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Biomedical subjects

M Mantle

Publications and source records attributed to M Mantle.

At least 37 records · Page 2Linked to original sources
PubMed1990

The disulphide-bond content and rheological properties of intestinal mucins from normal subjects and patients with cystic fibrosis.

The disulphide/thiol (S-S/SH) content and rheological properties of highly purified small-intestinal mucins from normal (N) subjects and patients with cystic fibrosis (CF) were investigated. (1) An assay was developed to measure free SH groups (before reduction) and total SH content (after reduction) using 4,4'-dipyridyl disulphide. S-S bonds were calculated by difference. Experimental values for the S-S and SH contents of well-characterized proteins obtained with the assay showed good agreement with expected values. (2) The S-S and free SH contents of nine N and six CF mucins were variable: 44.4 +/- 5.4 nmol of S-S and 4.3 +/- 1.1 nmol of free SH per mg of N mucin and 31.7 +/- 7.6 nmol of S-S and 7.5 +/- 3.7 nmol of SH per mg of CF mucin. N and CF mucins were not statistically different. In most mucins, approximately 90% of the SH groups were involved in S-S bonds. (3) Gels were reconstituted from the same N and CF mucins at concentrations between 8 and 25 mg/ml and their rheological properties were assessed by using a magnetic microrheometer. (4) Once formed, mucin gels were stable and maintained the same mechanical properties over a long period of time (3-14 days). (5) The rheological profiles of both N and CF samples did not vary with the concentration of mucin present and were characteristic of weak, visco-elastic gels. (6) Although variations were seen in the visco-elastic properties of individual mucins, no systematic differences were detected between N and CF preparations. (7) There was no apparent correlation between the rheological properties of a mucin and its S-S/SH content.

Cysteine↗
PubMed1989

The putative 'link' glycopeptide associated with mucus glycoproteins. Composition and properties of preparations from the gastrointestinal tracts of several mammals.

The existence of a discrete 'link' peptide in epithelial mucins has been debated for many years. There is evidence that at least some mucins contain a specific 'link' peptide (or glycopeptide) that enhances mucin polymerization by forming disulphide bridges to large mucin glycoprotein subunits. A major difficulty has been to know whether the reported differences in putative 'link' components represent artifacts generated by inter-laboratory differences in technical procedures used in mucin purification. The present paper outlines the results of a collaborative study involving five laboratories and 53 samples of purified gastrointestinal mucins (including salivary, gastric, small-intestinal and colonic mucins) prepared by five techniques from four different animal species. An early step in mucin purification in all cases was the addition of proteinase inhibitors. Representative mucins were analysed for their composition, electrophoretic mobility in SDS/polyacrylamide-gel electrophoresis before and after disulphide-bond reduction, and for their reactivity with monospecific antibodies developed against the 118 kDa putative 'link' glycopeptide isolated from either rat or human small-intestinal mucins. Our results indicate that, despite differences in laboratory techniques, preparative procedures, organs and species, each of the purified mucins contained a 'link' component that was released by disulphide-bond reduction and produced a band on SDS/polyacrylamide-gel electrophoresis at a position of approx. 118 kDa. After electroelution and analyses, the 118 kDa bands from the different mucins were found to have similar amino acid profiles and to contain carbohydrate. It would appear therefore that a 'link' glycopeptide of molecular mass approx. 118 kDa is common to all of the gastrointestinal mucins studied.

Animals↗
PubMed1989

Intestinal mucins from normal subjects and patients with cystic fibrosis. Variable contents of the disulphide-bound 118 kDa glycoprotein and different reactivities with an anti-(118 kDa glycoprotein) antibody.

