Cloning of a mucin-desulfating sulfatase gene from Prevotella strain RS2 and its expression using a Bacteroides recombinant system.

A gene encoding the mucin-desulfating sulfatase in Prevotella strain RS2 has been cloned, sequenced, and expressed in an active form. A 600-bp PCR product generated using primers designed from amino acid sequence data was used to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfating sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase proprotein was identified, and the deduced 517-amino-acid protein minus its signal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl- and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into Escherichia coli expression vectors. However, cloning the gene into a Bacteroides expression vector did produce active sulfatase. This is the first mucin-desulfating sulfatase to be sequenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direction. Its sequence has close homology to iron-sulfur proteins that posttranslationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary for enzymatic activity.

Functional protective role for mucin glycosylated repetitive domains.

Mucins carry out a number of protective roles, some of which are more easily studied than others. One mucin function is believed to be the protection of the mucosal epithelium against acidic and proteolytic damage in the stomach and intestines. In the present work, a portion of stomach mucin tandem repeat sequence (Muc6) was joined to the catalytic domain of a reporter enzyme [human milk cholesterol esterase (CE)] to determine whether the former can protect the latter protein from damage. This Muc6 domain replaced a unique series of glycosylated C-terminal repeats normally present in CE. The chimeric protein (CE/Muc6) was expressed in two different cell lines and its properties compared to recombinant full-length CE and a truncated version of CE which contained only the catalytic domain (CE/trunc). Results showed that both CE and CE/Muc6 were resistant to denaturation by acid and to proteolysis by pepsin at low pH values or by pancreatic proteases compared to CE/trunc. Thus, a stomach Muc6 domain is sufficient to confer stability on the CE catalytic domain, demonstrating a protective effect by a glycosylated mucin sequence.

The effect of pH on the inhibition of bacterial growth by physiological concentrations of butyric acid: implications for neonates fed on suckled milk.

Butyric acid is released from milk by pre-intestinal lipases during suckling. It is also known to inhibit bacterial growth. To investigate whether butyric acid may be a significant factor in controlling bacterial growth in the stomach of pre-weaned animals, the ability of butyric acid to inhibit growth of selected bacteria was tested over physiological ranges of pH and butyric acid concentrations. Six enteric and environmental strains of bacteria were used: two strains of Escherichia coli, Klebsiella pneumoniae, Enterococcus faecium, Enterococcus faecalis, and Enterococcus casseliflavus. At pH 4.5 and 5.0, the growth of all organisms was significantly inhibited in the presence of butyrate, and in some cases growth was completely arrested. At pH 6.0, butyric acid did not affect bacterial growth until the concentration reached 40 mM. The maximum concentration of butyric acid available in cow's milk after incubation with pre-gastric lipase is approximately 16 mM, which would be sufficient to prevent growth of the organisms tested at pH values occurring in the stomach. Therefore, butyric acid inhibition of bacterial growth may explain in part, the role of pre-intestinal lipases in young animals' natural defenses against bacteria in ingested food prior to weaning.

Adsorption of a hydrophobic mutagen to cereal brans and cereal bran dietary fibres.

The abilities of brans from the cereals barley, oats, maize, rice, and wheat to adsorb in vitro the hydrophobic, environmental mutagen 1,8-dinitropyrene (DNP) were investigated using a mutagenicity assay. These brans were obtained from known cultivars using defined milling conditions and were chemically characterised. The abilities of total and insoluble dietary fibre preparations obtained from these brans to adsorb DNP were also investigated. The predicted weight of each bran required to adsorb 50% of the added DNP was used to compare the adsorptive abilities of the different brans. The brans were ranked in the order (most effective to least effective): rice, wheat, maize, barley, and oats. The adsorptive abilities of the dietary fibre preparations were not significantly different from the bran from which they were prepared. However, if the dietary fibres (cell walls) were the only components adsorbing the DNP, we would have expected the dietary fibre preparations to have adsorbed more DNP than the equivalent unextracted bran. This suggests that other components, probably starch, also adsorb DNP in the unextracted brans. It is not known why brans from different cereal species differ in adsorptive ability but the lignified cell walls in wheat bran may be important in conferring good adsorptive properties to this bran. The possible relationship between adsorptive ability and ability of the bran from a particular species to protect against colorectal cancer is discussed.

