Degradation of pig gastric and colonic mucins by bacteria isolated from the pig colon.

Mucin degradation was studied with one Clostridium (RS42) and two Bacteroides (RS2 and RS13) strains isolated from the pig colon mucosa. Mucins from pig colon and stomach were prepared in their subunit forms for use as growth substrates, and the loss of the individual sugars from the mucins was measured after bacterial growth. Colonic mucin was more resistant to degradation than gastric mucin. The strains differed in their competence in degrading the mucins. Carbohydrate plus sulfate removal from gastric mucin varied from 63 to 76% for RS2, 37 to 46% for RS13, and 37 to 53% for RS42. All three strains removed more fucose (67 to 87%) and less sulfate (22 to 63%) than the average carbohydrate plus sulfate loss. Under the same conditions of growth, a mixed pig fecal culture removed 78% of sulfate and 96% of each sugar. Of the two major glycoprotein types present in the subunit pig gastric mucin preparation (R. A. Stanley, S. P. Lee, and A. M. Roberton, Biochim. Biophys. Acta 760:262-269, 1983), the less highly sulfated mucin was more susceptible to RS42 degradation. The degradation of gastric mucin by RS2 was not affected by glucose or high sulfate concentrations in the growth medium. The results show that the three strains of colon bacteria are capable of significant hydrolysis of mucin carbohydrate and that the extent of degradation seen with pure cultures is determined in part by the subunit glycoprotein type(s) present in the mucin.     

Degradation of pig gastric and colonic mucins by bacteria isolated from the pig colon

Mucin degradation was studied with one Clostridium (RS42) and two Bacteroides (RS2 and RS13) strains isolated from the pig colon mucosa. Mucins from pig colon and stomach were prepared in their subunit forms for use as growth substrates, and the loss of the individual sugars from the mucins was measured after bacterial growth. Colonic mucin was more resistant to degradation than gastric mucin. The strains differed in their competence in degrading the mucins. Carbohydrate plus sulfate removal from gastric mucin varied from 63 to 76% for RS2, 37 to 46% for RS13, and 37 to 53% for RS42. All three strains removed more fucose (67 to 87%) and less sulfate (22 to 63%) than the average carbohydrate plus sulfate loss. Under the same conditions of growth, a mixed pig fecal culture removed 78% of sulfate and 96% of each sugar. Of the two major glycoprotein types present in the subunit pig gastric mucin preparation (R. A. Stanley, S. P. Lee, and A. M. Roberton, Biochim. Biophys. Acta 760:262-269, 1983), the less highly sulfated mucin was more susceptible to RS42 degradation. The degradation of gastric mucin by RS2 was not affected by glucose or high sulfate concentrations in the growth medium. The results show that the three strains of colon bacteria are capable of significant hydrolysis of mucin carbohydrate and that the extent of degradation seen with pure cultures is determined in part by the subunit glycoprotein type(s) present in the mucin.     

Fermentation of mucin and plant polysaccharides by strains of Bacteroides from the human colon.

Ten Bacteroides species found in the human colon were surveyed for their ability to ferment mucins and plant polysaccharides ("dietary fiber"). A number of strains fermented mucopolysaccharides (heparin, hyaluronate, and chondroitin sulfate) and ovomucoid. Only 3 of the 188 strains tested fermented beef submaxillary mucin, and none fermented porcine gastric mucin. Many of the Bacteroides strains tested were also able to ferment a variety of plant polysaccharides, including amylose, dextran, pectin, gum tragacanth, gum guar, larch arabinogalactan, alginate, and laminarin. Some plant polysaccharides such as gum arabic, gum karaya, gum ghatti and fucoidan, were not utilized by any of the strains tested. The ability to utilize mucins and plant polysaccharides varied considerably among the Bacteroides species tested.     

