The precipitation of mucin by aluminium

The interactions of Al with a mucin glycopeptide have been studied. A number of specific reactions were identified the nature of which were dependent upon the Al chemistry in the hydration environment. In particular, Al was observed to precipitate mucin and it is suggested that this proceeded via the intercalation of the hydroxide within the hydrated macroreticular network of the mucin biopolymer. This precipitation of mucin was visible by eye and abolished the viscosity of native mucin. Viscometry indicated that Al was bound by mucin at low pH. At pH > 3 Al formed a low molecular weight complex with mucin which was hydrolytically stable and was not precipitated at pH up to 8. In an additional and competitive reaction Al was bound by mucin and the resultant mucin–Al complex was suggested to be the precursor to self-assembled mucin–Al spheres identified in solution, by photon correlation spectroscopy, and in precipitate using selective histochemistry. The majority of these spherical structures were of sub-micron diameter and, through their interaction with each other, were probably responsible for the observed pH-dependent peaks of mucin solution viscosity. The larger spheres, between 20 and 80 μm in diameter, were only identified in isolated mucin/Al precipitates and, being comparatively rare, were unlikely to have influenced solution viscosities. These large spheres were observed to act as possible nucleation sites for the flocculation of mucin/Al precipitate. Al at concentrations as low as 0.015 mM induced changes in the rheological properties of mucin. Considering the ubiquitous nature of mucin and the degree to which it is conserved within biota the interactions of Al with mucin may have wide ranging implications for biological systems.     

Rat small intestinal mucin: isolation and characterization of a water-soluble mucin fraction

A water-soluble fraction of sialoglycoprotein containing sulfate was isolated from the mucosal scrapings of the rat small intestine without prior treatment with proteolytic enzymes. Chromatography of the water-soluble mucin on a DEAE-cellulose column gave three main fractions: a major carbohydrate-rich fraction containing sulfate (IGP-A), one high in protein content, and a third with a composition similar to the starting material. Fraction IGP-A was resolved into two components by ultracentrifugation and disc-gel electrophoresis. The higher molecular-weight species of IGP-A was separated from the second component by Sepharose-4B chromatography. These two glycoprotein fractions designated IGP-A₁ and IGP-A₂ had the same chemical composition as IGP-A.     

Utilization of mucin by oral Streptococcus species

The ability of oral Streptococcus strains to utilize oligosaccharide chains in mucin as a source of carbohydrate was studied in batch cultures. Pig gastric mucin, as a substitute of human salivary mucin, was added to chemically defined medium containing no other carbohydrates. Strains of S. mitior attained the highest cell density, while mutans streptococci: S. mutans, S. sobrinus, S. rattus, grew very little in the medium with mucin. S. mitis, S. sanguis, and S. milleri in decreasing order, showed intermediate growth. Mucin break-down as measured by sugar analyses indicated that oligosaccharide chains were only partially degraded. Every strain produced one or more exoglycosidases potentially involved in hydrolysis of oligosaccharide. The enzyme activities occurred mainly associated with the cells, and very little activity was found in the culture fluids. The relationships between glycosidase activities and growth, or mucin degradation were not always clear.     

Wettability of polymers by mucin aqueous solutions

The wettability of poly(methyl methacrylate) and polyethylene by water and aqueous mucin solutions have been studied by sessile drop and under-water captive air bubble contact angles, respectively. From the sessile drop and octane under-water contact angles the polymer-water interfaces have been characterized in terms of works of adhesion and acid-base (polar) interactions. A large water-air contact angle hysteresis observed with poly(methyl methacrylate) surfaces has been attributed to side-chain beta relaxations of polymer ester methyl groups. The wettabilities of the polymers by mucin aqueous solutions have been studied as a function of protein concentration and related to the surface tensions. A positive slope of adhesion tension vs surface tension line, characteristic of polar surfaces, was found with poly(methyl methacrylate). By contrast, a change in the slope, explained as a change in mucin relative adsorption densities at solid/liquid and solid/vapour interfaces, was observed with polyethylene. This adhesion tension behavior appeared to be in agreement with previous data we have published concerning the quantity and state of mucin which are adsorbed to polymers characterized by different surface properties.     