1. A specific antibody was developed against the disulphide-bound 118 kDa glycoprotein of human intestinal mucin and used to establish an e.l.i.s.a. Fourteen purified mucins [eight normal (N) and six cystic fibrosis (CF)] had the same affinity for the antibody in the e.l.i.s.a., but their relative immunoreactivities varied widely (approx. 100,000-fold). In general, CF mucins were more antigenic than N mucins. 2. Variations (approx. 10-fold) were detected in the 118 kDa glycoprotein content of both N and CF mucins (assessed from Coomassie Blue-stained polyacrylamide gels), but these did not appear to be responsible for the differences in mucin immunoreactivity. 3. Variations (approx. 6-fold) were also observed in the size of the 118 kDa peak produced by N and CF mucins on Western blots. These were mostly due to differences in the 118 kDa glycoprotein content of mucins, although a small proportion resulted from changes in the number of antigenic determinants within individual 118 kDa glycoproteins. 4. After concanavalin A affinity chromatography of four reduced mucins (two N and two CF), purified 118 kDa glycoprotein was recovered in the bound fractions from the column, specifically eluted by methyl alpha-mannoside. 5. The amounts of 118 kDa glycoprotein isolated from the four mucins varied as predicted from the size of their 118 kDa bands on Coomassie Blue-stained gels. 6. Three 118 kDa glycoproteins (one N and two CF) showed almost identical reactivity in the e.l.i.s.a.; the fourth had fewer antigenic determinants. 7. Since differences in 118 kDa glycoprotein content and in the number of antigenic determinants within the 118 kDa glycoprotein did not account for variations in the reactivity of native mucins in the e.l.i.s.a., it appeared that accessibility of the 118 kDa glycoprotein to antibody binding may be critical in determining mucin immunoreactivity. This suggests that the three-dimensional conformation of CF mucins may differ from that of N mucins, leading to increased antigenicity.

Antibodies↗
PubMed1989

Effects of streptozotocin-diabetes on rat intestinal mucin and goblet cells.

Intestinal mucin and goblet cells were examined in streptozotocin-diabetic rats and age-matched controls. Mucin (tissue content and secretion) was measured using a highly specific enzyme-linked immunoassay. In contrast to the increased protein to deoxyribonucleic acid ratio, an absolute decrease was observed in the mucin to deoxyribonucleic acid ratio in mucosal homogenates of the diabetic intestine. This was not due to a loss of goblet cells as their numbers per crypt-villus unit increased in diabetic rats (in proportion to the rise in enterocyte numbers and crypt-villus length). Histochemically, goblet cell mucin was unchanged in diabetes. After a 90-min incubation of everted intestinal segments in Krebs' buffer, pH 7.4, at 37 degrees C, the amount of mucin released into the medium was the same in diabetic and control rats when expressed relative to tissue deoxyribonucleic acid. However, secreted mucin represented a significantly larger proportion of the total tissue mucin content in diabetic animals. Thus, to maintain mucin output at normal levels, the rate of mucin secretion is apparently increased in the diabetic intestine, despite (or perhaps causing) a large decrease in the tissue mucin content.

Animals↗
PubMed1989

Binding of Yersinia enterocolitica to rabbit intestinal brush border membranes, mucus, and mucin.

Mucus and its gel-forming glycoprotein component, mucin, are thought to protect the gastrointestinal tract from enteric pathogens by inhibiting their attachment to enterocytes. In this study, we investigated interactions between Yersinia enterocolitica (isogenic strains of virulent and nonvirulent organisms) and crude mucus, highly purified mucin, and brush border membranes (BBMs) isolated from the upper mid-, and distal small intestine and the proximal colon of the rabbit. Adherence of radiolabeled bacteria was assessed to BBMs, mucus, and mucin immobilized in polystyrene microtiter plate wells. Virulent Y. enterocolitica showed saturable binding to mucus, mucin, and BBMs from all four regions of the intestinal tract, although adherence to BBMs was appreciably greater than that to mucus or mucin. Maximal binding of bacteria was higher to BBMs from the distal small intestine and the proximal colon than to those from the upper and mid-small intestine, which may in part explain why the organism localizes to the ileo-caecal regions of the gut. Adherence of virulent Y. enterocolitica to BBMs was significantly reduced in the presence of homologous mucus or mucin preparations. Binding of virulent bacteria appears to depend on plasmid-encoded proteins located on the outer surface membrane, since (i) the isogenic strain lacking the virulence plasmid showed markedly less binding to all BBM, mucus, and mucin preparations; (ii) growth of the virulent strain at 25 degrees C, which inactivates its plasmid, significantly diminished binding to BBMs, mucus, and mucin; and (iii) mild proteolysis substantially decreased adherence of virulent bacteria to BBMs. Compared with rabbit intestinal and colonic mucins, binding of virulent Y. enterocolitica was significantly greater to purified human intestinal mucin and significantly less to rat intestinal mucin. These findings provide support for the role of mucus and mucin in host defense by preventing adherence of virulent Y. enterocolitica to epithelial cell membranes.

