A 70000-molecular-weight protein isolated from purified pig gastric mucus glycoprotein by reduction of disulphide bridges and its implication in the polymeric structure.

The glycoprotein of pig gastric mucus has been isolated free of non-covalently bound protein as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and equilibrium density-gradient centrifugation. After reduction with 0.2 M-mercaptoethanol, protein was released from the glycoprotein, which consisted of a major 70000-mol.wt. component and a minor 60000-mol.wt. component. The 70000-mol.wt. protein fraction was separated from the reduced glycoprotein by either density-gradient centrifugation in CsCl or by gel filtration. Analysis of the 70000-mol.wt. protein fraction showed that, within the limits of the analysis, it was non-glycosylated, and its amino acid analysis was quite different from that of the reduced glycoprotein, which is high in serine, threonine and proline. There was a ratio of one 70000-mol.wt. protein per native glycoprotein molecule of 2 X 10(6) mol.wt. Dissociation of the native glycoprotein into glycoprotein subunits (5 X 10(5) mol.wt.) by reduction or proteolysis results in the release or hydrolysis respectively of the 70000-mol.wt. protein. A similar 70000-mol.wt. protein is demonstrated in human gastric mucus glycoprotein. A structural role for the proteins in these mucus glycoproteins is proposed.     

Polymeric structure of pig small-intestinal mucus glycoprotein. Dissociation by proteolysis or by reduction of disulphide bridges.

Pig small-intestinal mucus glycoprotein, of molecular weight 1.72 X 10(6), is cleaved by Pronase digestion into glycoprotein subunits of molecular weight 4.5 X 10(5). Of the protein component of the native glycoprotein 29% by weight was lost on Pronase digestion, with no loss of carbohydrate. The non-glycosylated region of the protein that was lost with proteolytic digestion had a broad spectrum of amino acid residues, in contrast with the glycosylated region of the protein, which was resistant to proteolysis and was rich in serine, threonine and proline residues. Reduction with 0.2M-mercaptoethanol dissociated the Pronase-digested glycoprotein subunits into smaller glycoprotein subunits of molecular weight 2.7 X 10(5). On reduction, the native glycoprotein was dissociated into subunits of molecular weight 2.4 X 10(5), a similar size to those obtained from reduction of the Pronase-digested glycoprotein. On reductive dissociation of the native glycoprotein, in addition to glycoprotein subunits, protein was also released principally as a component of 90000 molecular weight. This protein was separated quantitatively from the reduced glycoprotein in amounts compatible with one 90000-mol.wt. protein molecule per 1.72 X 10(6)-mol.wt. native glycoprotein molecule. No 90000-mol.wt. protein was released on reduction of the isolated Pronase-digested glycoprotein. Pig small-intestinal mucus glycoprotein is therefore a covalent polymer of glycoprotein subunits joined by disulphide bridges. This polymeric structure differs in important respects from that previously shown for gastric mucus, in particular with respect to the size and number of component subunits per native molecule.     

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.     

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.     

The isolation and characterization of the high-molecular-weight glycoprotein from pig colonic mucus.

1. A high-molecular-weight glycoprotein constitutes over 80% by weight of the total glycoprotein from water-soluble pig colonic mucus. 2. It was isolated from from nucleic acid and non-covalently bound protein by nuclease digestion followed by equilibrium centrifugation in a CsCl gradient. 3. The glycoprotein has the following composition by weight: fucose 10.4%; glucosamine 23.9%; galactosamine 8.3%; sialic acid 9.9%; galactose 20.8%; sulphate 3.0%; protein 13.3%; moisture about 10%. 4. The native glycoprotein has the high mol.wt. of 15 X 10(6). 5. Reduction of the native glycoprotein with 2-mercaptoethanol results in a glycoprotein of mol.wt. 6 X 10(6). 6. Pronase digestion removes 29% of the protein (3% of the glycoprotein) but none of the carbohydrate. 7. The molecular weight of the Pronase-digested glycoprotein is 1.5 X 10(6), which is halved to 0.76 X 10(6) on reduction with 2-mercaptoethanol. 8. The contribution of non-covalent interactions, disulphide bridges and the non-glycosylated peptide core to the quaternary structure of the glycoprotein are discussed and compared with the known structure of pig gastric glycoportein.     

