhibition was proportional to the ethanol concentration up to 0.4 M and was of the competitive type. The K1 values were 0.80 microM for the intact mucus glycoprotein and 1.82 microM for the deglycosylated glycoprotein. Preincubation of the microsomal enzyme with low concentrations of ethanol (up to 0.5 M) did not seem to exert any additional deterrent effect on acyltransferase activity. Higher concentrations of ethanol (1.0 M and above), however, caused substantial reduction in the transferase activity due to denaturation of the enzyme.     

The action of proteolytic enzymes on the glycoprotein from pig gastric mucus.

A glycoprotein of mol.wt. 2x10(6) was isolated in homogeneous form from pig gastric mucus by isopycnic centrifugation in CsCl but without enzymic digestion or reductive cleavage of disulphide bonds. Digestion of the purified glycoprotein with trypsin, pepsin or Pronase resulted in the formation of glycoprotein subunits, of mol.wt. 5.2x10(5)-5.8x10(5), one-quarter that of the undigested glycoprotein. The glycoprotein subunits were isolated by gel filtration and shown to contain all the carbohydrate present in the undigested glycoprotein, but 18.6-25.6% of the total amino acids originally present were lost on digestion. The relative amount of threonine, serine and proline had increased from 41% (w/w) in the undigested glycoprotein to 61-67% of the total amino acids in the glycoprotein subunits after digestion. The results support the previously proposed structure for the glycoprotein, namely that of four subunits joined by disulphide bridges. These results show the presence of two distinct regions in the glycoprotein molecule, one rich in threonine, serine and proline, which is glycosylated and resistant to proteolyis, whereas the other, with an amino acid composition more characteristic of a globular protein, is not glycosylated and is susceptible to proteolysis. In addition, the region that is susceptible to proteolysis contains the disulphide bridges which join the glycoprotein subunits together to form the gastric glycoprotein.     

Enzymatic sulfation of mucus glycoprotein in rat submandibular salivary gland

1. The enzymic activity which catalyzes transfer of sulfate ester group from 3'-phosphoadenosine-5'-phosphosulfate to mucus glycoprotein was found associated with Golgi-rich membrane fraction of rat submandibular salivary gland. 2. Optimum enzyme activity was obtained with 0.5% Triton X-100, 4 mM MgCl2 and 25 mM NaF at a pH of 6.8 using desulfated submandibular salivary mucus glycoprotein. The apparent Km of the enzyme for mucus glycoprotein was 11.1 mg/ml. 3. Alkaline borohydride reductive cleavage of the synthesized 35S-labeled glycoprotein led to the liberation of the label into reduced oligosaccharides. A 75.4% of the label was found incorporated in four oligosaccharides. These were identified in order of abundance as sulfated penta-, tri-, hepta- and nonsaccharides. 4. Based on the results of chemical and enzymatic analyses of the intact and desulfated compounds the pentasaccharide was characterized as SO3H----GlcNAc beta----Gal beta----GlcNAc(NeuAc alpha----)GalNAc-ol and the trisaccharide as SO3H----GlcNAc beta----Gal beta----GalNAc-ol.     

Effects of fasting on mucus glycoprotein biosynthesis in rat stomach

Biosynthetic activity of gastrin mucus glycoprotein in rats after fasting for 24 and 72 hr was studied by the organ culture technique. Fasting produced a slight reduction in gastric mucus glycoprotein biosynthesis in the corpus and antrum (about 70-90% of fed rats). Sulfation of gastric mucus glycoprotein was restrained in the corpus (18% in control for 72 hr).     

Enzymatic sulfation of mucus glycoprotein in gastric mucosa. Effect of ethanol

A sulfotransferase activity that catalyzes the transfer of sulfate ester group from 3'-phosphoadenosine 5'-phosphosulfate to carbohydrate chains of gastric mucus glycoprotein has been demonstrated in the antral and body mucosa of rat stomach. Subcellular fractionation studies revealed that the enzyme is associated with Golgi-rich membrane fraction. The sulfotransferase activity of this fraction in antral mucosa was about 35% lower than that in the body. Optimum enzyme activity was obtained with 0.5% Triton X-100 and 30 mM NaF at a pH of 6.8 using desulfated mucus glycoprotein substrate. The enzyme was equally capable of sulfation of the proteolytically degraded and reduced forms of the desulfated glycoprotein, but the acceptor capacity of the intact mucus glycoprotein was about 60% lower than that of the desulfated preparation. The enzyme preparation also catalyzed the transfer of sulfate to galactosylceramide. The sulfation of mucus glycoprotein, however, was not affected by the presence of this glycolipid, suggesting that the sulfotransferase involved in mucus glycoprotein sulfation is different from that responsible for the synthesis of sulfatoglycosphingolipid. The mucus glycoprotein sulfotransferase activity was inhibited by ethanol. The rate of inhibition was proportional to the concentration of ethanol up to 0.3 M and was of the competitive type. The apparent Km value of the enzyme for mucus glycoprotein was 10.5 X 10(-6) M (21 mg/ml), and the KI in the presence of ethanol was 4.7 x 10(-1) M. The 35S-labeled mucus 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 led to the liberation of the label into reduced acidic oligo-saccharide fraction. Most of the label was found incorporated in three oligosaccharides. These were identified as tri-, tetra-, and pentasaccharides, each carrying a labeled sulfate ester group on the terminal N-acetyl-glucosamine residue. Based on the results of structural analyses, the most abundant oligosaccharide was characterized as SO3H----6GlcNAc beta 1----3Gal beta 1----3GalNAc-ol.     

Effects of aspirin on gastric mucus glycoprotein in fed and fasted rats

Gastric lesions induced by aspirin increased the ulcer index and incidence with prolongation of fasting time. Aspirin decreased gastric mucus glycoprotein in both the corpus and antrum. However, the rate of decrease in mucus glycoprotein induced by aspirin differed according to feeding habits and the gastric region. Qualitative change in corpus mucus glycoprotein was induced by aspirin.     

Cotranslational attachment of fatty acids to nascent peptides in gastric mucus glycoprotein

Using gastric mucous cells which are involved exclusively in the synthesis of secretory O-glycosidic glycoprotein (mucin), the relationship between protein core synthesis and its acylation with fatty acids was investigated. Labeling of the cells with [3H]palmitic acid and [35S]methionine followed by isolation of peptidyl-tRNA and release of nascent peptides, indicated that these peptides contain covalently bound fatty acids. The high performance thin layer chromatography, SDS-gel electrophoresis, and radioactivity scanning revealed that the preparation contained three fractions labeled with palmitate (Mr 15,000-3,600) and two (Mr 1,500 and less) without this label. Based on these data and the nascent peptides amino acid analysis, we conclude that the protein core of the O-glycosidic glycoprotein is acylated with fatty acids during translation, when the peptide chain is longer than 21 amino acid residues.