Recognition of mucin components by Pseudomonas aeruginosa

Pseudomonas aeruginosa remains one of the most important bacterial pathogens in lung diseases and especially in Cystic fibrosis. This unusual predilection is best explained by the existence of defects in host defense mechanisms as resulting from the genetic lesion and the presence of a specific colonization niche within the lungs. The niche has been identified as the mucus layer wherein mucin glycoproteins provide a substrate for binding and allows the persistence of this organism in this milieu by a number of possible mechanisms. While this organism is capable of binding to non CF mucins, it is perhaps a combination of factors e.g. increased binding and decreased mucociliary clearance that is responsible for this marked state of colonization in CF. The organism uses chiefly proteins of its flagellar apparatus to initiate this binding and recognizes a variety of oligosaccharides that have been identified in mucins. Among these are both, neutral oligosaccharides and several forms of acidic oligosaccharides derived from the Lewis antigens. There are more than likely a larger repertoire of receptors than those identified and certainly more adhesins present than those currently known. However, the information gathered to date provides an excellent example of the specificity of bacterial interactions with mucins that will certainly be expanded as we study more pulmonary pathogens.     

Analysis of cancer-associated colonic mucin by ion-exchange chromatography: evidence for a mucin species of lower molecular charge and weight in cancer

Cancer-associated mucins in the colon are antigenically distinct and glycosylated differently from their normal counterparts. Mucin-rich glycoconjugate preparations were made from nine non-neoplastic colons, seven colon cancers, and two different xenografts from mucin-producing human colon cancer cell lines, and radiolabeled with 3H. The preparation was applied to a DEAE-cellulose ion-exchange column, and eluted with a discontinuous ascending NaCl gradient resulting in seven discrete fractions or 'species'. Over half of the 3H-labeled glycoconjugates from specimens of non-neoplastic colonic epithelium eluted in fraction V (eluted with 0.25 NaCl). Significantly less of the 3H-labeled glycoconjugates from specimens of colon cancer eluted in fraction V (34%, P less than 0.0005), and there were significant increases in glycoconjugates eluted in fractions IV (P less than 0.008), III (P less than 0.0005), and II (P less than 0.028). Additional samples were prepared without the radiolabeling procedures, chromatographed on a DEAE-cellulose ion-exchange column, and analyzed for monosaccharide content. Each of the fractions contained the monosaccharides expected in mucin-type glycoproteins, but only sialic acid was differentially expressed in the seven fractions or 'species', occurring principally in the more charged species. However, differences in sialic acid content were not sufficient to explain the differences in retention on the ion-exchange column, nor were differences in O-acetylation of the mucins. Mucin-type glycoconjugates from colon cancers are relatively less charged than those from the normal colon, and elute at lower ionic strengths. Of interest, cancer-associated mucins appear to be of lower molecular weight than their normal counterparts. Additional studies of oligosaccharide and apomucin structure will be required to explain the molecular basis of these differences in charge.     

MUC17, a novel membrane-tethered mucin

Membrane mucins have several functions in epithelial cells including cytoprotection, extravasation during metastases, maintenance of luminal structure, and signal transduction. In this paper we describe a large membrane mucin expressed in the normal intestine. This novel mucin, designated MUC17, contains an extended, repetitive extracellular glycosylation domain and a carboxyl terminus with two EGF-like domains, a SEA module domain, a transmembrane domain, and a cytoplasmic domain with potential serine and tyrosine phosphorylation sites. RNA blot analysis and in situ hybridization indicates that MUC17 is expressed in select pancreatic and colon cancer cell lines and in intestinal absorptive cells. Radiation hybrid mapping localized MUC17 to chromosome 7q22 where it resides in close proximity with three other membrane mucin genes, MUC3A, MUC3B, and MUC12. Thus, these membrane mucins reside together in a gene cluster, but are expressed in different tissues and are likely to have different functions as well.     

