Mucus and mucins in diseases of the intestinal and respiratory tracts.

This review describes the organization and importance of mucus in the intestine and lungs in relation to the diseases cystic fibrosis (CF), ulcerative colitis and chronic obstructive pulmonary disease (COPD). The inner surfaces of the body are protected by mucus built around polymeric glycoproteins called mucins. In the disease CF, the small intestinal mucus is in contrast the normal attached to the epithelium, explaining the intestinal problems at this disease. The inner of the two mucus layers of colon is normally impenetrable to bacteria, keeping the commensals away from and protecting the epithelium. This impenetrable property is dependent on the bacterial composition and the host diet, observations that can explain the increased incidence of inflammatory bowel diseases in the western world as bacteria reach the epithelial cells in active ulcerative colitis. The respiratory tract is normally cleared by thick mucus bundles that moved by the cilia sweep the epithelial surface. In CF, the bundles are nonmoving already at birth. Cholinergic stimulations stop the bundle movement explaining some of the beneficial effect of anticholinergic treatment in COPD. In this disease as well as in more developed CF, an attached mucus layer is formed. This mucus has features similar to the protective inner colon mucus and is by this able to separate bacteria from the epithelial surface. When formed in healthy individuals this mucus can be coughed up, but in chronically diseased lungs, bacteria colonizing the mucus will remain in the lungs and the resulting inflammation contribute to the destruction of the lungs.

Functional mucous layer and healing of proximal colonic anastomoses in an experimental model.

BACKGROUND: Anastomotic leakage (AL) is the most dreaded complication after colorectal surgery, causing high morbidity and mortality. Mucus is a first line of defence against external factors in the gastrointestinal tract. In this study, the structural mucus protein Muc2 was depleted in genetically engineered mice and the effect on healing of colonic anastomoses studied in an experimental model. METHODS: Mice of different Muc2 genotypes were used in a proximal colonic AL model. Tissues were scored histologically for inflammation, bacterial translocation was determined by quantitative PCR of bacterial 16S ribosomal DNA, and epithelial cell damage was determined by assessing serum levels of intestinal fatty acid-binding protein. RESULTS: Of 22 Muc2-deficient (Muc2-/- ) mice, 20 developed AL, compared with seven of 22 control animals (P < 0·001). Control mice showed normal healing, whereas Muc2-/- mice had more inflammation with less collagen deposition and neoangiogenesis. A tendency towards higher bacterial translocation was seen in mesenteric lymph nodes and spleen in Muc2-/- mice. Intestinal fatty acid-binding protein levels were significantly higher in Muc2-/- mice compared with controls (P = 0·011). CONCLUSION: A functional mucous layer facilitates the healing of colonic anastomoses. Clinical relevance Colorectal anastomotic leakage remains the most dreaded complication after colorectal surgery. It is known that the aetiology of anastomotic leakage is multifactorial, and a role is suggested for the interaction between intraluminal content and mucosa. In this murine model of proximal colonic anastomotic leakage, the authors investigated the mucous layer at the intestinal mucosa, as the first line of defence, and found that a normal, functioning mucous layer is essential in the healing process of colonic anastomoses. Further research on anastomotic healing should focus on positively influencing the mucous layer to promote better postoperative recovery.

Importance and regulation of the colonic mucus barrier in a mouse model of colitis.