Animals↗
PubMed1989

Effect of Yersinia enterocolitica on intestinal mucin secretion.

Mucin and glycoprotein synthesis and secretion were evaluated in the upper, mid, and distal small intestine and in the proximal colon of rabbits infected with Yersinia enterocolitica (YE). Infected (INF) animals were examined on day 6 and compared with pair-fed controls and unmanipulated weight-matched rabbits. Tissue mucin content in vivo and mucin secretion in vitro, measured by a specific immunoassay, were significantly elevated in all four regions of the gut of INF rabbits compared with both control groups. In vitro secretion of stored glycoprotein, prelabeled with [3H]glucosamine, was not increased in the upper and mid small intestine of INF animals but was significantly elevated in the distal small intestine and proximal colon. In vitro incorporation of [14C]glucosamine was increased in all four regions of the gut of INF rabbits, but secretion of newly synthesized [14C]glycoprotein was only significantly elevated in the distal small intestine and proximal colon. A graded response was observed down the intestinal tract of INF rabbits, with the greatest increase in mucin content, synthesis and secretion occurring in the distal small intestine and proximal colon where the morphological impact of disease is also most severe.

Animals↗
PubMed1989

Disulphide bonds and the 118,000 Mr glycoprotein of human intestinal mucin.

Purified intestinal mucins from eight normal subjects (N) and six patients with cystic fibrosis (CF) were compared for: (1) their content of free thiol groups (-SH) and disulphide bonds (S-S), (2) their immunoreactivity in a specific enzyme-linked immunoassay (ELISA) for the disulphide-bound 118,000 Mr glycoprotein and (3) their content of 118 kDa glycoprotein. Results showed considerable variability in the S-S/-SH content of individual mucin preparations but no differences were observed between N and CF samples. In the ELISA, all mucins were found to have identical antigenic determinants with the same affinity for the anti-118 kDa glycoprotein antibody but marked differences (ranging from 0.2 to 120,000 ng mucin) were observed in their relative immunoreactivities, indicating variable numbers of antigenic determinants. In general, CF mucins appeared to react more strongly in the immunoassay than N mucins. By SDS/polyacrylamide-gel electrophoresis under reducing conditions and densitometric analysis of Coomassie blue-stained gels, differences (up to six-fold) were also detected in the amount of 118 kDa glycoprotein in individual mucin preparations. However, this did not appear to be the cause of the variations in mucin immunoreactivity because not all strongly antigenic mucins contained large amounts of 118 kDa glycoprotein. Thus, accessibility of the 118 kDa glycoprotein to the antibody or the number of functional antigenic determinants within the 118 kDa glycoprotein may vary among different mucin preparations. The greater antigenicity of CF mucins may therefore reflect changes in their macromolecular conformation or in their 118 kDa glycoprotein.(ABSTRACT TRUNCATED AT 250 WORDS)

Cystic Fibrosis↗
PubMed1988

Rabbit intestinal and colonic mucins: isolation, partial characterization, and measurement of secretion using an enzyme-linked immunoassay.