Heterogeneity in gastrointestinal mucins

Pig digestive tract mucins have often been used as model mucins for studying mucin structure, function and metabolism. In the present study pig gastric mucin and pig colonic mucin in the subunit form have been characterised and compared. Following Sepharose 4B or 2B-CL gel chromatography, the mucin eluant fractions were assayed colorimetrically by both the periodic acid-Schiff and the Alcian blue binding assays. Subunit colonic mucin eluted as a single unimodel peak that was easily detected by both assays. In contrast, subunit gastric mucin gave a peak primarily detected by periodic acid-Schiff that was overlapped by, but partially separated from, another peak primarily detected by Alcian blue. Subunit gastric mucin was separated into two periodic acid-Schiff staining spots when electrophoresed on cellulose acetate. Cetylpyridinium chloride (CPC) was able to precipitate only about half the subunit gastric mucin. The CPC-precipitable subunit gastric mucin corresponded to the faster running spot on electrophoresis, and the subunit gastric mucin in the CPC supernatant (which may have more than one subunit mucin type) to the slower spot(s). The former had a higher sulphate content and stained with Alcian blue. The latter had a lower sulphate content and showed very little Alcian blue reactivity. These results indicate that subunit pig gastric mucin is heterogeneous with respect to both size and charge. The differences between the types may be important in biological and physiochemical behaviour of gastric mucin. It seems likely that different laboratories may have worked on one or other of the pig gastric mucin types or a mixture, depending on the preparation method.     

Fermentation of mucins and plant polysaccharides by anaerobic bacteria from the human colon.

A total of 154 strains from 22 species of Bifidobacterium, Peptostreptococcus, Lactobacillus, Ruminococcus, Coprococcus, Eubacterium, and Fusobacterium, which are present in high concentrations in the human colon, were surveyed for their ability to ferment 21 different complex carbohydrates. Plant polysaccharides, including amylose, amylopectin, pectin, polygalacturonate, xylan, laminarin, guar gum, locust bean gum, gum ghatti, gum arabic, and gum tragacanth, were fermented by some strains from Bifidobacterium, Peptostreptococcus, Ruminococcus, and Eubacterium species. Porcine gastric mucin, which was fermented by some strains of Ruminococcus torques and Bifidobacterium bifidum, was the only mucin utilized by any of the strains tested.     

Fermentation of mucins and plant polysaccharides by anaerobic bacteria from the human colon

A total of 154 strains from 22 species of Bifidobacterium, Peptostreptococcus, Lactobacillus, Ruminococcus, Coprococcus, Eubacterium, and Fusobacterium, which are present in high concentrations in the human colon, were surveyed for their ability to ferment 21 different complex carbohydrates. Plant polysaccharides, including amylose, amylopectin, pectin, polygalacturonate, xylan, laminarin, guar gum, locust bean gum, gum ghatti, gum arabic, and gum tragacanth, were fermented by some strains from Bifidobacterium, Peptostreptococcus, Ruminococcus, and Eubacterium species. Porcine gastric mucin, which was fermented by some strains of Ruminococcus torques and Bifidobacterium bifidum, was the only mucin utilized by any of the strains tested.     

Digestion of mucin polysaccharides in vitro by bacteria isolated from the rabbit cecum

The chemical composition of mucin prepared from rabbit small intestines was compared with that of commercial pig gastric mucin. Changes in carbohydrate structure of both mucins after degradation by rabbit cecal bacteria were monitored with the periodic acid-Schiff's reaction (PAS), gas-liquid chromatography, and blood group serology. Out of 220 bacterial isolates from the rabbit cecal microflora, 37 were able to remove more than 25% of PAS-reactive mucin material from pig gastric mucin, which was more easily digested than the rabbit preparation.Bacteroides spp. were most active in mucin digestion, but nonmucinolytic cecal isolates could also use the oligosaccharides likely to be released by this activity.     