Release of oligosaccharides possessing reducing-end N-acetylgalactosamine from mucus glycoprotein in Streptomyces sp. OH-11242 culture medium through action of endo-type glycosidase

A crude enzyme preparation from a culture medium of Streptomyces sp. OH-11242 contained endo-alpha-N-acetylgalactosaminidase activity. The activity could be induced by the addition of purified porcine gastric mucin to the culture medium. Oligosaccharides corresponding to approximately 2-14 glucose units were detected in the culture medium and also in an incubated reaction mixture of crude enzyme preparation and mucus glycoprotein. The resulting product with N-acetylgalactosamine at the reducing terminal implied the presence of a new type of endo-glycosidase liberating not only Gal beta 1-3GalNAc but also other larger oligosaccharides by hydrolysis of the O-glycosidic linkage between GalNAc and Ser (Thr).     

Preparation and characterization of armadillo submandibular glycoproteins.

The nine-banded armadillo (Dasypus novemcinctus mexicanus Peters) was chosen for this study so that a comparison could be made of the salivary mucus glycoproteins of an ancient mammalian species with those derived from previously studied, more highly evolved species. Two mucus glycoproteins, armadillo submandibular glycoprotein A and armadillo submandibular glycoprotein B, were prepared from the armadillo submandibular gland by a modification of the method of Tettamanti & Pigman (1968) (Arch. Biochem. Biophys. 124, 41-50). The composition of glycoprotein A is the simplest one among the known mucus glycoproteins. Six amino acids constitute 98.5 mol/100mol of the protein of glycoprotein A and 82 mol/100 mol of that of glycoprotein B. These are serine and threonine (which make up 40-50% of the molar amino acid composition), glutamic acid, glycine alanine and valine. Proline is absent from glycoprotein A and comprises only 2.3% of glycoprotein B. For both glycoproteins, the protein content, as determined by the method of Lowry, Rosebrough, Farr & Randall (1951) (J. Biol. Chem 193, 265-275), with bovine serum albumin as standard, was nearly 60% higher than when determined by the sum of the amino acids. The ratios of total mol of amino acid/total mol of carbohydrate are 1:0.63 for glycoprotein A and 1:0.68 for glycoprotein B, N-Acetylneuraminic acid and N-acetylgalactosamine, in a molar ratio of about 0.35:1.00, are the principal carbohydrates present in both glycoproteins. Neutral sugars seem to be absent from glycoprotein A, but galactose and fucose are present in glycoprotein B. The carbohydrate side chains in glycoprotein A are composed of about two-thirds monosaccharide and one-third disaccharide residues, whereas those of glycoprotein B are more complex. For both glycoproteins, essentially all of the N-acetylgalactosamine was attached O-glycosidically to the hydroxyamino acid residues of the protein core. The linkage of N-acetylneuraminic acid glycoprotein A was extremely sensitive to dilute acid and neuraminidase. Glycoprotein B has chemical properties similar to those of glycoprotein A. However, whereas glycoprotein A was susceptible to both Clostridium perfringens and Vibrio cholerae neuraminidases, only the latter enzyme had an effect on glycoprotein B at pH 4.75. Both glycoproteins were homogeneous by cellulose acetate electrophoresis and ultracentrifugal analyses. The apparent mol.wts. of glycoprotein A and glycoprotein B were 7.8 X 10(4) and 3.1 X 10(4) respectively.     

Control of mucin molecular forms expression by salivary protease: Differences with caries

• 1.1. A protease activity capable of degradation of the high mol. wt salivary mucus glycoprotein to a low mol. wt glycoprotein form was identified in human submandibular gland secretion.
• 2.2. The protease exhibited optimum activity at pH 7.0–7.4, and gave on SDS-PAGE under reducing conditions two major protein bands of 48 and 53 kDa. The enzyme showed susceptibility to PMSF, α₁antitrypsin, and egg white and soybean inhibitors, a characteristic typical to serine proteases.
• 3.3. The activity of the protease towards the high mol. wt mucus glycoprotein was found to be 3.8-fold higher in submandibular gland secretion of caries-resistant individuals than that of caries-susceptible. Furthermore, the enzyme from both groups displayed greater activity against the mucus glycoprotein of caries-resistant subjects.
• 4.4. Since the low mol. wt salivary mucus glycoprotein form is more efficient in bacterial clearance than the high mol. wt mucin, the enhanced expression of this indigenous salivary protease activity towards mucin may be the determining factor in the resistance to caries.

     

A neutral collagenase from human gastric mucosa.

