Colonic mucins in ulcerative colitis: evidence for loss of sulfation.

Colonic tissue obtained at surgery from control individuals and patients with ulcerative colitis was used to isolate mucins and to prepare mucin glycopolypeptides by pronase digestion. These were compared with mucins labelled with [35S] sulfate and [3H]-glucosamine after organ culture tissue samples from the same patients. A significant loss of mucin sulfation was detected in the colitis patients by both metabolic labelling and chemical analysis of the glycopolypeptides. A change in the size distribution of purified mucin oligosaccharides fractionated on BioGel P6 after release by beta-elimination was seen in both radiolabelled and non-labelled colitis mucins compared with controls. Amino acid analysis of the glycopolypeptides showed a close similarity to the expected ratio of serine:threonine:proline for MUC2 and did not vary between control and colitis groups. Analysis of the mucins confirmed > 90% purity in the labelling experiments, characteristic behaviour on density gradient centrifugation and agarose gel electrophoresis in control and ulcerative colitis groups and differences in sulfation and turnover at various sites in the normal colon.

Influence of blood group and secretor status on carbohydrate structures in human gastric mucins: implications for peptic ulcer.

1. The content and distribution of carbohydrate was examined in mucus glycopolypeptides from human antral mucosae. 2. The mean amount of carbohydrate per 1000 amino acid residues was found to be similar in glycopolypeptides with A, B or H activity. It was slightly, though significantly, less in glycopolypeptides lacking these determinants, because carbohydrate chains were of a shorter average length than in the A-, B- or H-active preparations. This difference was reflected in the sizes of oligosaccharide-alcohols released from representative glycopolypeptides with alkaline borohydride. 3. Differences between A-, B- or H-active and non-secretor glycopolypeptides in terms of the mean number of carbohydrate chains per 1000 amino acid residues were found to be small, and without significance. 4. The average number of peripheral monosaccharide units per 1000 amino acid residues was greater in A-active than in H-active, and least in non-secretor, glycopolypeptides. This order was reversed for monosaccharide units incorporated into skeletal (core plus backbone) structures. The difference in each case was statistically significant. 5. These findings suggest that the increased risk of peptic ulcer associated with blood group O and non-secretor status is unlikely to be attributable to an inherent deficiency in the protective mucus layer, linked to differences between mucins that are associated with A, B or H activity. Other hypotheses linked to infection with Helicobacter pylori are examined.

Sialic acids in human gastric aspirates: detection of 9-O-lactyl- and 9-O-acetyl-N-acetylneuraminic acids and a decrease in total sialic acid concentration with age.

1. The total sialic acid content of human gastric aspirates was measured using a colorimetric assay. Care was taken to optimize the assay and to eliminate interference. 2. The sialic acid content of gastric aspirates collected under resting conditions from 77 patients with non-ulcer dyspepsia was found to decrease with age from > 100 micrograms/ml at 25 years and younger to < 20 micrograms/ml above 70 years of age. 3. Analysis of the sialic acids by gas chromatography, mass spectrometry and thin-layer chromatography showed the presence of N-acetylneuraminic acid and two O-acylated derivatives, 9-O-acetyl- and 9-O-lactyl-N-acetylneuraminic acids. These forms were predominantly glycosidically bound. 4. Thin-layer chromatographic analysis of individual aspirate samples showed that the O-acetylated sialic acids were present in all samples, with a maximum of 25% and a minimum of 5% of the total sialic acids.

The roles of enteric bacterial sialidase, sialate O-acetyl esterase and glycosulfatase in the degradation of human colonic mucin.

Sialidase activity in normal faecal extracts showed a preference for mucin-related glycoprotein and oligosaccharide substrates, but the presence of two or more O-acetyl esters at positions C7-C9 on the sialic acids retarded the rate of hydrolysis. A specific sialate O-acetyl esterase was detected with a lower total activity relative to sialidase with mucin substrates and having a pH optimum of 7.8 and a KM of approximately 1 mM sialate O-acetyl ester. A specific glycosulfatase activity was found in faecal extracts using the substrate lactit-[3H]ol 6-O-sulfate with a pH optimum of pH 5.0 and a KM of approximately 1 mM. Faecal extracts from ulcerative colitis (UC) patients had higher sialate O-acetyl esterase and glycosulfatase activity, while mucin sialidase activity was unchanged. Metabolically labelled mucin isolated from UC patients contained less sulfate and had lower sialic acid O-acetylation compared with normal mucin. Colonic mucin was degraded more efficiently by faecal extracts from UC patients compared with normal extracts. The UC mucin was degraded more rapidly than the normal mucin by faecal enzyme extracts from both normal and UC subjects.

Mucin changes in ileoanal pouches monitored by metabolic labelling and histochemistry.