MUC3 human intestinal mucin. Analysis of gene structure, the carboxyl terminus, and a novel upstream repetitive region.

MUC3 is a large mucin glycoprotein expressed by the human intestine and gall bladder. In this manuscript, we present details of the deduced protein structure of MUC3. The MUC3 carboxyl-terminal domain is 617 residues in length, including 511 residues of a non-repetitive mucin-like domain (27% Thr, 22% Ser, and 11% Pro) and a 106-residue Cys-rich domain with homology to the epidermal growth factor (EGF) -like structural motifs found in many proteins. The region of MUC3 located upstream of the previously described 51-base pair (bp) tandem repeats, which encode a major Ser and Thr-rich domain, consists of a second type of repetitive structure with an imperfect periodicity of approximately 1125 bp. This domain is also mucin-like and appears to be considerably larger than 2000 residues (6000 bp). The MUC3 gene itself is large and complex. Using pulse field gel electrophoresis and blot analysis, the smallest fragment found that contained all human genomic DNA hybridizing to the 51-bp tandem repeat probe was 200 kilobases with restriction enzyme SwaI. Both PvuII and PstI produced two sets of hybridizing fragments that were hypervariable within the human population with a pattern suggestive of both a variation in the number of tandem repeats (VNTR) and sequence polymorphism. These fragments varied independently of each other, but no genetic recombination was detected in a study of 40 human families. Thus, the MUC3 gene encodes a very large glycoprotein with a structure very different from that of any mucin currently described.

Aspirin changes the secretion rate and amino acid composition of human small intestinal mucin in subjects with ileal conduits.

The effect of aspirin on the rate of secretion and amino acid composition of human ileal mucin was studied, using subjects with ileal conduits as a model system in which mucin secreted from the ileal conduit tissue is flushed out in the urine and can be measured and analysed. Aspirin (600 mg per day, administered orally) increased the daily mucin output by 37-104% in subjects by days 3 or 4, but thereafter the mucin output declined to below the baseline level by day 10. Mucin samples, purified from the ileal conduit urine during the control period and during aspirin administration, were compared. There were no discernible changes in the degree of polymerisation or the density, but during aspirin administration the amino acid composition was significantly changed, and in particular threonine and proline were enriched. One possible explanation, consistent with the compositional analyses, is that the N- and C-terminal regions of the mucin subunits have been cleaved off and lost during aspirin administration. The observed changes in mucin secretion may have implications for the mechanism of the toxic effects of aspirin on the small intestine by altering the barrier properties of the mucus layer.

The adsorption of heterocyclic aromatic amines by model dietary fibres with contrasting compositions.

It is often recommended that consumption of dietary fibre should in increased to protect against colorectal cancer. However, although more than 95% of dietary fibre is contributed by whole plant cell walls, very little experimental work has been done using whole plant cell walls. These may protect by adsorbing carcinogens, thus lowering their effective concentration in the alimentary tract, and by carrying the carcinogens out of the body in the faeces. However, plant cell walls vary widely in their composition and physical properties, and not all cell walls will necessarily have protective properties. We therefore isolated 4 plant cell-wall preparations with contrasting compositions as models of the types of cell walls that occur in the diet. We investigated the abilities of these preparations to adsorb in vitro 6 heterocyclic aromatic amines (HAAs). HAAs occur in the human diet and several are colon carcinogens, at least in rats. We found that the ability of the HAAs to adsorb to the plant cell walls increased with increasing hydrophobicity of the HAA, measured as the calculated logarithm of the partition coefficient between 1-octanol and water (C logP). A cell-wall preparation containing mainly the walls of parenchyma cells (the most common cell type in food plants) had only poor adsorptive ability. A cell-wall preparation from commercial cork had the best adsorptive ability. This preparation was the most hydrophobic of those examined because the cell walls contained the polymer, suberin, together with associated waxes. The preparation modelled suberized cell walls which occur in the diet, for example in potato skins. The other two cell-wall preparations contained another hydrophobic polymer, lignin, and had intermediate adsorptive abilities which were not significantly different from one another. These preparations modelled lignified cell walls which occur in the diet, for example in wheat bran. Our results indicate that suberized and lignified cell walls may be important in protecting against colorectal cancer.