The colonic mucus layer serves as an important barrier and prevents colonic bacteria from invading the mucosa and cause inflammation. The regulation of colonic mucus secretion is poorly understood. The aim of this study was to investigate the role of the mucus barrier in induction of colitis. Furthermore, regulation of mucus secretion by luminal bacterial products was studied. The colon of anesthetized Muc2(-/-), Muc1(-/-), wild-type (wt), and germ-free mice was exteriorized, the mucosal surface was visualized, and mucus thickness was measured with micropipettes. Colitis was induced by DSS (dextran sodium sulfate, 3%, in drinking water), and disease activity index (DAI) was assessed daily. The colonic mucosa of germ-free and conventionally housed mice was exposed to the bacterial products LPS (lipopolysaccharide) and PGN (peptidoglycan). After DSS induction of colitis, the thickness of the firmly adherent mucus layer was significantly thinner after 5 days and onward, which paralleled the increment of DAI. Muc2(-/-) mice, which lacked firmly adherent mucus, were predisposed to colitis, whereas Muc1(-/-) mice were protected with significantly lower DAI by DSS compared with wt mice. The mucus barrier increased in Muc1(-/-) mice in response to DSS, whereas significantly fewer T cells were recruited to the inflamed colon. Mice housed under germ-free conditions had an extremely thin adherent colonic mucus layer, but when exposed to bacterial products (PGN or LPS) the thickness of the adherent mucus layer was quickly restored to levels observed in conventionally housed mice. This study demonstrates a correlation between decreasing mucus barrier and increasing clinical symptoms during onset of colitis. Mice lacking colonic mucus (Muc2(-/-)) were hypersensitive to DSS-induced colitis, whereas Muc1(-/-) were protected, probably through the ability to increase the mucus barrier but also by decreased T cell recruitment to the afflicted site. Furthermore, the ability of bacteria to regulate the thickness of the colonic mucus was demonstrated.

Receptor analogs and monoclonal antibodies that inhibit adherence of Bordetella pertussis to human ciliated respiratory epithelial cells.

The adherence of Bordetella pertussis to human respiratory cilia is critical to the pathogenesis of whooping cough. To explore the development of agents that could interrupt adherence, the structure of the receptor on the ciliary surface was investigated. Using an in vitro adherence assay to human ciliated epithelial cells, galactose, lactose, and complex carbohydrates containing lactose eliminated adherence when preincubated with the bacteria. 10(-2) M galactose eluted adherent bacteria from cilia. B. pertussis and its two purified adhesins bound specifically to natural lactose-containing glycolipids in a TLC assay. mAbs to eukaryotic glycoconjugates with specificity for substituted galactose-glucose moieties blocked adherence when preincubated with ciliated cells. The carbohydrates that serve as receptors for B. pertussis on human cilia are galactose-glucose-containing glycolipids. Receptor analogs and anti-receptor antibodies effectively block adherence of B. pertussis to cilia and thus should be considered candidates for therapeutic intervention against disease.

Core 1- and 3-derived O-glycans collectively maintain the colonic mucus barrier and protect against spontaneous colitis in mice.

Core 1- and 3-derived mucin-type O-glycans are primary components of the mucus layer in the colon. Reduced mucus thickness and impaired O-glycosylation are observed in human ulcerative colitis. However, how both types of O-glycans maintain mucus barrier function in the colon is unclear. We found that C1galt1 expression, which synthesizes core 1 O-glycans, was detected throughout the colon, whereas C3GnT, which controls core 3 O-glycan formation, was most highly expressed in the proximal colon. Consistent with this, mice lacking intestinal core 1-derived O-glycans (IEC C1galt1-/-) developed spontaneous colitis primarily in the distal colon, whereas mice lacking both intestinal core 1- and 3-derived O-glycans (DKO) developed spontaneous colitis in both the distal and proximal colon. DKO mice showed an early onset and more severe colitis than IEC C1galt1-/- mice. Antibiotic treatment restored the mucus layer and attenuated colitis in DKO mice. Mucins from DKO mice were more susceptible to proteolysis than wild-type mucins. This study indicates that core 1- and 3-derived O-glycans collectively contribute to the mucus barrier by protecting it from bacterial protease degradation and suggests new therapeutic targets to promote mucus barrier function in colitis patients.

Discrimination of MUC1 mucins from other sialyl-Le(a)-carrying glycoproteins produced by colon carcinoma cells using a novel monoclonal antibody.