New monoclonal antibodies against gastric gland mucous cell-type mucins: a comparative immunohistochemical study

 The immunohistochemical reactivity of monoclonal antibodies raised against rat and pig gastric mucins (HIK1083, PGM36, and PGM37) was investigated in normal gastrointestinal tracts obtained from fish, amphibians, reptiles, birds, and mammals (including humans). These monoclonal antibodies exhibited highly selective reactivity with class III mucins, as identified by paradoxical concanavalin A stain, in the gastrointestinal tract of vertebrates. All three monoclonal antibodies reacted with the mucous neck cells and pyloric gland cells of amphibians, reptiles and mammals, the cardiac glands of reptiles and mammals, and Brunner’s glands of mammls. The deep crypt secretory cells of the rat colon and certain goblet-type cells deep in crypts in the pig colon differed from the above pattern only in that they did not show immunoreactivity with monoclonal antibody PGM36. These data suggest that the development of class III mucin is a fundamental evolutionary characteristic of vertebrate gastric mucins. These monoclonal antibodies should prove useful for the investigation of cell differentiation among gastrointestinal mucous cells and for the biochemical analysis of gastrointestinal mucins in different species. Accepted: 17 February 1998     

Chromatographic measurement of mucin production in cultures of gastric mucous cells.

The use of a miniature column chromatographic assay (using Sepharose CL-4B columns) for measuring mucin production in guinea pig gastric mucous cell cultures is described. The assay was based upon the ability of radiolabelled precursors ([14C]serine and [3H]galactose) to incorporate with high specificity into mucins which thereby appeared in the excluded material. Rates of excluded material radiolabelling by both precursors were constant for incubations up to 24 hours, and substantially reduced by cycloheximide co-incubation (25 microM). Labelled excluded material was completely degraded by mild alkaline borohydride treatment, only partially degraded by HNO2 (pH 1.5), and not degraded by chondroitinase ABC. Thus the major radiolabelled product measured in this system was mucin, although we found that it was less glycosylated than gastric mucins obtained from other sources. In addition, the technique employed to separate and measure mucin production proved rapid and consistent.     

CFTR expression does not influence glycosylation of an epitope-tagged MUC1 mucin in colon carcinoma cell lines

The cause of the mucus clearance problems associated with cystic fibrosis remains poorly understood though it has been suggested that mucin hypersecretion, dehydration of mucins, and biochemical abnormalities in the glycosylation of mucins may be responsible. Since the biochemical and biophysical properties of a mucin are dependent on O-glycosylation, our aim was to evaluate the O-glycosylation of a single mucin gene product in matched pairs of cells that differed with respect to CFTR expression. An epitope-tagged MUC1 mucin cDNA (MUC1F) was used to detect variation in mucin glycosylation in stably transfected colon carcinoma cell lines HT29 and Caco2. The glycosylation of MUC1F mucin was evaluated in matched pairs of Caco2 cell lines that either express wild-type CFTR or have spontaneously lost CFTR expression. The general glycosylation pattern of MUC1F was evaluated by determining its reactivity with a series of monoclonal antibodies against known blood group and tumor-associated carbohydrate antigens. Metabolic labeling experiments were used to estimate the gross levels of glycosylation and sulfation of MUC1F mucin in these matched pairs of cell lines. Expression of CFTR in this experimental system did not affect the gross levels of glycosylation or sulfation of the MUC1F mucin nor the types of carbohydrates structures attached to the MUC1F protein.     

Identification and characterization of a sulfoglycosidase from Bifidobacterium bifidum implicated in mucin glycan utilization

Human gut symbiont bifidobacteria possess carbohydrate-degrading enzymes that act on the O-linked glycans of intestinal mucins to utilize those carbohydrates as carbon sources. However, our knowledge about mucin type O-glycan degradation by bifidobacteria remains fragmentary, especially regarding how they decompose sulfated glycans, which are abundantly found in mucin sugar-chains. Here, we examined the abilities of several Bifidobacterium strains to degrade a sulfated glycan substrate and identified a 6-sulfo-β-d-N-acetylglucosaminidase, also termed sulfoglycosidase, encoded by bbhII from Bifidobacterium bifidum JCM 7004. A recombinant BbhII protein showed a substrate preference toward 6-sulfated and 3,4-disulfated N-acetylglucosamines over non-sulfated and 3-sulfated N-acetylglucosamines. The purified BbhII directly released 6-sulfated N-acetylglucosamine from porcine gastric mucin and the expression of bbhII was moderately induced in the presence of mucin. This de-capping activity may promote utilization of sulfated glycans of mucin by other bacteria including bifidobacteria, thereby establishing the symbiotic relationship between human and gut microbes.     