Biopsy specimens of human gastric mucosa, maintained in culture for 7 days in the absence of serum, released a collagen-degrading enzyme into the medium. The yield of active enzyme reached a maximum after 2-3 days, and viable tissue, capable of protein synthesis, was essential for its production. 2. At 25 degrees C the enzyme attacked undenatured collagen in solution, resulting in a 55% loss of specific viscosity and producing the two products TCA and TCB characteristic of neutral-collagenase action. 3. Electron microscopy of segment-long-spacing crystallites of these reaction products showed the exact cleavage locus of the collagen molecules to be between bands 43 and 44 (I-43). The larger TCA and smaller TCB products were fragments representing 77 and 23% respectively of the length of the collagen molecule. 4. Optimal enzyme activity was observed over the pH range 7.5-8.5 and a mol.wt. of approx. 38000 was derived from gel-filtration studies. 5. The enzyme was shown to be inhibited by the human serum proteins alpha2-macroglobulin and a smaller component of mol.wt. approx. 40000; alpha1-anti-trypsin was not inhibitory. 6. EDTA, 1, 10-phenanthroline, cysteine and dithiothreitol all inhibited collagenase activity. 7. The gastric enzyme has properties similar to other well characterized collagenases, but differences exist with respect to its molecular size and the site of attack on the collagen molecule.     

Subunit Structure of Castor Bean Hemagglutinin

Two constituent polypeptide chains of castor bean hemagglutinin (CBH-A) were isolated from the performic acid-oxidized or reduced-carboxymethylated CBH-A by chromatography on DEAE-cellulose or Sepharose 4B. From the analyses of the N-terminal amino acids, the amino acid compositions and the tryptic peptides of each chain, it was found that the larger chain with mol. wt. 34,000 and the smaller chain with mol. wt. 31,000 were homologous with the Ala and He chains of ricin D, respectively, and the subunit structure of CBH-A is represented as (α′/β′)₂ in relation to αβ of ricin D.     

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.     

Purification and characterization of alpha-L-fucosidase from Bacillus circulans grown on porcine gastric mucin.

Bacillus circulans isolated from soil was found to produce two types of alpha-L-fucosidase differing in substrate specificity. One was able to liberate L-fucose from porcine gastric mucin (PGM), but not from artificial substrates, including p-nitrophenyl and methyl alpha-L-fucosides, while the other acted not on PGM but on p-nitrophenyl alpha-L-fucoside. The production of the former enzyme was enhanced about 150 times as much by PGM added to the medium as by glucose. The alpha-L-fucosidase acting on PGM was purified from the culture fluid obtained with PGM medium by ammonium sulfate fractionation and subsequent column chromatography. The purified enzyme was found to be homogeneous by PAGE and its molecular weight was estimated to be approximately 285,000. The optimum pH was found to be 5.5 to 6.5 and the stable pH range was 4.5 to 9.0. The enzyme decomposed various blood group O(H) active substances such as PGM, human milk and human saliva, and moreover acted on A-, B-, and O-erythrocytes. The enzyme was shown to cleave alpha-(1----2)-, (1----3)-, and (1----4)-L-fucosidic linkages in various glycoproteins and oligosaccharides, but failed to hydrolyze alpha-(1----6)-L-fucosic linkages in 6-O-alpha-L-fucopyranosyl-N-acetylglucosamine and intact bovine thyroglobulin.     

Structural studies on dissociated porcine gastric mucus gel

Reduction of the insoluble porcine mucus gel with dithiothreitol, followed by molecular sieve chromatography, resulted in the recovery of the two fragments of molecular size 2.54 X 10(6) and 1.75 X 10(6). Both gastric mucus glycoproteins have been cleaved by pronase, papain or trypsin into fragments whose molecular sizes range from 5.60 X 10(5) to 1.87 X 10(6) daltons. On reduction, the enzyme-treated fragments were further dissociated into smaller fragments which have been characterized by SDS-PAGE. The evidence indicates that the pol: neric structure of gastric mucus differs from that of mucus of varying origins, in particular with respect to the size and number of component subunits per native molecule.     

The glycoprotein of measles virus. External radioactive labelling of its carbohydrate and partial characterization of the glycopeptide.

Measles virus was propagated in VERO cells and purified from the culture supernatants by two successive tartrate-density-gradient centrifugations. Surface carbohydrates were labelled both in vitro and in vivo with 3H after treatment with galactose oxidase/NaB3H4 or with [3H]glucosamine. The major labelled glycoprotein in measles virions had a mol.wt. of 79 000. After labelling with periodate/NaB3H4, which would result in specific labelling of sialic acid residues, the 79 000-mol.wt. glycoprotein was very weakly labelled. This suggests that there is no or a very low amount of sialic acid in the virions. Further analysis of the glycoprotein showed that galactose is the terminal carbohydrate unit in the oligosaccharide, and the molecular weight of the glycopeptide obtained after Pronase digestion is about 3000. The oligosaccharide is attached to the polypeptide through an alkali-stable bond, indicating a N-glycosidic asparagine linkage.     