Expression cloning of gastric mucin complementary DNA and localization of mucin gene expression.

BACKGROUND & AIMS: Secretory mucins play an important role in gastric cytoprotection and are derived from a heterogeneous family of genes. The aim of this study was to determine the specific type and location of mucin gene expression in the human stomach. METHODS: Expression cloning was performed by screening a human gastric complementary DNA expression library with antisera against deglycosylated gastric mucin. RNA analysis and immunohistochemistry were used to quantify and localize mucin gene expression. RESULTS: Sequencing of positive clones revealed two clones containing tandem repeats. The first contained a 169-amino acid repeat and was named MUC6 (as previously described). The second contained the same 8-amino acid repeat consensus sequence (APTTSTTS) as complementary DNAs previously isolated from a tracheobronchial complementary DNA library and was labeled MUC5 (or MUC5AC). RNA analysis indicated that the gastric epithelium contains high levels of MUC5 and MUC6 messenger RNA with little or no MUC2, MUC3, and MUC4 messenger RNA. Immunohistochemical analysis showed that surface mucous cells of the cardia, fundus, and antrum expressed MUC5 peptide. In contrast, MUC6 peptide expression was limited to mucous neck cells of the fundus, antral-type glands of the antrum and cardia, and Brunner's glands of the duodenum. CONCLUSIONS: MUC5 and MUC6 represent major secretory mucins in the stomach and are localized to distinct cell types.

The effects of a soluble-fibre polysaccharide on the adsorption of carcinogens to insoluble dietary fibres.

Epidemiology and animal experiments indicate that dietary fibres protect against the development of colorectal cancer. However, insoluble dietary fibres appear to be more effective than soluble dietary fibres and one mechanism by which they may protect is by adsorbing dietary carcinogens. We found previously that the ability of a carcinogen to adsorb in vitro to alpha-cellulose (a model insoluble dietary fibre) was strongly related to the hydrophobicity of the carcinogen, measured as the calculated logarithm of the partition coefficient between 1-octanol and water (C log P). Furthermore, soluble dietary fibres (soluble-fibre polysaccharides), including gum arabic, reduced the adsorption of the hydrophobic carcinogen, DNP, to alpha-cellulose. In the present study we tested the ability of gum arabic to reduce the adsorption in vitro of the carcinogens BaP (C log P = 6.124), DNP (C log P = 4.384), and the heterocyclic amines, Trp-P-1 (C log P = 3.230) and MeIQx (C log P = 1.078). Gum arabic reduced the adsorption to alpha-cellulose of BaP and DNP, but not the adsorption of Trp-P-1 or MeIQx. Gum arabic also reduced the adsorption of BaP to an insoluble, dietary-fibre preparation from commercial cork which contains the hydrophobic component, suberin, but did not affect the adsorption of DNP, Trp-P-1 or MeIQx. It also did not affect the adsorption of DNP to an insoluble, dietary-fibre preparation from wheat straw, which contains the hydrophobic component, lignin. The results are discussed in terms of hydrophobic interactions between carcinogens and insoluble dietary fibres. In vivo, it is likely that soluble dietary fibres reduce the adsorption of only highly hydrophobic carcinogens to some insoluble dietary fibres.

Colorectal mucin histochemistry in health and disease: a critical review.

Neoplastic, inflammatory and regenerative processes affecting colorectal mucosa are associated with alterations in structure of epithelial mucin. This review collates mucin-, lectin-, and immuno-histochemical observations on colorectal mucins and introduces recent molecular genetic insights into the structure of the protein backbone of mucins. The numerous structural modifications uncovered by the various technical approaches have been reduced to a few manageable principles that are of relevance to both researcher and diagnostic pathologist. Particular attention is drawn to the need to appreciate the limited specificities of probes, the confounding influences of anatomical site and genetic factors (necessitating the use of appropriate positive and negative control tissues) and the precise location of secretory material. In the past, insufficient attention has been given to the effects of altered differentiation including metaplasia and differing lineage expression in epithelial disorders of growth. It is likely that certain changes loosely ascribed to goblet cell mucin, such as neo-expression of blood group antigens and anomalous expression of core carbohydrate structures, do not occur at all. Critical examination of available data point to only two consistent and unequivocal changes affecting goblet cell mucin in pathological processes: loss of O-acetyl substituents at sialic acid C4 and C7,8,9 and increased sialylation. Furthermore, there are no neoplasia-specific alterations in mucins documented to date. All neoplasia-associated changes have been described in non-neoplastic lesions also.