Cysteine-rich regions of pig gastric mucin contain von willebrand factor and cystine knot domains at the carboxyl terminal(1).

In order to sequence the cysteine-rich regions of pig gastric mucin (PGM), we used our previously identified pig gastric mucin clone PGM-2A to screen a pig stomach cDNA library and perform rapid amplification of cDNA ends to obtain two cysteine-rich clones, PGM-2X and PGM-Z13. PGM-2X has 1071 base pairs (bp) encoding 357 amino acids containing five serine-threonine-rich 16 amino acid tandem repeats, downstream from a cysteine-rich region similar to human and mouse MUC5AC. PGM-Z13 encodes the complete 3'-terminus of PGM and is composed of 3336 bp with a 2964 bp open reading frame encoding 988 amino acids with four serine-threonine-rich tandem repeats upstream from a cysteine-rich region similar to the carboxyl terminal regions of human and rat MUC5AC and human MUC5B. This region is homologous to von Willebrand factor C and D domains involved in acid induced polymerization, and to the carboxyl terminal cystine-knot domain of various mucins, TGF-beta, vWF and norrin, which is involved in dimerization. These newly sequenced cysteine-rich regions of pig gastric mucin may be critical for its gelation and for its observed increased viscosity induced by low pH.     

A monoclonal antibody, PGM34, against 6-sulfated blood-group H type 2 antigen, on the carbohydrate moiety of mucin

Mucin, a major component of mucus, is a highly O-glycosylated, high-molecular-mass glycoprotein extensively involved in the physiology of gastrointestinal mucosa. To detect and characterize mucins derived from site-specific mucous cells, we developed a monoclonal antibody, designated PGM34, by immunizing a mouse with purified pig gastric mucin. The reactivity of PGM34 with mucin was inhibited by periodate treatment of the mucin, but not by trypsin digestion. This suggests that PGM34 recognizes the carbohydrate portion of mucin. To determine the epitope, oligosaccharide-alditols obtained from pig gastric mucin were fractionated by successive gel-filtration, ion-exchange, and normal-phase HPLC, and tested for reactivity with PGM34. Two purified oligosaccharide-alditols that reacted with PGM34 were obtained. Their structures were determined by NMR spectroscopy as Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta1-6(Fucalpha1-2Galbeta1-3)GalNAc-ol and Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta1-6(Galbeta1-3)GalNAc-ol. None of the defucosylated or desulfated forms of these oligosaccharides reacted with PGM34. Thus, the epitope of PGM34 was determined as the Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta- sequence. Immunohistochemical examination of rat gastrointestinal tract showed that PGM34 stained surface mucous cells close to the generative cell zone in the gastric fundus and goblet cells in the small intestine, but only slightly stained antral mucous cells in the stomach. These data, taken together, show that PGM34 is a very useful tool for elucidating the role of mucins with characteristic sulfated oligosaccharides.     

Quantitation of lectin binding sites in human colon mucins by use of peanut and wheat germ agglutinins

We have developed a novel method for quantitation of lectin binding sites in mucins derived from colon tissues. Binding of peanut agglutinin and wheat germ agglutinin was measured in extracts from normal and malignant human colon epithelium. Binding of wheat germ agglutinin was used as an estimate of the total mucin present in the tissue extract. Peanut agglutinin was found to bind to mucin from normal colon, but at levels that may be difficult to appreciate by fluorescence microscopy. The yield of mucin extracted from colon cancer was more variable than that from normal colon, and the binding ratio of peanut agglutinin to wheat germ agglutinin was greater in extracts from tumors than in normal tissues. Our findings confirm the histological observation that peanut agglutinin binds more avidly to mucins from colon cancer than to those from normal colon. The finding of peanut agglutinin binding sites in mucins front normal colon was not expected. The quantitative technique may have detected small numbers of binding sites not readily appreciable by fluorescence microscopy. Alternatively, the chromatographic method for measuring lectin binding may be sufficiently sensitive to detect nonspecific binding of the lectin to terminal galactose residues other than the Thomsen-Friedenreich antigen.     