Characterization of a high-molecular-weight glycoprotein isolated from the pulmonary secretions of patients with alveolar proteinosis

A high-molecular-weight glycoprotein was isolated, purified and partially characterized from the insoluble pulmonary secretions accumulating in lungs of patients suffering from pulmonary alveolar proteinosis. On electrophoresis in 5% polyacrylamide gel in the presence of sodium dodecyl sulphate and 2-mercaptoethanol, the purified protein gave one major band as detected by Coomassie Blue as well as with periodic acid/Schiff staining. An apparent mol.wt. of 250000 was estimated for this glycoprotein. Amino acid analysis showed that it contains hydroxyproline, and relatively high amounts of glycine, glutamic acid, aspartic acid and leucine. It contains approx. 6% hexose, 3% sialic acid and 2% glucosamine. The neutral sugars are galactose, mannose and fucose. An antiserum prepared in rabbits against this high-molecular-weight glycoprotein cross-reacted with two smaller glycoproteins (mol.wts. 62000 and 36000) isolated from the same pulmonary secretions of these patients. A complementary observation was also made when this large alveolar glycoprotein cross-reacted with an antiserum prepared in rabbits against the smaller glycoprotein (mol.wt. 36000). It appears that this high-molecular-weight glycoprotein may be the precursor of the two smaller glycoproteins present in the same diseased pulmonary secretions.     

Isolation of transferrin from porcine gastric mucosa: comparison with porcine serum transferrin

1. An iron-binding glycoprotein has been purified to homogeneity from porcine gastric mucosa. 2. The molecular weight (80,000), amino acid composition, carbohydrate content, N-terminal amino acid sequence, tryptic map, stoichiometry of iron binding (2 mol/mol), visible absorption spectrum of the ferric complex and chromatographic behaviour of the gastric protein are all strikingly similar to the corresponding properties of porcine serum transferrin. 3. The quantity of the gastric protein (1.3 mg/g wet weight) present in the gastric mucosa suggests that it is not serum transferrin (plasma concentration 1.8 mg/ml) contaminating the tissue. 4. A role for transferrin in the uptake of dietary iron by the gastrointestinal tract is proposed.     

Purification and properties of porcine platelet-derived growth factor.

The purification to homogeneity of a potent growth factor from porcine platelets is described. This cationic mitogen is named porcine platelet-derived growth factor (PDGF) on the basis of close structural, functional and immunological similarities to human PDGF. Porcine PDGF, like its human homologue, is a hydrophobic, disulphide cross-linked protein, which is stable to heat, acid, sodium dodecyl sulphate (SDS), and guanidine. The purified protein has an apparent mol. wt. on SDS-polyacrylamide gels of 38 000, similar to those reported for human PDGF (27 500-35 000). Amino terminal sequence analysis of native porcine PDGF gave a single 15 amino acid residue sequence, of which 11 residues were identical to the amino terminal sequence of the B chain of human PDGF. Gel permeation h.p.l.c. in guanidine solutions of the reduced protein revealed a single species of mol. wt. 17 000 suggesting that native porcine PDGF may be a homodimer of a 17 000 mol. wt. chain. Since porcine PDGF can be purified at low cost from large quantities of fresh platelets, it provides an alternative source of PDGF for structural and functional studies, and could be of use in preparing defined media for cell culture.     

Isolation and partial characterization of serine- and threonine-rich porcine gastric mucus glycopeptides.

1. Two subfractions from purified porcine gastric mucus glycopeptide were found to separate from each other by cesium chloride equilibrium centrifugation. The highest density fraction and two lower density fractions separated were designated VHD, HD and LD, respectively. A comparative study of these components was made. 2. The high and low density fractions, HD and LD, appeared almost the same or identical, while VHD differed completely from either of them in the following respects: (1) VHD exhibited strong alcian blue binding activity. (2) 57% of VHD bound to the DEAE-Toyopearl column equilibrated with 0.2 M NaCl. (3) VHD eluted from the Sephacryl S-400 column as a lower molecular subunit. (4) One third of the sialic acid as a minor component in VHD was constituted by N-glycolylneuraminic acid. (5) Carbohydrate composition showed typical mucus glycoprotein with slightly higher fucose content. (6) Amino acid compositions of the anionic components prepared from VHD showed the highest Ser/Thr ratio, 1.92 compared to 0.46 for LD and 0.62 for HD. (7) Oligosaccharide released from VHD by alkaline-sodium borohydride treatment was larger than that from HD or LD. 3. The above results indicate the minor component, VHD, separated from the major components, to be a quite similar but not identical component to the so-called sulfated mucus glycoprotein reported previously [Slomiany et al. (1972) J. biol. Chem. 247, 5062-5070].     

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.     

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.