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.     

Glycosylation of human fetal mucins: a similar repertoire of <Emphasis Type="Italic">O</Emphasis>-glycans along the intestinal tract

Intestinal mucins are very high molecular weight glycoproteins secreted by goblet cells lining the crypt and the surface of the colonic mucosa. Profound alterations of mucin O-glycans are observed in diseases such as cancer and inflammation, modifying the function of the cell and its antigenic and adhesive properties. Based on immunohistochemical studies, certain cancer- and inflammation- associated glycans have been defined as oncofetal antigens. However, little or no chemical analysis has allowed the structural elucidation of O-glycans expressed on human fetal mucins. In this paper, mucins were isolated from different regions of the normal human intestine (ileum, right, transverse and left colon) of eight fetuses with A, B or O blood group. After alkaline borohydride treatment, the released oligosaccharides were investigated by nanoESI Q-TOF MS/MS (electrospray ionization quadrupole time-of-flight tandem mass spectrometry). More than 117 different glycans were identified, mainly based on core 2 structures. Some core 1, 3 and 4 oligosaccharides were also found. Most of the structures were acidic with NeuAc residues mainly α2–6 linked to the N-acetylgalactosaminitol and sulphate residues 3-linked to galactose or 6-linked to GlcNAc. In contrast to adult human intestinal mucins, Sda/Cad determinants were not expressed on fetal mucin O-glycans and the presence of an acidic gradient along the intestinal tract was not observed. Similar patterns of glycosylation were found in each part of the intestine and the level of expression of the major oligosaccharides was in the same order of magnitude. This study could help determining new oncofetal antigens, which can be exploited for the diagnosis or the treatment of intestinal diseases.     

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.     

The broad diversity of neutral and sialylated oligosaccharides derived from human salivary mucins.

Mucin glycopeptides were prepared from the salivary mucins of 20 healthy donors with blood group O. The carbohydrate chains of the high-molecular-weight mucins were released by alkaline borohydride treatment. Neutral and monosialylated oligosaccharide-alditols were purified by ion-exchange chromatography, gel filtration, and HPLC. The structures of the oligosaccharide-alditols were determined by high-resolution 1H-NMR spectroscopy in combination with fast atom bombardment mass spectrometry and methylation analysis. Thirty-seven oligosaccharide-alditols were characterized and illustrate the extreme diversity of the salivary mucins carbohydrate chains. This diversity might represent a mosaic of bacterial adhesion sites and be involved in the early events of the nonimmune defense of the oral cavity. Among these 37 oligosaccharide-alditols, 31 have not been previously described in human saliva and five of these are novel structures: [formula: see text]     

Behavior of aldehyde moieties involved in the activation of suppressor cells by sodium periodate

The treatment of mouse spleen cells with periodate at the optimal mitogenic concentration (1 mM) induces the activation of suppressor cells of the in vitro antibody response and leads to the formation of aldehydes on the carbohydrate termini of the surface sialoglycoconjugates. These aldehyde moieties are found on the C8 (N-AN 8) and the C7 (N-AN 7) derivatives of sialic acid. Immediate borohydride reduction prevents the activation of the suppressor cells. Data from this work show that borohydride reduction must be performed within the first 6 hr to prevent the generation of suppressor cells; 18 hr after the initial periodate oxidation, borohydride treatment did not reverse the in vitro suppressive activity of periodate-treated cells. The kinetics of the disappearance of aldehydes from the cell surface were studied by using [3H]borohydride labeling and chromatographic analysis of sialic acid derivatives. About 70 to 80% of the aldehyde moieties were found to be present 6 hr after periodate oxidation. After 18 hr, 50 to 70% of the aldehyde had disappeared from the lymphocyte membrane. Oxidized sialyl residues disappear completely after 60 hr of culture. This period corresponds to the de novo synthesis of sialic acid residues on the surface of periodate-activated cells. The two classes of oxidized sialyl-glycoconjugates were found to behave in different ways. In effect, our data showed that the aldehydes remaining at 18 hr are mainly located on the gangliosides, whereas the aldehyde moieties located on high m.w. glycoproteins disappear from the cell surface between 9 and 18 hr. This would suggest that the remaining aldehydes located on gangliosides are not directly involved in the expression of suppressive activity.     