Campylobacter pyloridis degrades mucin and undermines gastric mucosal integrity

The role of Campylobacter pyloridis, a spiral bacteria associated with gastritis and peptic ulcers in weakening the mucus component of gastric mucosal barrier was investigated. The colonies of bacteria, cultured from antral mucosal biopsies of patients undergoing gastroscopy, were washed with saline, passed through sterilization filter and the filtrate was examined for protease and glycosylhydrolase activities. The obtained results revealed that the filtrate exhibited a strong proteolytic activity not only towards the typical protein substrates such as albumin but also towards gastric mucin. Optimum enzymatic activity for degradation of mucin was attained at pH 7.0 and the protease activity was found in a low m.w. (less than 50K) protein fraction. The filtrate showed little glycosylhydrolase activity and did not cause the hydrolysis of mucin carbohydrates. The data suggest that C pyloridis infection weakens the gastric mucosal defense by causing proteolytic degradation of mucin component of the protective mucus layer.     

Evaluation of bifidobacterial adhesion to acidic sugar chains of porcine colonic mucins

The aim of this study was to assess the adhesion of Bifidobacterium strains to acidic carbohydrate moieties of porcine colonic mucin. Mucins were extracted and purified via gel filtration chromatography followed by density-gradient ultracentrifugation. The presence of sulfated and sialylated carbohydrates in mucins was shown by enzyme-linked immunosorbent assays using PGM34 and HMC31 monoclonal antibodies (mAbs), respectively. Adhesion of Bifidobacterium strains to mucin preparations was markedly affected by the degree of purification. In eight of 22 strains, we observed increased adhesion to mucin preparations purified by ultracentrifugation. Moreover, in some of these eight strains, adhesion to mucin was reduced by pretreatment with sulfatase and/or sialidase, and competitively inhibited by pretreatment with PGM34 and/or HCM31 mAbs. Our results showed that some Bifidobacterium strains adhered to sulfo- and/or sialomucin and were able to recognize carbohydrate structures of the mAbs epitopes.     

Changes in colonic mucins of germfree rats in response to the introduction of a "normal" rat microbial flora. Rat colonic mucin.

In order to determine the influence of bacterial colonization on amount and composition of colonic mucins, germfree male AS/Ztm rats were colonized with a rat specific intestinal flora for different times (2, 7, 14, 21, 28, 35, 120 days). The amount of colonic mucins was determined by gel filtration on Sepharose CL-4B; the relative amount of acidic mucins was calculated after ion exchange chromatography. In addition, cecal weight and dry matter of feces were monitored. While germfree and SPF rats revealed similar amounts of colonic mucins (7.0 vs. 7.2 mg mucin/300 g body weight), the initial phase of association was characterized by considerably decreasing values. After four weeks of association, the total amount of colonic mucins had almost equalized in the two groups. The amount of acidic mucins, having decreased during the first three weeks of colonization, rendered values comparable to the SPF mucins after four months of adaptation. Cecomegaly in germfree rats disappeared within the first two days, while solidification of the intestinal content occurred within four months. Mucin losses during initial phase of association are attributed 1. to the disappearance of the cecal mucin pool, and 2. to the mucin degrading activity of some bacterial strains known to be present in the intestinal flora. Further development is conducted by a stimulation of mucin secretion, described to follow the colonization. The initially increased secretion of neutral mucins is attributed to a pronounced release of immature mucin glycoproteins, while the shift to more acidic mucins is considered to result from stimulated secretion as well as from a selective bacterial degradation of neutral mucin components.     