A constituent of human small intestinal mucus that copurifies with mucin and can interfere with raising antibodies to the mucin.

The accuracy of an immunoassay for mucin depends on the antibodies' specificity. During human small intestinal mucin purification on a CsCl gradient, a very antigenic non-mucin contaminant was found at 1.48 g.ml-1 density. This was separated from the mucin (1.42 g.ml-1 density) by dividing the gradient into 26 fractions, but not by dividing it into 8 fractions. Polyclonal antibodies raised against mucin obtained using 8 fractions reacted with mucin and contaminant, but antibodies raised against mucin obtained using 26 fractions reacted only with mucin. The identity of the contaminant is unknown. However, it contained nearly equal amounts of galactose, N-acetylglucosamine and glucose, but did not react strongly with periodate-Schiff reagent. Immunofluorescence microscopy showed it was located in the small intestinal goblet cells and mucus layer. Unless the contaminant is removed before raising polyclonal antibodies to mucin, immunoassays will give inaccurate results.

The adsorption of a range of dietary carcinogens by alpha-cellulose, a model insoluble dietary fiber.

One of the ways dietary fibers may protect against colorectal cancer is by adsorbing carcinogens and carrying them out of the digestive tract, thus lessening interaction of the carcinogens with the colonic tissue. We investigated this mechanism of action by testing in vitro the abilities of a range of carcinogens, including known animal colon carcinogens, to adsorb to alpha-cellulose, which we have used as a model insoluble dietary fiber. The carcinogens were N-nitroso-N-methylurea (NMU), benzo[a]pyrene (B[a]P) and a number of heterocyclic aromatic amines which have been found in heated foods. It was found that the ability of a carcinogen to adsorb to alpha-cellulose is strongly related to the hydrophobicity of the carcinogen measured as the calculated logarithm of the partition coefficient between 1-octanol and water (C log P). The hydrophilic carcinogen, NMU, (C log P = -0.204), adsorbed only poorly, whereas the very hydrophobic carcinogen, B[a]P, (C log P = 6.124), adsorbed strongly. Carcinogens with intermediate hydrophobicities showed intermediate abilities to adsorb.

Roles of endogenous substances and bacteria in colorectal cancer.

Because the environment of the human colon is so complex, factors which lead to the development of colorectal cancer are difficult to identify. The effects of 3 endogenous components that affect development of colorectal cancer--colonic bacteria, the mucus layer and bile acids--will be reviewed in this article. The major effects of the bacteria are deconjugation and reduction of bile acids, activation of mutagen precursors, fermentation and production of volatile fatty acids, formation of endogenous mutagens and physical adsorption of hydrophobic chemicals. The mucus layer covering the surface acts as a barrier, and its composition changes in premalignant and malignant colon tissue. Its secretion is elevated by certain plant cell wall components in the diet. Mucus has some hydrophobic properties, and its presence may alter the distribution of hydrophobic molecules. Bile acid concentration in faecal water, rather than the total bile acid concentration, determines the toxicity to epithelial cells and increased concentrations stimulate cell proliferation rates. There is evidence that elevated bile acids in the lumen can activate cellular protein kinase C, which stimulates cell proliferation. These effects are consistent with bile acids acting as tumour promoters.

A glycosulphatase that removes sulphate from mucus glycoprotein.