Analysis of serine/threonine-linked oligosaccharides derived by alkaline-borohydride treatment of mucin glycoproteins electroblotted onto membranes: comparison of the saccharide profiles of the 390 kDa and 350 kDa forms of epitectin

Alkaline borohydride treatment is widely used for the release of carbohydrate moieties from O-glycosylated glycoproteins and mucins. We have adapted this procedure to micro quantities of glycoproteins blotted on membranes. After electrophoresis and transfer to nitrocellulose, nylon or polyvinylidene difluoride membrane, alkaline borohydride treatment was done directly on glycoprotein containing areas of membrane which were cut out with the aid of guide strips stained with Coomassie Blue or lectin-digoxigenin. In combination with standard saccharide fractionation techniques, this procedure can be used to characterize the oligosaccharides of mucins or mucin-type glycoproteins that are separated by gel electrophoresis from crude sources. Using this approach we have characterized the saccharides derived from the two species of epitectin, a malignancy-associated mucin type glycoprotein, isolated from metabolically labelled H.Ep2 cells.     

Binding of Shigella to rat and human intestinal mucin

Invasion of epithelial cells by Shigella is an early step in their pathogenesis. Adherence is generally presumed to be a prerequisite for invasion. This study examined the possibility of intestinal mucins serving as initial binding sites for clinical isolates of S. boydii and S. sonnei. The interactions of Shigella with rat and human small intestinal and colonic mucin were investigated. In solid phase binding assays, [³⁵S] labelled Shigella did not show any preferential binding to rat/human small intestinal mucin or to rat colonic mucin. On the other hand, Shigella bound specifically to human colonic mucin in a concentration-dependent manner. This specific binding to human colonic mucin was not by weak hydrophobic interactions and could not be attributed to the presence of contaminating glycolipids in the mucin preparation. The human colonic mucin receptor was sensitive to periodate treatment suggesting the involvement of the carbohydrate portion of the mucin. Reduction and alkylation of mucin enhanced adherence probably by exposing buried binding sites. The monosaccharides present in mucins were ineffective as hapten inhibitors as was the lectin wheat germ agglutinin suggesting that the mucin receptor is a more complex one. This study identifies, for the first time, the presence of a specific Shigella-binding site on the carbohydrate portion of human colonic mucin, which is not present in rat colonic mucin or in rat/human small intestinal mucin.     

Butyrate enemas upregulate Muc genes expression but decrease adherent mucus thickness in mice colon

Colonic mucosal protection is provided by the mucus gel, mainly composed of mucins. Several factors can modulate the formation and the secretion of mucins, and among them butyrate, an end-product of carbohydrate fermentation. However, the specific effect of butyrate on the various colonic mucins, and the consequences in terms of the mucus layer thickness are not known. Our aim was to determine whether butyrate modulates colonic MUC genes expression in vivo and whether this results in changes in mucus synthesis and mucus layer thickness. Mice received daily for 7 days rectal enemas of butyrate (100 mM) versus saline. We demonstrated that butyrate stimulated the gene expression of both secreted (Muc2) and membrane-linked (Muc1, Muc3, Muc4) mucins. Butyrate especially induced a 6-fold increase in Muc2 gene expression in proximal colon. However, butyrate enemas did not modify the number of epithelial cells containing the protein Muc2, and caused a 2-fold decrease in the thickness of adherent mucus layer. Further studies should help understanding whether this last phenomenon, i.e. the decrease in adherent mucus gel thickness, results in a diminished protective function or not.     

Black Periodic and Black Bauer Methods for Tissue Polysaccharides

m-Aminophenol gives a more rapid and more complete blockage of the Schiff reaction of aldehydes produced in tissue by oxidation with periodic or chromic acid than does aniline. Moreover, the phenol thus attached to aldehyde sites in tissue can then be azo coupled with alkaline solutions of fresh or stabilized diazonium salts. When so used at pH 8, fast black K, C.I. 37190, shows glycogen, mucins, basement membranes, muscle stroma, fungus cell walls, etc. stained black, on a somewhat grayish pink to red background.     

Black Periodic and Black Bauer Methods for Tissue Polysaccharides

m-Aminophenol gives a more rapid and more complete blockage of the Schiff reaction of aldehydes produced in tissue by oxidation with periodic or chromic acid than does aniline. Moreover, the phenol thus attached to aldehyde sites in tissue can then be azo coupled with alkaline solutions of fresh or stabilized diazonium salts. When so used at pH 8, fast black K, C.I. 37190, shows glycogen, mucins, basement membranes, muscle stroma, fungus cell walls, etc. stained black, on a somewhat grayish pink to red background.     