Marked upregulation of cholesterol 25-hydroxylase expression by lipopolysaccharide

During screening of genes upregulated by lipopolysaccharide (LPS; endotoxin) treatment of bone marrow-derived mouse macrophages, it was unexpectedly found that cholesterol 25-hydroxylase (Ch25h) was strongly upregulated. Treatment of macrophages with 10 ng/ml of LPS for 2 h resulted in a 35-fold increase in the expression of Ch25h. In contrast, LPS treatment did not increase the expression of Cyp27a1 or Cyp7b1. The increased Ch25h expression was found to be independent of Myeloid differentiation protein 88 signaling but dependent on Toll-like receptor 4 signaling. LPS treatment of macrophages caused a 6- to 7-fold increase in cellular 25-hydroxycholesterol concentration. When macrophages were treated with increasing concentrations of 25-hydroxycholesterol, a dose-dependent release of CCL5 into the culture medium was observed. Intravenous injection of LPS in eight healthy volunteers resulted in an increase in plasma 25-hydroxycholesterol concentration. The possibility is discussed that 25-hydroxycholesterol may have a role in the inflammatory response, in addition to its more established role in the regulation of cholesterol homeostasis.     

Intestinal mucin is a chaperone of multivalent copper

1. Download : Download high-res image (262KB)
2. Download : Download full-size image

     

Human MUC1 mucin: a multifaceted glycoprotein

Human MUC1 mucin, a membrane-bound glycoprotein, is a major component of the ductal cell surface of normal glandular cells. MUC1 is overexpressed and aberrantly glycosylated in carcinoma cells. The role MUC1 plays in cancer progression represents two sides of one coin: on the one hand, loss of polarity and overexpression of MUC1 in cancer cells interferes with cell adhesion and shields the tumor cell from immune recognition by the cellular arm of the immune system, thus favoring metastases; on the other hand, MUC1, in essence a self-antigen, is displaced and altered in malignancy and induces immune responses. Tumor-associated MUC1 has short carbohydrate sidechains and exposed epitopes on its peptide core; it gains access to the circulation and comes into contact with the immune system provoking humoral and cellular immune responses. Natural antibodies to MUC1 present in the circulation of cancer patients may be beneficial to the patient by restricting tumor growth and dissemination: early stage breast cancer patients with a humoral response to MUC1 have a better disease-specific survival. Several MUC1 peptide vaccines, differing in vectors, carrier proteins and adjuvants, have been tested in phase I clinical trials. They are capable of inducing predominantly humoral responses to the antigen, but evidence that these immune responses may be effective against the tumor in humans is still scarce.     

Regulation of mucin gene expression by CREB via a nonclassical retinoic acid signaling pathway

Vitamin A and its metabolite retinoic acid (RA) are essential elements for normal lung development and the differentiation of lung epithelial cells. We previously showed that RA rapidly activated cyclic AMP response element-binding protein (CREB) in a nonclassical manner in normal human tracheobronchial epithelial (NHTBE) cells. In the present study, we further demonstrated that this nonclassical signaling of RA on the activation of CREB plays a critical role in regulating the expression of airway epithelial cell differentiation markers, the MUC2, MUC5AC, and MUC5B genes. We found that RA rapidly activates the protein kinase Calpha isozyme and transmits the activation signal to CREB via the Raf/MEK/extracellular signal-regulated kinase/p90 ribosomal S6 kinase (RSK) pathway. Activated RSK translocated from the cytoplasm to the nucleus, where it phosphorylates CREB. Activated CREB then binds to a cis-acting replication element motif on the promoter (at nucleotides [nt] -878 to -871) of the MUC5AC gene. The depletion of CREB using small interfering RNA abolished not only the RA-induced MUC5AC but also RA-induced MUC2 and MUC5B. Taken together, our findings demonstrate that CREB activation via this nonclassical RA signaling pathway may play an important role in regulating the expression of mucin genes and mediating the early biological effects of RA during normal mucous differentiation in NHTBE cells.     