The pattern of mucin synthesis and secretion in mucosal biopsies from the proximal ileum, distal ileum, rectum and pouch before and after ileostomy closure was monitored in patients undergoing restorative proctocolectomy by metabolic labelling with [3H]glucosamine and [35S]sulphate and compared with the mucin histochemistry in each patient. Metabolic labelling clearly demonstrated a reduction in sulphation associated with the rectal mucosa in colitis. Significant differences in the turnover of isolated secreted mucin between proximal and distal ileum and rectum were also found, as was a metaplastic change towards a colonic-type mucosa in the pouch before and after ileostomy closure relative to the proximal and distal ileum, and rectum. This technique can be used to monitor colonic metaplasia in the pouch mucosa and is suitable for comparative studies where mucin changes are implicated.

Mucin degradation in the human colon: production of sialidase, sialate O-acetylesterase, N-acetylneuraminate lyase, arylesterase, and glycosulfatase activities by strains of fecal bacteria.

Oligosaccharide side chains of human colonic mucins contain O-acetylated sialic acids and glycosulfate esters. Although these substituents are considered to protect the chains against degradation by bacterial glycosidases, sialate O-acetylesterase, N-acetylneuraminate lyase, and glycosulfatase activities have been found in fecal extracts. To better define the source of these activities, we measured extracellular and cell-bound sialidase, sialate O-acetylesterase, N-acetylneuraminate lyase, arylesterase, and glycosulfatase activities produced by 23 isolates of human fecal bacteria grown anaerobically in a hog gastric mucin culture medium; these represented dominant populations of fecal anaerobes, facultative anaerobes, and the subset of mucin oligosaccharide-degrading bacteria. Every strain produced sialidase and high levels of arylesterase, and all but five facultative anaerobes produced sialate O-acetylesterase. Sialic acids containing 2 mol or more of O-acetyl ester per mol of sialic acid were cleaved from mucin glycoproteins more slowly by sialidases of mucin oligosaccharide-degrading stains than were sialic acids containing 1 or 0 mol, and only N-acetyl- and mono-O-acetylated sialic acids were recovered from enzyme digests of a mucin containing di-O-acetylated sialic acids. No detectable N-acetylneuraminate lyase activity was produced by any strain, but low activity was induced by increasing the glycoprotein-bound sialic acid concentration in the culture medium of six Escherichia coli strains. Using lactitol-6-sulfate as a substrate, we found weak glycosulfatase activity in the partially purified, concentrated enzyme mixture in the culture supernatants of four mucin oligosaccharide-degrading strains but in none of the unconcentrated culture fractions. We conclude that the presence of two or more O-acetyl groups on sialic acids inhibits enteric bacterial sialidases but that production of sialate O-acetylesterases by several populations of enteric bacteria lessens the likelihood that mucin oligosaccharide chains terminating in O-acetylated sialic acids are protected from degradation. Sialate O-acetylesterases have a role in bacterial degradation of mucin glycoproteins in the human colon.

The glycoconjugate content of human faeces and ileostomy effluents.

Normal human faeces and effluent from terminal ileostomies in patients with ulcerative colitis were collected. The results for ileostomy effluent given as g/24 h (mean +/- SD; n = 12) were as follows: wet weight, 897 +/- 120; total dry weight, 84 +/- 8; non-diffusible dry weight 46 +/- 5. The corresponding results for faeces (n = 6) were 104 +/- 24; 26 +/- 6; 19 +/- 5. Whereas approximately 15% of the total dry weight of ileostomy effluent appeared to consist of mucin-derived material, only trace amounts of such material could be detected in faeces.

Characterization of the major and minor mucus glycoproteins from bovine submandibular gland.

Two mucins were isolated from bovine submandibular glands and termed major and minor on a quantitative basis. The major mucin representing over 80% of the total glycoprotein fraction contained 37% of its dry weight as protein in contrast to 62% for the minor mucin. Differences in the amino acid composition reflected the higher proportion of typically non-glycosylated peptide in the minor mucin. The molar ratio of N-acetylgalactosamine to serine plus threonine was 0.82 in major and 0.65 in minor mucins, indicating a lower degree of substitution of potential glycosylation sites in the minor mucin. Differences in the carbohydrate composition were found largely related to the sialic acids, with higher relative amounts of N-glycoloylneuraminic acid in the minor mucin. In addition, the proportion of di-O-acetylated sialic acids was higher in the major mucin. The rate of sialidase action on the two mucins could be correlated with the content of N-glycoloylneuraminic acid in each glycoprotein. There was no difference in the type of oligosaccharide found in each mucin and the differences in relative proportions reflected the monosaccharide composition for the two mucins. Gel filtration on Sepharose CL 2B showed a lower molecular weight distribution for the minor in contrast to the major mucin which was partially excluded. Density gradient centrifugation reflected this variation. SDS-PAGE demonstrated a regular banding pattern for the major mucin with a lowest subunit size of 1.8 x 10(5) Da and aggregates in excess of 10(6) Da, while the minor mucin ranged from 3.0 x 10(5) to 10(6) Da. The chemical composition of the isolated mucins was compared with previous histochemical analysis of mucin distribution in bovine submandibular glands and indicates a possible cellular location for each mucin.

Characterization of a sialic-acid-rich mucus glycoprotein secreted by a premalignant human colorectal adenoma cell line.