Rapid and simple single nanogram detection of glycoproteins in polyacrylamide gels and on electroblots

The fluorescent hydrazide, Pro-Q Emerald 300 dye, may be conjugated to glycoproteins by a periodic acid Schiff's (PAS) mechanism. The glycols present in glycoproteins are initially oxidized to aldehydes using periodic acid. The dye then reacts with the aldehydes to generate a highly fluorescent conjugate. Reduction with sodium metabisulfite or sodium borohydride is not required to stabilize the conjugate. Though glycoprotein detection may be performed on transfer membranes, direct detection in gels avoids electroblotting and glycoproteins may be visualized within 2-4 h of electrophoresis. This is substantially more rapid than PAS labeling with digoxigenin hydrazide followed by detection with an antidigoxigenin antibody conjugate of alkaline phosphatase, or PAS labeling with biotin hydrazide followed by detection with horseradish peroxidase or alkaline phosphatase conjugates of streptavidin, which require more than eight hours to complete. Pro-Q Emerald 300 dye-labeled gels and blots may be poststained with SYPRO Ruby dye, allowing sequential two-color detection of glycosylated and nonglycosylated proteins. Both fluorophores are excited with mid-range UV illumination. Pro-Q Emerald 300 dye maximally emits at 530 nm (green) while SYPRO Ruby dye maximally emits at 610 nm (red). As little as 300 pg of alpha 1-acid glycoprotein (40% carbohydrate) and 1 ng of glucose oxidase (12% carbohydrate) or avidin (7% carbohydrate) are detectable in gels after staining with Pro-Q Emerald 300 dye. Besides glycoproteins, as little as 2-4 ng of lipopolysaccharide is detectable in gels using Pro-Q Emerald 300 dye while 250-1000 ng is required for detection with conventional silver staining. Detection of glycoproteins may be achieved in sodium dodecyl sulfate-polyacrylamide gels, two-dimensional gels and on polyvinylidene difluoride membranes.     

The use of a transesterification technique to distinguish between certain neuraminidase resistant epithelial mucins.

The effects of potassium hydroxide and soidum methoxide treatments upon the Alcian blue staining and neuraminidase lability of certain neuraminidase resistant epithelial mucins have been studied. The results were interpreted as indicating that while the mucins of rat colon and rabbit Brunner's gland contain only 4-0-acetyl sialic acid, human colonic epithelial mucins may contain some sialic acid with esters at the C1 carboxyl group.     

Monoclonal antibodies directed against the galactose-binding lectin of Entamoeba histolytica enhance adherence.

The Entamoeba histolytica galactose-binding lectin is a surface glycoprotein composed of 170- and 35-kDa subunits. Inhibition of this lectin with galactose or anti-170 kDa subunit polyclonal antibody blocks amebic adherence to target cells and colonic mucin glycoproteins. We describe the properties of 10 mAb with specificity for the 170-kDa subunit. Based on competitive binding studies, six nonoverlapping antigenic determinants on the lectin were identified. The effect of the mAb on adherence of amebic trophozoites to both Chinese hamster ovary (CHO) cells and human colonic mucins was measured. Antilectin antibodies directed against epitopes 1 and 2 enhanced adherence, with the number of amebae having at least three adherent CHO cells increasing with the addition of epitope 1 mAb from 26 +/- 9 to 88 +/- 2% and the binding of colonic mucins increasing from 34 +/- 1 to 164 +/- 3 pg/10(5) amebae. Antibody-enhanced adherence remained 90 to 100% galactose inhibitable, occurred at 4 degrees C and was not Fc mediated. Univalent Fab fragments of epitope 1 mAb augmented mucin binding by 238% and CHO cell adherence by 338%. The binding of purified lectin to CHO cells was increased from 1.1 +/- 0.1 to 2.4 +/- 0.3 ng/10(3) CHO cells by mAb directed to epitope 1, demonstrating that enhanced adherence was due to direct activation of the lectin. mAb to epitope 3 bound to the lectin only upon its solubilization from the membrane and had no effect on adherence. Adherence to CHO cells and mucins was inhibited from 50 to 75% by mAb to epitopes 4 and 5; epitope 6 mAb inhibited amebic adherence to CHO cells but not mucins. The pooled sera from 10 patients with amebic liver abscess blocked the binding to the 170-kDa subunit of mAb directed to all six epitopes. Striking individual variations in the effects of immune sera on adherence were observed. Although the sera of all 44 South African patients with amebic liver abscess had high titer anti-lectin antibodies, 16 patients' sera significantly (more than 3 SEM) enhanced adherence whereas 25 patients' sera significantly inhibited adherence. Antilectin antibodies exert profound functional effects on the interaction of E. histolytica with target cells and human colonic mucins. Exploration of the clinical consequences of adherence-enhancing and inhibitory antibody responses may give insight into the role of antilectin antibodies in immunity to invasive amebiasis.     