Earlier studies (Baeckström et al., J. Biol. Chem., 266: 21537-21547, 1991) have revealed that the colon carcinoma cell line COLO 205 produces two different proteins carrying the carcinoma-associated sialyl-Le(a) carbohydrate epitope. One is the MUC1 mucin apoprotein, and the other protein is smaller and has not been characterized in detail. This paper describes a comparison of COLO 205 with three other colon carcinoma cell lines, aided by the use of a novel MUC1-reactive monoclonal antibody, Ma552. Ma552 reacted with H-CanAg, the MUC1 mucin purified from COLO 205, the binding increasing greatly upon partial deglycosylation of H-CanAg with trifluoromethanesulfonic acid. This pattern of reactivity was in contrast with that of the MUC1-reactive monoclonal antibody HMFG-2, which did not recognize H-CanAg without prior deglycosylation. The nature of the epitope of Ma552 was further investigated using a synthetic peptide corresponding to the tandem-repeat sequence of the MUC1 protein. When the peptide was used as an inhibitor of antibody binding to immobilized, partially deglycosylated H-CanAg mucin, Ma552 was inhibited, as was HMFG-2. Using short, immobilized synthetic peptides identical to parts of the MUC1 tandem repeat, the reactivity of Ma552 was mapped to the hexapeptide TRPAPG. Ma552 and C50, a monoclonal antibody reactive with sialyl-Le(a), were used in immunofluorometric assays to analyze gel filtration fractions of extracts and spent media from the colorectal carcinoma cell lines COLO 205, SW1116, LoVo, and LS 174T. Using C50 in a homologous assay, all sialyl-Le(a)-carrying glycoproteins were detected. Among these, mucins based on the MUC1 apoprotein were identified using Ma552 and C50 in a combination assay. The results showed that sialyl-Le(a) was present on more than one glycoprotein not only in COLO 205 but also in SW1116 and LoVo. The Ma552/Eu-C50 assay revealed the presence of sialyl-Le(a)-carrying MUC1 in COLO 205 as expected, as well as in SW1116. The presence of MUC1 in Ma552-reactive fractions was confirmed by deglycosylation followed by assaying with the monoclonal antibody HMFG-2. Furthermore, Northern blots revealed the presence of MUC1 mRNA only in the two Ma552-positive cell lines.

A blood group B-like pentaglycosylceramide is the major complex glycosphingolipid of the Madin-Darby canine kidney epithelial cell line I (MDCK I).

Two sublines of the epithelial cell line MDCK differ in glycosphingolipid composition (Hansson, G.C. et al. (1986) EMBO J. 5, 483-489). The Forssman pentaglycosylceramide was an abundant glycolipid in the MDCK II subline, but was absent in the MDCK I subline. The MDCK I line instead contained another five-sugar glycolipid in relatively large amounts. This component has now been isolated and characterized with mass spectrometry, methylation analysis, exoglycosidase digestion, and proton NMR spectroscopy. The structure was concluded to be Gal alpha 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1 Cer. This is a blood group B-like glycolipid lacking fucose, earlier found in rabbit and bovine erythrocytes.

The subcellular localization of the glycosphingolipids in the epithelial cells of rat small intestine.

Analysis of brush-border and basolateral membrane prepared from the epithelial cells of rat small intestine showed that the brush border contained less of some phospholipids and more glycosphingolipids per protein. The N-glycoloylneuraminosyllactosylceramide was enriched in the brush border, while cholesteryl sulphate and fucosyllactosylceramide were more prominent in the basolateral membrane. By periodate-NaB3H4 labelling of the intact small intestine, it was shown that N-glycoloylneuraminosyllactosylceramide is exposed at the luminal cell surface of the epithelial cell.

Structure and genetic polymorphism of blood group A-active glycosphingolipids of the rat large intestine.