The human colonic cell line PC/AA, derived from an adenoma, retains in vitro colonic cell differentiation, notably the production of mucus glycoproteins. The PC/AA adenoma cells produce an extracellular gel layer in culture. The PC/AA gel could be isolated by extraction of the cell cultures with guanidine hydrochloride. The extracted material was purified by gel filtration and caesium chloride density-gradient centrifugation and showed properties typical of mucus glycoproteins, namely, a carbohydrate content above 60% of dry weight rich in N-acetylgalactosamine and sialic acid and low in mannose; an amino acid composition with high serine threonine and proline content; a molecular weight above 1,000 kDa on Sepharose CL 4B chromatography and on SDS-polyacrylamide gel electrophoresis under reducing conditions (greater than 200 kDa); a buoyant density of approximately 1.48 g/ml and the release of oligosaccharides by the alkaline beta-elimination reaction. Comparison of the gel mucus glycoprotein purified from premalignant PC/AA cells with normal human colon mucin showed that it has a higher sialic acid content. This suggests that higher sialic acid levels may precede the development of malignancy.

A comparison of the results of sequential hydrazinolysis-nitrosation and alkali-mediated cleavage-nitrosation of the O-linked oligosaccharides of gastric mucus glycoproteins.

Analysis of the oligosaccharides released from pig gastric mucus glycopolypeptides by hydrazinolysis showed that degradation had occurred. Nitrosation of the products followed by reduction gave a mixture that had a low content of 2,5-anhydro-D-talitol, which implied destruction of much of the terminal reducing 2-amino-2-deoxy-D-galactose. Under the conditions of hydrazinolysis, cellobiose was largely unchanged but laminaribiose gave a complex mixture that probably contained glucose hydrazone (13C-n.m.r. data). In order to avoid degradation, the hydrazinolysis-nitrosation sequence should be applied to the reduced oligosaccharides released on cleavage with alkali.

Measurement of sialyl-transferase activity in isolated colonic mucosal cells of the rat.

An assay for sialyl-transferase activity in isolated rat colonic mucosal cells has been developed. Total and specific activity with asialobovine sub-mandibular gland glycoprotein and endogenous substrates was approximately two-fold higher in the proximal, relative to the distal, colon. These activities were similar when asialo-alpha-acid glycoprotein was used as substrate. Endogenous activity was approximately 10-fold lower than with exogenous substrates in both proximal and distal colonic segments. Analysis of total and specific sialyl-transferase activity up to 7 weeks after jejunoileal bypass (JIB) and sham operation showed a marked increase at the first week, decreasing toward normal by week 7. Similar differences between proximal and distal colon, and with the type of substrate, were found with both groups of operated animals. Histochemical analysis showed small elevation after 1 week for sialo and sulphomucins after the JIB operation only.

The gastric mucosal barrier.

The gastric mucosal barrier is a complex system made up of submucosal, epithelial and mucus elements. The mucus gel layer is a thick tenacious organized layer adherent to the epithelium. Despite these properties it is composed of more than 95% water, the organization being provided by long interacting glycoprotein molecules (mucus glycoprotein or mucin). These molecules are largely made up of carbohydrate which is present in large numbers of relatively small oligosaccharide units packed around the polypeptide core. This structure provides clues to the nature of the protection afforded by the mucus layer. For example, it is relatively resistant to proteolysis in the gastrointestinal tract; it retains water in an unstirred layer; the tangled glycoproteins exclude large molecules and the carbohydrate of the oligosaccharide units mirror that at the surface of the epithelial cell. Few biochemical studies have been carried out on the effect of ulcer-healing drugs on gastric mucus. Normal subjects were, therefore, given two weeks treatment with cimetidine, carbenoxolone or misoprostol and the secretions aspirated from the unstimulated and pentagastrin-stimulated stomach. The volume of secretion and weight and carbohydrate content of non-diffusable glycoconjugates were determined for each specimen, together with the proportion of high molecular mass mucin and qualitative and quantitative analyses of the glycopolypeptide. There were no significant differences between the results for each drug or without drug. This may be because normal subjects were studied who already have an effective mucosal barrier. In addition, it is likely that the process of mucus biosynthesis and secretion in a healthy individual is relatively resistant to the action of ulcer healing drugs.

Effects of cimetidine and carbenoxolone on gastric mucus.

The effect of cimetidine on normal human gastric mucus has been compared with that of carbenoxolone, a drug believed to enhance mucus production. Each drug was given for two weeks, the gastric contents aspirated over a timed period and the results assessed in unstimulated and pentagastrin stimulated secretions. The volume, dry weight and the carbohydrate contents of non-diffusable glycoconjugates, high molecular mass glycoproteins and glycopolypeptides were investigated. Both drugs reduced the volumes of stimulated secretions. This was statistically significant after cimetidine. More importantly neither drug affected the amount of non-diffusable glycoconjugates, so that the concentration remained the same or increased. Both drugs reduced the monosaccharide content of the high molecular mass fractions. This reached significance for the stimulated secretion after cimetidine. As the carbohydrate content of the glycopolypeptides was unchanged this indicated the presence of a non-mucin glycoprotein or protein. Overall there was no fundamental difference between the results for cimetidine and carbenoxolone.