High performance anion exchange chromatography of reduced oligosaccharides from sialomucins

High performance anion exchange chromatography on pellicular ion exchange resins under high pH conditions with detection of sugars using a pulsed amperometric detector has been developed as a method for the separation and analysis of reduced oligosaccharides liberated from mucins by alkaline borohydride treatment. Ovine, bovine and porcine submaxillary mucins were used as models to develop the method. Although neutral reduced di-to tetraoligosaccharides were poorly retained on the column, a variety of sialylated reduced oligosaccharides could be separated efficiently. Treatment of the samples with sialidase and rechromatography identified the sialylated compounds in the elution profile. A striking finding was the greatly delayed elution times given byN-glycolylneuraminic acid containing compounds in comparison with the correspondingN-acetylneuraminic acid containing analogues. The elution profiles for the product from the mucins closely corresponded to those expected for the major oligosaccharides from these mucins. The procedures described will be useful for analysing sialomucins on a microscale without resorting to radiolabelling procedures.     

Reactivity of mucin-specific lectin from Sambucus sieboldiana with simple sugars, normal mucins and tumor-associated mucins. Comparison with other lectins.

Mucin-specific lectin from Sambucus sieboldiana (SSA-M) reacts in Western blotting and ELISA with mucins from porcine stomach, bovine and ovine submaxillary glands, the human milk fat globule membrane, in vitro human ovarian, breast and colonic tumor cell lines, and mucins produced in vivo in the ascites of patients with endometrial and ovarian tumors, but not with fetal bovine fetuin or human transferrin. Sialidase treatment of these mucins led to an increase in the binding of SSA-M, suggesting that sialic acid is not part of the binding site for this lectin. Furthermore, sialic acid did not inhibit lectin binding. Treatment of asialomucin with O-glycanase decreased the binding of SSA-M, confirming the reactivity of the lectin with an O-linked carbohydrate. Treatment of mucins with trifluoromethanesulfonic acid, which removes all but core carbohydrate, led to an increase in the binding of SSA-M, suggesting that the lectin reacts with O-linked core glycans. Indeed, the increased reactivity after sialidase treatment of ovine submaxillary mucin suggests the lectin reacts with peptide-linked N-acetylgalactosamine (GalNAc), since more than 98% of the glycan chains attached to this mucin are sialylated GalNAc. The binding of SSA-M to sialidase-treated porcine mucin was inhibited strongly by GalNAc and disaccharides containing galactose (lactose, melibiose, and N-acetyllactosamine) but not by free galactose (Gal), suggesting that the glycan for optimum binding is Gal beta(1-3)GalNAc. This pattern of inhibition was different to other core glycan-reactive lectins tested, indicating that SSA-M is distinct, and should be of use in the isolation and characterisation of mucins and O-linked glycans.     