Study of blood group A- and B-active glycosphingolipid content of the epithelium of the large intestine of 16 strains of inbred rats led to the discovery of two related strains, SHR and WKY, devoid of A-active glycolipids, whereas all strains expressed B-active glycolipids. This finding evidenced a new A/non-A genetic polymorphism in the rat. Blood group A-active glycolipids were isolated from the large intestine of F344 rats and purified by affinity chromatography on immobilized Helix pomatia lectin. Three glycolipid fractions were separated by preparative thin-layer chromatography and characterized by electron-impact mass spectrometry of their permethylated and permethylated-LiAlH4-reduced derivatives. They were identified as a tetraglycosylceramide (A-4), a hexaglycosylceramide (A-6), and a difucosylated heptaglycosylceramide (A-7) with small amounts of monofucosylated octaglycosylceramide (A-8). Methylation analysis and fragmentation indicated that A6 and A-8 had a lacto- and A-7 a neolactotetraosylceramide core, respectively, identical to the core structures of B-6 and B-7 previously characterized in the large intestine of WF rats (Angström et al. (1987) Biochim. Biophys. Acta 926, 79-86). Upon methylation analysis, B-6 and B-7 purified from SHR (A-deficient) and F344 (A-expressing) were found identical to those of WF rats. This result indicated that precursor substrates for the synthesis of A-active glycolipids were available in SHR rats and thus the genetic deficiency of A-active glycolipid expression probably originated in a defect of the termination of the blood group A determinant by the alpha-3-N-acetylgalactosaminyltransferase.

Glycolipids of rat large intestine. Characterization of a novel blood group B-active tetraglycosylceramide absent from small intestine.

A novel blood group B-active tetraglycosylceramide has been isolated from rat large intestine. It is probably present only in the epithelial cells. Although the compound was not obtained pure, the structure was conclusively established by mass spectrometry, proton NMR spectroscopy and degradative studies to be Galp alpha 1 leads to 3Galp(2 comes from 1 alpha Fucp)beta 1 leads to 4Glcp beta 1 leads to 1Cer. The ceramide was composed of mainly trihydroxy long-chain base (18:0) and non-hydroxy fatty acids (16:0-24:0). The glycolipid was absent from small intestine.

Glycosphingolipid patterns of the epithelial and non-epithelial compartments of rat large intestine.

The epithelial cells and the non-epithelial residue from large intestine of two inbred rat strains were separated and the glycosphingolipids characterized in comparison with earlier detailed data from small intestine of the same strains. Total acid and non-acid glycolipids were prepared and the non-acid glycolipids were further fractionated into subgroups as acetylated derivatives on silicic acid. The fractions obtained were characterized mainly by thin-layer chromatography, including binding of monoclonal anti-A and anti-B antibody to the chromatogram, and by direct-inlet mass spectrometry after derivatization. This combined technology allowed an overall conclusion from a small number of animals concerning relative amounts of glycolipids, microheterogeneity of blood group glycolipids and carbohydrate sequence and lipophilic components of major species of each subfraction. As for the small intestine, the two separated compartments differed distinctly in composition, with blood group fucolipids being confined to the epithelial cells, and a series of glycolipids with probably internal Gal alpha being restricted to the non-epithelial part. The main difference between large and small intestine concerned fucolipids of the epithelium. Three blood group B active glycolipids with four, six and seven sugars were detected which were absent from the small intestine. The four-sugar glycolipid was a major glycolipid with the structure Gal alpha 1----3Gal(2----1 alpha Fuc) beta 1----4Glc beta 1----1Cer. as reported before. The six-sugar glycolipid was shown by mass spectrometry and NMR spectroscopy to have the probable structure Gal alpha 1----3Gal(2----1 alpha Fuc)beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1Cer. The seven-sugar glycolipid had an additional fucose linked to N-acetylhexosamine, as shown by mass spectrometry. Three blood group A active glycolipids with four, six and seven sugars were found in both rat strains, with sequences analogous to the B glycolipids but with a terminal GalNAc instead of Gal. The four- and six-sugar blood group A compounds, but not the seven-sugar glycolipid, have been found before in the small intestine of one of the rat strains. In the small intestine, on the other hand, a branched-chain twelve-sugar blood group A active glycolipid has been found which was absent from the large intestine. Therefore large intestine of both rat strains expressed glycolipid-based blood group A and B activity, while small intestine lacked B activity and showed A activity only in one of the strains.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies on differentiating epithelial cells of rat small intestine. Alterations in the lipophilic part of glycosphingolipids during cell migration from crypt villus tip.