Mucin was purified separately from the upper, mid, and distal small intestine and the proximal colon of the rabbit. The carbohydrate profiles of the three intestinal mucins were the same but differed from that of the colonic mucin, which contained less N-acetylgalactosamine but more sialic acid. All four mucins had similar polymeric structures composed of large, heterogeneous glycoprotein monomers and a smaller protein of Mr approximately 120,000, held together by disulphide bonds. The three intestinal mucins, however, were more resistant to dissociation by thiol reduction or degradation by proteolysis than colonic mucin. An antibody against a pool of the purified mucins was developed in guinea pigs and used to establish an enzyme-linked immunosorbent assay (ELISA). The antibody was highly specific for rabbit intestinal and colonic mucins, showing no cross-reactivity with nonmucin components of rabbit intestinal and colonic tissue, and very little or no reactivity with purified intestinal mucins from other species. The four purified rabbit mucins had the same affinity for the antibody and similar numbers of antigenic determinants. Antigenicity was apparently associated with the protein moeity of the mucins and was critically dependent on three-dimensional conformation, since proteolysis decreased the number of antigenic determinants while thiol reduction abolished antigen-antibody affinity. Using the ELISA, the tissue mucin content and the rate of mucin secretion were found to be significantly higher in the proximal colon that in the three regions of the intestine, which were the same.

Animals↗
PubMed1986

Radiation inactivation of human intestinal mucin: determination of the size of the functional antigenic unit.

Previously we have shown that the major antigenic determinant of human intestinal mucin is associated with its glycopeptide monomers and not the 118 kDa 'link' component. In the present study, the size and nature of the functional unit containing the antigenic determinant has been assessed by radiation inactivation and immunological assays. Increasing doses of radiation led to a monoexponential decay in antigenic reactivity due to a progressive loss of antigenic determinants. From three independent mucin preparations, a value of 78500 +/- 7000 was determined for the Mr of the functional antigenic unit. Prolonged pronase digestion of native mucin released large degraded glycopeptide monomers containing all the mucin carbohydrate, and low molecular weight peptides. The antigenicity of the glycopeptides decreased with digestion but could not be recovered in the peptide fractions, suggesting that determinants were released and destroyed by the enzyme. Treatment of native mucin with trifluoromethanesulphonic acid caused a major loss of carbohydrate (approx. 70%), but the protein component was unchanged in amino acid profile and remained antigenic. Subsequent thiol reduction, however, abolished the antigenicity of the deglycosylated mucin. We conclude that antigenicity is associated with a non-glycosylated segment of the peptide backbone of the glycopeptides and that a large functional unit of Mr 78500 which is stabilized by disulphide bonds is important for full antigenic activity.

Amino Acids↗
PubMed1986

The effects of delipidation on the major antigenic determinant of purified human intestinal mucin.

To investigate whether the antigenicity of purified human intestinal mucin was dependent on the presence of associated lipid, native mucin (purified by equilibrium density gradient centrifugation in CsCl (twice) and gel filtration on Sepharose 2B) was extracted five times with organic solvents to remove any noncovalently bound lipid and, subsequently, treated with hydroxylamine to release any covalently bound fatty acids. The first organic extract contained cholesterol, phosphatidylethanolamine, and phosphatidylserine, with lesser amounts of phosphatidylcholine, triglycerides, fatty acids, sphingomyelin, and glycolipids. In total, this noncovalently bound lipid amounted to less than 5% by weight of the native mucin preparation. Further organic extracts were free of lipid. Removal of noncovalently bound lipid had essentially no effect on mucin antigenicity, as assessed by radioimmunoassay. Treatment of the delipidated mucin with hydroxylamine caused no detectable changes in mucin antigenicity or composition and the release of covalently (ester) bound fatty acids could not be demonstrated. We therefore conclude that although purified human intestinal mucin contains small amounts of noncovalently bound lipid this lipid is not involved in mucin antigenicity.

Amino Acids↗
PubMed1985

Neutral and acidic species of human intestinal mucin. Evidence for different core peptides.