Sulfation of Colonic Mucins by N-Acetylglucosamine 6-O-Sulfotransferase-2 and Its Protective Function in Experimental Colitis in Mice

N-Acetylglucosamine 6-O-sulfotransferase-2 (GlcNAc6ST-2) catalyzes the sulfation of mucin-like glycoproteins, which function as ligands for a lymphocyte homing receptor, L-selectin, in the lymph node high endothelial venules (HEVs). We previously showed that GlcNAc6ST-2 is expressed not only in lymph node HEVs but also in the colonic epithelial cells in mice. Here we investigated the regulatory mechanism and physiological significance of colonic expression of GlcNAc6ST-2 in mice. Treatment of a mouse colonic epithelial cell line with butyrate, a short-chain fatty acid produced by anaerobic bacteria, induced GlcNAc6ST-2 expression in the presence of epidermal growth factor. Administration of butyrate in the drinking water stimulated GlcNAc6ST-2 expression in the mouse intestine, indicating that butyrate could serve as a regulatory molecule for the GlcNAc6ST-2 expression in vivo. Immunohistochemical analysis indicated that the sulfation of colonic mucins was greatly diminished in GlcNAc6ST-2-deficient mice. Liquid chromatography coupled to electrospray ionization tandem mass spectrometry of the colonic-mucin O-glycans from wild-type and GlcNAc6ST-2-deficient mice showed that GlcNAc-6-O-sulfation was the predominant sulfate modification of these mucins, and it was exclusively mediated by GlcNAc6ST-2. After colitis induction by dextran sulfate sodium, significantly more leukocyte infiltration was observed in the colon of GlcNAc6ST-2-deficient mice than in that of wild-type mice, indicating that the sulfation of colonic mucins by GlcNAc6ST-2 has a protective function in experimental colitis. These findings indicate that GlcNAc6ST-2, whose expression is regulated by butyrate, is a major sulfotransferase in the biosynthesis of sulfomucins in the mouse colon, where they serve as a mucosal barrier against colonic inflammation.     

Mucin-carbohydrate directed monoclonal antibody

To raise monoclonal antibodies recognizing cancer-associated alterations of the carbohydrate structure of glycoproteins, Balb/c mice were immunized with human colonic cancer cells (LS 180 from ATCC). One of the generated hybridomas produced a monoclonal antibody that bound to the carbohydrate moiety of mucin-type glycoproteins from LS 180. The antibody did not bind to glycoproteins from another colonic cancer cell line, SW 1116, or to glycolipids from any of the colonic cancer cell lines. The antibody bound to ovine and bovine submaxillary mucins (OSM and BSM). NeuAc alpha 2----6Ga1NAc seemed to be involved in the epitope.     

The use of a transesterification technique to distinguish between certain neuraminidase resistant epithelial mucins

Summary The effects of potassium hydroxide and sodium methoxide treatments upon the Alcian blue staining and neuraminidase lability of certain neuraminidase resistant epithelial mucins have been studied. The results were interpreted as indicating that while the mucins of rat colon and rabbit Brunner's gland contain only 4-O-acetyl sialic acid, human colonic epithelial mucins may contain some sialic acid with esters at the C₁ carboxyl group. Supported by the Medical Research Council of Canada Grant MA 4376.     

Functional Heterogeneity of Colonic Adenocarcinoma Mucins for Inhibition of Entamoeba histolytica Adherence to Target Cells1

Mucins secreted from the gastrointestinal epithelium form the basis of the adherent mucus layer which is the host's first line of defense against invasion by Entamoeba histolytica. Galactose and N-acetyl-D-galactosamine residues of mucins specifically inhibit binding of the amebic 170 kDa heavy subunit Gal-lectin to target cells, an absolute prerequisite for pathogenesis. Herein we characterized the secretory mucins isolated from the human colon and from three human colonic adenocarcinoma cell lines: two with goblet cell-like (LS174T and T84) and one with absorptive cell-like morphology (Caco-2). By Northern blot analysis the intestinal mucin genes MUC2 and MUC3 were constitutively expressed by confluent LS174T and Caco-2 cells, whereas T84 cells only transcribed MUC2 and not MUC3 mRNA. ³H-glucosamine and ³H-threonine metabolically labeled proteins separated as high M_r mucins in the void (V_o > 10⁶ Da) of Sepharose-4B column chromatography and remained in the stacking gel of SDS-PAGE as depicted by fluorography. All mucin preparations contained high amounts of N-acetyl-glucosamine, galactose, N-acetyl-galactosamine, fucose and sialic acid, saccharides typical of the O-linked carbohydrate side chains. Mucin samples from the human colon and from LS174T and Caco-2 cells inhibited E. histolytica adherence to Chinese hamster ovary cells, whereas mucins from T84 cells did not. These results suggest that genetic heterogeneity and/or posttranslational modification in glycosylation of colonic mucins can affect specific epithelial barrier function against intestinal pathogens.     

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.