Self-association of mucin

Aggregation phenomena in aqueous solutions of purified human tracheobronchial mucin have been studied by rheological methods, steady-state fluorescence, quasielastic light scattering, and spin probe techniques. At temperatures below 30 degrees C and concentrations above 15 mg/mL and in the absence of chaotropic agents, mucin solutions are viscoelastic gels. A gel-sol transition is observed at temperatures above 30 degrees C that is manifested by the diminishing storage modulus and a loss tangent above unity throughout the studied frequency range of the oscillatory shear. No decline in the mucin molecular weight is observed by size-exclusion chromatography above 30 degrees C in the absence of redox agents or proteolytic enzymes. Aggregation of hydrophobic protein segments of the mucin chains at 37 degrees C is indicated by QELS experiments. The decreasing polarity of the microenvironment of pyrene solubilized into mucin solutions at temperatures above 30 degrees C, concomitant with the gel-sol transition, shows the hydrophobicity of the formed aggregates. ESR spectra of the fatty acid spin probe, 16-doxylstearic acid indicate that the aggregate-aqueous interface becomes more developed at elevated temperatures.     

In vitro utilization of mucin by Bacteroides fragilis.

A method for isolating pig colon mucin in a soluble high-molecular-weight form, suitable for addition to bacterial growth media, is described. This preparation was utilized as a sole carbohydrate energy source by two strains of Bacteroides fragilis. The extent of degradation was compared with that of commercial pig gastric mucin by the same strains. Gas-liquid chromatographic analysis of the mucin carbohydrates and gel chromatography of the preparations were carried out before and after in vitro degradation. The mucin carbohydrates were utilized only to a very limited extent, colon mucin being more resistant to degradation than gastric mucin. Both mucins chromatographed at or near the excluded volume on Sepharose 4B, and only in the case of ATCC 25285 grown on gastric mucin was a significant degradation peak detected. If mucins are degraded in vivo by the sequential action of several bacteria, a pure culture in vitro might be expected to degrade mucins to a limited extent only. Techniques previously used to examine mucin utilization by pure cultures may have overlooked limited mucin degradation demonstrated by the methods used in this work.     

Enhancement of UDP-galactose:mucin galactosyltransferase activity by spermine

A microsomal preparation prepared from the mucosal lining of canine trachea catalyzed the transfer of galactose from its uridine diphosphate derivative to sialidase-treated ovine submaxillary mucin. Maximal incorporation occurred at 30 mm mn²⁺. When the concentration of mn²⁺ in the reaction mixture was reduced to 2.5 mm, approximately two-thirds of the enzymatic activity was lost, but full activity could be restored by the addition of 1 mm spermine. Under these conditions spermine did not affect the K_m for UDP-galactose, but lowered the K_m for sialidase-treated ovine submaxillary mucin and Mn²⁺ by a factor of 10. The effect of spermine was abolished with increasing concentrations of Mn²⁺, and in the absence of the metal, enzymatic activity was lost and could not be restored by the addition of spermine. Spermidine also stimulated activity at low levels of Mn²⁺, but to a lesser degree than spermine. A slight stimulatory effect was consistently derived from putrescine as well, while cadaverine, putreanine, and monoamines were ineffectual. Spermine had a similar effect on the enzymatic transfer of GalNAc to a protein core acceptor but had little or no effect on the enzymatic transfer of sialic acid to sialidase-treated ovine submaxillary mucin, galactose to N-acetylglucosamine, or fucose to sialidase-galactosidase-treated fetuin. Similar results were obtained with enzyme preparations prepared from canine submaxillary glands. Other polycationic compounds such as protamine, histone, and polylysine also stimulated enzymatic activity at suboptimal concentrations of mn²⁺.