Highly purified human mucins from postmortem intestinal tissue were fractionated on anion exchange columns to generate separate neutral and acidic species. The neutral mucin (less than 1.0 mol % sialic acid) was the major species (greater than 80% by weight) and contained a higher molar proportion of fucose, galactose, and N-acetylglucosamine, and a lower proportion of sialic acid and N-acetylgalactosamine than the acidic species (greater than 10 mol % sialic acid). Amino acid analyses revealed a highly significant enrichment in serine, aspartate, and alanine in the neutral species and proline, threonine, and glycine in the acidic species. Thiol reduction of each species to remove their integral 118,000-dalton component did not alter the essential interspecies differences. Differences in threonine, proline, and serine also remained after removal of all "naked" or pronase-susceptible peptide regions from each species. These results indicate that neutral and acidic mucins contain glycopeptide segments exclusive of their 118,000-dalton and naked peptide components, which differ in amino acid composition. The key amino acid markers are similar to those observed for fuco- and sialoglycopeptides obtained after proteolytic digestion of human colonic mucin by Gold et al. (Gold, D.V., Schochat, D., and Miller, F. (1981) J. Biol. Chem. 256, 6354-6358). The oligosaccharide composition of small intestinal and colonic mucin may therefore depend upon transcriptional control of the synthesis of specific mucin peptides as well as the post-translational activity of glycosyltransferases. These findings may have significance for the quality and functions of mucus produced in a variety of pathological states.

Amino Acids↗
PubMed1984

Biochemical characterization of the component parts of intestinal mucin from patients with cystic fibrosis.

Previous studies have shown that human small-intestinal mucin consists of high-Mr glycoproteins and a smaller S-S-bonded protein of 118 kDa. The major antigenic determinants of the mucin were associated with the large glycoproteins, but depended for stability on intact disulphide bonds, and were destroyed by digestion with Pronase. In the present study we isolated and analysed the component parts of mucin from patients with cystic fibrosis with special attention being paid to the peptide constituents. After reduction with 0.2 M-beta-mercaptoethanol [5 min, 100 degrees C in 1% SDS (sodium dodecyl sulphate)], the large glycoproteins and smaller peptide with an apparent molecular size of 118 kDa were separated by equilibrium density-gradient centrifugation in CsCl, Sepharose 4B chromatography or preparative SDS/polyacrylamide-gel electrophoresis. The large glycoproteins contained about 70% of the protein of the native mucin. Digestion with Pronase resulted in a further loss of 'naked' protein (10% of the native mucin protein) from the C-terminal end of the glycoprotein peptide core, and left behind highly glycosylated proteins comprised mainly (70 mol%) of threonine, serine and proline. The 118 kDa component, which contained about 30% of the native mucin protein, consisted mainly of aspartic acid, serine, glutamic acid and glycine (40 mol%), plus threonine, proline, alanine, valine and leucine (35 mol%). Together with the 'naked' protein segment, the 118 kDa component contained most of the cysteine residues of the native mucin. Surprisingly, the peptide also contained carbohydrate (less than or equal to 5% of the native mucin carbohydrate but 50% by weight of the 118 kDa component), which included 9 mol% mannose, suggesting the presence of N-linked oligosaccharides. The peptide exhibited strong non-covalent interactions with the high-Mr glycoproteins and a tendency to self-aggregate in the absence of dissociating agents. Our findings therefore suggest that native mucin consists of large glycoproteins capable of forming disulphide bridges from their C-terminal 'naked' (antigenic) regions to a smaller glycopeptide having an Mr of 118 000.

Amino Acids↗
PubMed1984

Antigenic and structural features of goblet-cell mucin of human small intestine.

With the use of a newly developed solid-phase radioimmunoassay method, the major antigenic determinants of human small-intestinal goblet-cell mucin were investigated and related to the overall tertiary structure of the mucin. Preliminary hapten inhibition studies with various oligosaccharides of known sequence and structure suggested that the determinants did not reside in carbohydrate. Exhaustive thiol reduction, however, almost abolished antigenicity, caused breakdown of the mucin into small heterogeneous glycopeptides, and liberated a 'link' peptide of Mr 118000. Western 'blots' of reduced mucin from polyacrylamide gels on to nitrocellulose sheets showed that a small amount of residual antigenicity remained in large-Mr glycopeptides (Mr greater than 200000). The 'link' peptide was not antigenic. Timed Pronase digestion of native mucin resulted in a progressive loss of antigenic determinants. Gel electrophoresis revealed that after 8h of digestion the 118000-Mr peptide had disappeared, whereas antigenicity, which was confined to large-Mr glycopeptides, was destroyed much more slowly with time (70% by 24h, 100% by 72h). Despite the loss of antigenicity, 72h-Pronase-digested glycopeptides retained all of the carbohydrate of the native mucin. Therefore the antibody to human small-intestinal mucin appears to recognize a 'naked' (non-glycosylated and Pronase-susceptible) peptide region(s) of mucin glycopeptides. For full antigenicity, however, disulphide bonds are required to stabilize a specific three-dimensional configuration of the 'naked' region.