Enhanced self-association of mucins possessing the T and Tn carbohydrate cancer antigens at the single-molecule level

Mucins are linear O-glycosylated glycoproteins involved in inflammation, cell adhesion, and tumorigenesis. Cancer-associated mucins often possess increased expression of the T (Galβ1,3GalNAcαThr/Ser) and Tn (GalNAcαThr/Ser) cancer antigens, which are diagnostic markers for several cancers, including colon cancer. We have used AFM based single-molecule forced unbinding under near physiological conditions to investigate the self-interactions between porcine submaxillary mucin (PSM) as well as between PSM analogs possessing various carbohydrates including the T- and Tn-antigen. Distributions of unbinding forces and corresponding force loading rates were determined for force loading rates from 0.18 nN/s to 39 nN/s, and processed to yield most probable unbinding forces f* and lifetimes of the interactions. Parameter f* varied in the range 27 to 50 pN at force loading rates of about 2 nN/s among the various mucins. All mucin samples investigated showed self-interaction, but the tendency was greatest for PSM displaying only the Tn-antigen (Tn-PSM) or a mixture of Tn-, T-antigen, and the trisaccharide Fucα1,2Galβ1,3GalNAc (Tri-PSM). Weaker self-interactions were observed for native PSM (Fd-PSM), which consists of a nearly equal mixture of the longer core 1 blood group A tetrasaccharide (GalNAcα1,3(Fucα1,2)Galβ1,3GalNAcαSer/Thr) and Tn-antigen. The data are consistent with the truncated Tn and T glycans enhancing self-interaction of the mucins. These carbohydrate cancer antigens may, thus, play an active role in the disease by constitutively activating mucin and mucin-type receptors by self-association on cells.     

Co-localization of TFF2 with gland mucous cell mucin in gastric mucous cells and in extracellular mucous gel adherent to normal and damaged gastric mucosa

Trefoil factor 2 (TFF2) is mucin associated peptide that has a mucosal barrier function in addition to participating in repair and healing. We examined the localization of TFF2 and gastric mucins in gastric mucous cells, the surface mucous gel layer (SMGL) adherent to normal gastric mucosa, and in the mucoid cap covering gastric erosions. Carnoy’s solution, or formalin/picric acid-fixed paraffin embedded materials from resected stomachs and formalin-fixed paraffin embedded gastric biopsy materials were used. Sections were immunostained for the TFF2 and histochemically stained for gastric mucins. In addition, thick sectioned gastric mucosa fixed in Carnoy’s solution were stained with FITC-labeled GSA-II lectin specific for gland mucous cell mucin and examined for three-dimensional images of the SMGL using a confocal laser scanning microscope. The TFF2 and gland mucous cell mucin were found intermixed together in the gastric gland mucous cells, in the SMGL in laminated layers, and in the mucoid cap. A laminated arrangement of continuous sheets of gland mucous cell mucin in the SMGL was demonstrated in the three-dimensional images. Co-localization of the TFF2 with gland mucous cell mucin suggests a physical interaction between the TFF2 and gland mucous cell mucin. The TFF2 trapped in the adherent mucins may be responsible for mucosal defense, healing, and repair.     

Tumor cell MUC1 and CD43 are glycosylated differently with sialyl-Lewis a and x epitopes and show variable interactions with E-selectin under physiological flow conditions

The mucins secreted from the colon carcinoma cell line COLO 205 have the MUC1 and CD43 (leukosialin) as core proteins, where both carry sialyl-Lewis a and MUC1 sialyl-Lewis x epitopes. The adhesion of E-selectin expressing CHO cells to the coated mucins was analyzed in a flow system revealing that the MUC1 mucin adhered better than the CD43 mucin. One reason could be their different glycosylation, a difference that was explored by analyzing the biosynthesis of MUC1 and CD43 in COLO 205 cells. Both the MUC1 and CD43 mucins became sialyl-Lewis a reactive, but after different times as revealed by pulse-chase studies. However, only MUC1 became sialyl-Lewis x reactive. These differences suggest that MUC1 and CD43 are synthesized in different compartments of the cell. It was also observed that the mucins from colon carcinoma patients had MUC1-type mucins that carried both sialyl-Lewis a and x epitopes and CD43-type sialyl-Lewis a mucins with only low levels of sialyl-Lewis x epitopes. One could hypothesize that colon carcinoma derived MUC1 is decorated with potent E-selectin epitopes, and that this could be one of several reasons for the involvement of MUC1 in cancer development.