Epithelial cells of rat small intestine have been separated into three intervals of different maturity correlated to cell migration from the crypt to the villus tip. The total acid and non-acid glycosphingolipids were isolated and analysed by thin-layer chromatography. The amount of glucosylceramide an N-glycoloylneuraminosyllactosylceramide was higher, while the amount of globotriaosylceramide and tetrahexosylceramide was lower in villus tip cells (more differentiated) compared to crypt cells (less differentiated). In addition to these alterations the lipophilic composition changed, as shown by a comparison by mass spectrometry of permethylated and LiAlH4-reduced, permethylated derivatives of two of the non-acid glycolipid mixtures (crypt cells and villus tip cells). The components of ceramide were mainly trihydroxy 18:0 long-chain base (phytosphingosine) and hydroxy and non-hydroxy fatty acids. The only significant change concerned the fatty acids. In the crypt cell glycolipids the most abundant fatty acid was 20:0 non-hydroxy fatty acid. In the villus tip cells there was a relative increase of hydroxy fatty acids, with the 24:0 species in dominance. This change occurred for most glycolipids, but the fatty acids of glucosylceramide were villus tip-like already in the crypt cells. The blood group A-active tetraglycosylceramide, and probably the hematoside, did not show any alteration in the lipophilic part. The results indicate that the turnover of some glycolipids (or only their lipophilic part) is more rapid than the epithelial cell turnover.

Glycolipids of rat small intestine. Characterization of a novel blood group H-active triglycosylceramide.

A novel blood group H-active triglycosylceramide has been isolated from rat small intestine. It was present exclusively in the epithelial cells. The structure was established by mass spectrometry, NMR spectroscopy and degradative methods to the Fucp alpha 1 leads to 2Galp beta 1 leads to 4Glcp beta 1 leads to 1Cer. The lipophilic part was made up of mainly trihydroxy base (phytosphingosine) and 16 : 0--24 : 0 fatty acids.

Hydrodynamic properties of solubilized (Na+ + K+)-ATPase from rectal glands of Squalus acanthias.

(1) (Na+ + K+)-ATPas from the rectal glands of Squalus acanthias, solubilized in octaethylene glycol dodecyl monoether (2 mg detergent/mg protein), retains its activity for days when stored at 0 degrees C both with and without 20% glycerol. Glycerol protects partially against inactivation at higher temperatures. (2) Solubilization leads to a decrease in the amount of lipids bound per mg protein. 50 mol phospholipids and 40 mol cholesterol are bound per 265 000 g protein. 90% of the phospholipid is phosphatidylcholine (72%) and phosphatidylethanolamine (18%) and there is about 1 mol acidic phospholipid per 265 000 g. In addition, the protein has about 27 000 g carbohydrate as hexose, hexosamine and sialic acid bound per 265 000 g. (3) The calculation of the molecular weight from an In C vs. r2 plot obtained by sedimentation equilibrium centrifugaton in the presence of 560 microM detergent gives a molecular weight of the protein part of the active solubilized enzyme of 265 000 using the measured values for bound detergent, lipid (phospholipid + cholesterol) and carbohydrate. The sedimentation coefficient (S20,w) is 10.1 S, giving a Stokes' radius of 77 A. (4) An increase in detergent concentration to 56 mM dissociates the 10.1 S particle into particles with a sedimentation coefficient of 5.8 S and a molecular weight of 139 000 (Stokes' radius, 66 A). In the presence of this detergent concentration the enzyme is inactive. (5) The molecular weights of the soijm dodecyl sulphate-solubilized, isolated alpha- and beta-chains are found to be 106 000 and 40 000, respectively. (6) It is concluded that the active solubilized enzyme is an (alpha beta)2 structure and that it dissociates into an enzymatically inactive alpha beta structure when the detergent-to-protein ratio is increased.

The mono- and difucosyl blood group B glycosphingolipids of rat large intestine differ in type of core saccharide.