Amino Acids↗
PubMed1984

Abnormal mucus: nominated but not yet elected.

Mucus secretions of the intestine may contribute to plug formation in CF, through several mechanisms acting alone or simultaneously. Excessive mucin secretion and relative dehydration of secretions would elevate mucin concentration and enhance mucus gel formation. Deficient pancreatic enzymes combined with intrinsic hyperglycosylation of mucin or an increase in covalently bound fatty acid groups on mucins, would retard normal proteolytic degradation of mucins. Excessive calcium ions in the ducts would tend to decrease mucin solubility, while excessive hydrogen ions and protein (especially albumin) together with mucins, may be responsible for the appearance of proteinaceous ductal 'plugs' characteristic of CF pathology.

Albumins↗
PubMed1983

Human intestinal mucin in cystic fibrosis.

Human intestinal mucins from six subjects with Cystic Fibrosis (CF) and eight subjects without CF were prepared from tissue obtained at surgery (one case) and postmortem. Subjects were not age-matched, but the nonCF mucin was obtained from subjects with ages which bracketed those of the CF subjects. Cesium chloride analytical gradient ultracentrifugation showed that CF mucins were generally denser than nonCF mucins. Sedimentation coefficients were also higher in the CF samples. CF mucins were enriched in fucose, galactose, N-acetylglucosamine and total carbohydrate per mg protein and per oligosaccharide chain (mole/mole GalNAc). Fucose/sialic acid molar ratios were significantly higher in CF mucins, and the average oligosaccharide chain length was approximately three residues greater in CF as compared with nonCF mucins. There was no difference in amino acid profiles or the number of side chains per molecule. The mean sulfate content was higher in the CF mucins but not to a level of significance; however, in the eight mucins, sulfate content correlated positively with total carbohydrate, N-acetylglucosamine and galactose, and therefore increased with oligosaccharide chain length. CF intestinal mucin was therefore denser and more highly glycosylated than nonCF musin and probably contained more sulfate. The increase in glycosylation resulted from a rise in fucose, galactose, and N-acetylglucosamine without a concomitant rise in sialic acid.

Acetylglucosamine↗
PubMed1981

Isolation and characterization of the native glycoprotein from pig small-intestinal mucus.

Glycoprotein from pig small-intestinal mucus was isolated free of non-covalently bound protein and nucleic acid with a yield of over 60%. No non-covalently bound protein could be detected by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis or by equilibrium centrifugation in a density gradient of CsCl with 4 M-guanidinium chloride. The intrinsic viscosity and reduced viscosity of the glycoprotein preparations rose with the removal of non-covalently bound protein and nucleic acid from the glycoprotein, evidence that non-covalently bound protein does not contribute to the rheological properties of the glycoprotein in the mucus. The pure glycoprotein, in contrast with impure preparations, gelled at the same concentration of glycoprotein as that present in the gel in vivo. The glycoprotein was a single component, as judged by gel filtration and analytical ultracentrifugation. The distribution of sedimentation coefficients was polydisperse but unimodal with an s025,w of 14.5S and a molecular weight of 1.72 X 10(6). The chemical composition of the glycoprotein was 77% carbohydrate and 21% protein, 52% of which was serine, threonine and proline. The glycoprotein had a strong negative charge and contained 3.1% and 18.3% by weight ester sulphate and sialic acid respectively. The molar proportion of N-acetylgalactosamine was nearly twice that of any of the other sugars present, the glycoprotein had A and H blood-group activity and the average maximum length of the carbohydrate chains was deduced to be six to eight sugar residues.

Amino Acids↗