A novel glycosulphatase has been purified from a mucus glycopeptide-degrading Prevotella from the colon. The purified enzyme removed inorganic [35S]sulphate from 35S-labelled native rat gastric mucus glycoprotein. Desulphation of mucus glycoprotein was initially rapid (19% complete after 10 min) but then plateaued, reaching only 33% after 3 h. Crude periplasmic extracts could remove 79% of the radioactivity as inorganic sulphate. These results suggest that steric hindrance may limit the access of the purified glycosulphatase to the mucus glycoprotein oligosaccharide chains in the absence of glycosidases, and/or that the enzyme may have the wrong specificity for some of the remaining sulphated sugars in the chains. The apparent molecular mass of the enzyme was 111 kDa as judged from gel exclusion chromatography, and it appeared to be composed of two identical subunits. The enzyme was localized in the periplasm of the bacterium, and using pig gastric mucus glycopeptide as a growth substrate markedly increased enzyme levels. Enzymic activity increased at the end of the growth phase. The substrate specificity of the enzyme was tested against low-molecular-mass sulphated molecules. The monosaccharides glucose 6-sulphate and N-acetylglucosamine 6-sulphate were rapidly desulphated, the latter being the major sulphated sugar in some mucus glycoproteins. Lactose 6-sulphate, galactose 6-sulphate, sulphated steroids and unsaturated disaccharide sulphate breakdown products from chondroitin sulphate were not desulphated. Glycosulphatases which can remove sulphate from mucus glycoproteins may play an important role in the degradation of highly sulphated mucus glycoproteins in the digestive tract, and could modify the effectiveness of mucus glycoproteins in mucosal protection.

Human gastric mucin. Identification of a unique species by expression cloning.

Gastric mucin is a large glycoprotein which is thought to play a major role in the protection of the gastrointestinal tract from acid, proteases, pathogenic microorganisms, and mechanical trauma. In this paper we describe the isolation by expression cloning and characterization of cDNAs which code for human gastric mucin. The cDNA sequence is characterized by a tandem repeat region whose individual repeat unit is 507 base pairs (169 amino acids) long. The translated sequence is rich in threonine, serine, and proline (31, 18, and 15%, respectively) and contains a relatively large amount of histidine (7.1%) and alanine (5.6%). RNA blot analysis shows a polydisperse pattern which is characteristic of mucins. Expression of this gene is highest in the stomach and gall bladder, with weaker expression in the terminal ileum and right colon. This expression pattern is different from other human mucins and indicates that this gene codes for a unique mucin. Fluorescence in situ hybridization techniques have localized this gene to chromosome 11p15.4-11p15.5. This is the third mucin to be localized to the 11p15 region and suggests a clustering of secretory mucin genes. We propose that this gene for human gastric mucin be called MUC6.

The effects of soluble-fiber polysaccharides on the adsorption of a hydrophobic carcinogen to an insoluble dietary fiber.

Dietary fiber is believed to decrease the incidence of colorectal cancer, but not all types of fiber are equally protective. Dietary fibers may be divided broadly into insoluble and soluble fibers, and there is evidence from animal experiments that the latter not only fails to protect against colorectal cancer but may enhance its development. Adsorption of carcinogens to insoluble dietary fiber in the intestinal tract is one of the mechanisms by which dietary fiber is believed to protect against colorectal cancer. In previous in vitro experiments, we showed that the hydrophobic carcinogen 1,8-dinitropyrene (DNP) adsorbs to insoluble plant cell wall components (insoluble dietary fibers). Soluble polysaccharides (pectic polysaccharides) extracted from the walls of parenchyma cells of dicotyledonous plants were found to maintain DNP in aqueous solutions and decrease its adsorption to insoluble wall components. In the present study, we examined a commercial preparation of pectin and seven other soluble-fiber polysaccharides with diverse structures for their effects on the distribution of DNP. Many of these are used as emulsifiers and stabilizers in the food industry. They all maintained DNP in aqueous solution and decreased its adsorption to alpha-cellulose, which we used as an example of an insoluble dietary fiber. Gum arabic was the most effective and kappa-carrageenan the least. The capacity of the polysaccharides to act as emulsifiers and stabilizers may explain their effects on DNP distribution. The monosaccharide glucose and the disaccharide cellobiose had no effect on the distribution of DNP. These results indicate three possible mechanisms by which soluble-fiber polysaccharides may enhance the development of colorectal cancer. First, because they reduce the ability of insoluble dietary fibers to adsorb hydrophobic carcinogens, more carcinogens may enter the colon maintained in solution than adsorbed onto insoluble fibers. Second, if soluble-fiber polysaccharides are maintaining hydrophobic carcinogens in solution and these polysaccharides are degraded by bacterial enzymes in the colon, then the carcinogens may come out of solution and be deposited onto the mucosal surface of the colon. Third, soluble-fiber polysaccharides may cross the intestinal epithelium and carry with them carcinogens maintained in solution. These studies have important consequences for nutrition, because soluble-fiber polysaccharides represent a common component of foods.