Two blood group B-active glycosphingolipids were isolated from rat large intestine and characterized by mass spectrometry, proton NMR spectroscopy and methylation analysis. The following structures were concluded: Gal alpha 1----3(Fuc alpha 1----2)Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1Cer and Gal alpha 1----3(Fuc alpha 1----2)Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1Cer. The two glycolipids thus differ in their core saccharides (type 1 and type 2 chain, respectively) and therefore must have different pathways for biosynthesis.

New developments in goblet cell mucus secretion and function.

Goblet cells and their main secretory product, mucus, have long been poorly appreciated; however, recent discoveries have changed this and placed these cells at the center stage of our understanding of mucosal biology and the immunology of the intestinal tract. The mucus system differs substantially between the small and large intestine, although it is built around MUC2 mucin polymers in both cases. Furthermore, that goblet cells and the regulation of their secretion also differ between these two parts of the intestine is of fundamental importance for a better understanding of mucosal immunology. There are several types of goblet cell that can be delineated based on their location and function. The surface colonic goblet cells secrete continuously to maintain the inner mucus layer, whereas goblet cells of the colonic and small intestinal crypts secrete upon stimulation, for example, after endocytosis or in response to acetyl choline. However, despite much progress in recent years, our understanding of goblet cell function and regulation is still in its infancy.

Rapid characterization of mucin oligosaccharides from rat small intestine with gas chromatography-mass spectrometry.

Mucin glycopeptides were isolated from rat small intestinal mucosa after reduction/alkylation, trypsin digestion and gel chromatography. The oligosaccharides were released by using alkaline-NaBH4, separated into neutral and acidic species and permethylated. The derivatized mixtures were analysed with fast atom bombardment mass spectrometry and gas chromatography-mass spectrometry using thin film columns. Permethylated neutral oligosaccharides with up to seven sugars could be chromatographed and detected with mass spectrometry. The complex mixture revealed was partly due to the linkage GalNAc being substituted at both position 3 and 6. The approach will be very useful when analysing small amounts of mucins and mucin fragments.

A novel approach to the study of glycolipid receptors for viruses. Binding of Sendai virus to thin-layer chromatograms.

A method for the binding of virus to a silica gel thin-layer chromatogram is presented. After development the chromatogram is overlayed with the 125I-labelled virus and the bound virus is autoradiographed. Alternatively, the unlabelled virus may be detected after exposure to monoclonal antibody and labelled anti-antibody. The Sendai virus strain used did not bind to brain gangliosides earlier proposed to be receptors, but bound to human erythrocyte gangliosides. This finding may be explained by the existence of Sendai virus variants with different receptor specificities.

Detection of blood group type glycosphingolipid antigens on thin-layer plates using polyclonal antisera.

The conditions for binding of antibodies to glycosphingolipids separated on a thin-layer plate have been optimized for polyclonal antisera. The method has a broad detection range with low background staining. Examples are shown for the detection of blood group A and B active glycosphingolipids.

The specific glycosphingolipid composition of human ureteral epithelial cells.

Total non-acid and acid glycolipid fractions were isolated from epithelial cell scrapings and the non-epithelial residue of a human upper ureter. The glycolipid fractions were structurally characterized as total mixtures by thin-layer chromatography, mass spectrometry, and proton NMR spectroscopy. Selected structural information was also obtained on binding of monoclonal antibodies and bacteria to the thin-layer chromatograms. The major epithelial cell glycolipids were Glc beta 1-1ceramide (75%), dihexosylceramide (10%) and NeuAcLacceramide (10%). In addition, 8 minor glycolipids belonging to the blood group P, Lewis and ABO systems were identified. The major glycolipids of the non-epithelial residues were mono- and dihexosylceramides together with globotriaosyl- and globotetraosylceramides. The epithelial mono- and diglycosylceramide compounds had an unusual ceramide composition with mainly C18 and C20 trihydroxy long chain bases in combination with C22-C24 hydroxy fatty acids in contrast to the non-epithelial glycolipids which contained mainly C18 dihydroxy long chain bases in combination with C16-C24 non-hydroxy fatty acids.