In vitro adsorption of a hydrophobic mutagen to gastrointestinal mucus glycoprotein (mucin) and dietary fibre.

The adsorption of mutagens by some dietary fibres has been suggested as one mechanism by which dietary fibres protect against colorectal cancer. It is thought that these dietary fibres carry the mutagen out of the digestive tract, decreasing the effective mutagen concentration to which epithelial cells are exposed. The ability of gastrointestinal mucin to alter the extent to which the hydrophobic mutagen 1,8-dinitropyrene (DNP) adsorbs in vitro onto the insoluble dietary fibre alpha-cellulose, was investigated. It was found that crude and purified human ileal mucins themselves adsorbed DNP and decreased the adsorption of DNP onto alpha-cellulose. Purified mucin which had been treated with trypsin also adsorbed DNP. These studies suggest that in the digestive tract there would be competition for the adsorption of DNP between mucin and insoluble dietary fibres, such as alpha-cellulose. This factor must be considered in predictions about the distribution of hydrophobic, mutagenic carcinogens in the digestive tract and their role in the etiology of colorectal cancer.

Adsorption of a hydrophobic mutagen to dietary fiber from taro (Colocasia esculenta), an important food plant of the South Pacific.

The incidence of colorectal cancer is lower in Polynesian populations of the South Pacific than in European populations. This difference in incidence of the disease may be, at least partly, related to diet. Dietary fiber is believed to protect against colorectal cancer, and one of the ways it may act is by adsorbing mutagens that are carcinogenic. Very little is known about the chemical composition or the ability to adsorb mutagens of these dietary fibers from South Pacific food plants. In contrast to European food plants, which are mostly dicotyledons, South Pacific food plants are mainly monocotyledons. We isolated cell walls (dietary fiber) from the three edible parts of taro (Colocasia esculenta), which is a monocotyledon and a major South Pacific food plant. The ability of these three unlignified cell-wall preparations to adsorb the hydrophobic environmental mutagen 1,8-dinitropyrene was studied. The greatest adsorption occurred with walls from leaf blade, followed by petiole and corm walls, although the differences were not major. The amount of adsorption was intermediate between the low adsorption previously found with unlignified dicotyledon walls (from the flesh of potato tubers and immature cabbage leaves) and the much higher adsorption found with unlignified walls from monocotyledons of the grass and cereal family (Poaceae) (from leaves of seedling Italian ryegrass). These data are consistent with the monosaccharide compositions of the taro wall preparations, which were more similar to those of unlignified walls of dicotyledons than to unlignified walls of the Poaceae. These findings are consistent with the hypothesis that the composition of the dietary fiber determines its adsorptive properties and that there may be important differences between the major dietary fibers of South Pacific and European food plants.

Use of the ileal conduit as a model for studying human small intestinal mucus glycoprotein secretion.

To establish a human model system for investigating control of mucus secretion by the small intestine, we have studied the output of mucus glycoprotein in the urine of patients with ileal conduits. A highly specific enzyme-linked immunosorbent assay (ELISA) was used to measure mucus glycoprotein with a sensitivity down to 0.3 ng protein of mucus glycoprotein. The assay detects human ileal mucus glycoprotein in the urine of ileal conduit patients; for an individual subject, the amount hardly varied from day to day, although there were substantial differences between individuals in the amounts secreted. Control urine from persons with normal bladders did not contain any mucus glycoprotein that reacted in the ELISA. The model has many advantages over other experimental systems for studying the rate of mucus glycoprotein secretion from the human ileum and can also be used as a reproducible resource of undegraded mucus glycoprotein.