Molecular organization of the mucins and glycocalyx underlying mucus transport over mucosal surfaces of the airways.

Mucus, with its burden of inspired particulates and pathogens, is cleared from mucosal surfaces of the airways by cilia beating within the periciliary layer (PCL). The PCL is held to be "watery" and free of mucus by thixotropic-like forces arising from beating cilia. With radii of gyration ~250 nm, however, polymeric mucins should reptate readily into the PCL, so we assessed the glycocalyx for barrier functions. The PCL stained negative for MUC5AC and MUC5B, but it was positive for keratan sulfate (KS), a glycosaminoglycan commonly associated with glycoconjugates. Shotgun proteomics showed KS-rich fractions from mucus containing abundant tethered mucins, MUC1, MUC4, and MUC16, but no proteoglycans. Immuno-histology by light and electron microscopy localized MUC1 to microvilli, MUC4 and MUC20 to cilia, and MUC16 to goblet cells. Electron and atomic force microscopy revealed molecular lengths of 190-1,500 nm for tethered mucins, and a finely textured glycocalyx matrix filling interciliary spaces. Adenoviral particles were excluded from glycocalyx of the microvilli, whereas the smaller adenoassociated virus penetrated, but were trapped within. Hence, tethered mucins organized as a space-filling glycocalyx function as a selective barrier for the PCL, broadening their role in innate lung defense and offering new molecular targets for conventional and gene therapies.

Interaction between mycobacteria and mucus on a human respiratory tissue organ culture model with an air interface.

Mycobacteria adhere specifically to extracellular matrix (ECM) and mucus with a fibrous, but not globular, appearance, in organ cultures of human respiratory mucosa examined by scanning electron microscopy. Previously, light microscopy sections made of tissue infected for 7 days demonstrated mycobacteria associated with mucus on the organ culture surface, and within submucosal glands in areas of damaged epithelium. The authors have now investigated the interactions between Mycobacterium avium complex (MAC), Mycobacterium tuberculosis (MTB), and Mycobacterium smegmatis (MS) and mucus by preincubating bacteria with purified mucins MUC5AC and MUC5B prior to inoculation onto the organ culture mucosal surface. They have also measured mucin production by the organ culture after mycobacterial infection. Mucus did not cause clumping of mycobacteria. There was a significant (P=.03) increase in the amount of fibrous mucus, but not globular mucus, observed on tissue inoculated with mucins compared to controls. The number of bacteria adhering to ECM was markedly reduced after incubation with mucins, which could indicate a protective effect. Mycobacterial infection did not increase mucin production by the organ culture. Mycobacterial adherence to mucins may play a role in the pathogenicity of mycobacteria in diseases such as cystic fibrosis, bronchiectasis, and chronic obstructive pulmonary disease (COPD), in which there are changes in mucus composition and clearance.

Identification of a nonmucin glycoprotein (gp-340) from a purified respiratory mucin preparation: evidence for an association involving the MUC5B mucin.

Rate-zonal centrifugation of a reduced and alkylated respiratory mucin preparation identified a protein-rich fraction. This was subjected to trypsin treatment and one of the many liberated peptides was purified and its N-terminal sequence determined. The peptide was identical to a 14 amino acid sequence from the scavenger receptor cysteine-rich domain containing glycoprotein gp-340. A polyclonal antiserum, raised against the peptide, stained the serous cells in the submucosal glands of human tracheal tissue. The glycoprotein was purified from respiratory mucus by density-gradient centrifugation, gel chromatography, and anion exchange chromatography. The molecule exhibited a heterogeneous distribution of buoyant density (1.28-1.46 g/ml) that overlapped with the gel-forming mucins, was included on Sepharose CL-2B and was quite highly anionic. SDS-PAGE indicated a mass greater than 208 kDa and measurements performed across the molecular size distribution indicated an average M(r) of 5 x 10(5) with a range of M(r) from 2 x 10(5) to 1 x 10(6). Gel chromatography of respiratory mucus extracts ("associative" and "dissociative") indicated that this glycoprotein forms complexes that may involve the large gel-forming mucins MUC5AC and MUC5B. Rate zonal centrifugation suggested such complexes are more likely to involve MUC5B rather than MUC5AC mucins.

Phosphorylation by cdc2-CyclinB1 kinase releases cytoplasmic dynein from membranes.

Movement of various cargoes toward microtubule minus ends is driven by the microtubule motor cytoplasmic dynein (CD). Many cargoes are motile only during certain cell cycle phases, suggesting that CD function may be under cell cycle control. Phosphorylation of the CD light intermediate chain (DLIC) has been suggested to play a crucial role in modulating CD function during the Xenopus embryonic cell cycle, where CD-driven organelle movement is active in interphase but greatly reduced in metaphase. This down-regulation correlates with hyperphosphorylation of DLIC and release of CD from the membrane. Here we investigate the role of the key mitotic kinase, cdc2-cyclinB1, in this process. We show that DLIC within the native Xenopus CD complex is an excellent substrate for purified Xenopus cdc2-glutathione S-transferase (GST) cyclinB1 (cdc2-GSTcyclinB1) kinase. Mass spectrometry of native DLIC revealed that a conserved cdc2 site (Ser-197) previously implicated in the metaphase modulation of CD remains phosphorylated in interphase and so is unlikely to be the key regulatory site. We also demonstrate that incubating interphase membranes with cdc2-GSTcyclinB1 kinase results in substantial release of CD from the membrane. These data suggest that phosphorylation of DLIC by cdc2 kinase leads directly to the loss of membrane-associated CD and an inhibition of organelle movement.

Hyaluronan: polysaccharide chaos to protein organisation.

Molecular dynamics simulations of hyaluronan have revealed the inherent flexibility of this glycosaminoglycan in solution. Crystal structures of hyaluronan-digesting enzymes have provided the first direct insights into the molecular basis of hyaluronan-protein interactions. Various studies on hyaluronan-binding proteins suggest there is considerable diversity in their mode of interaction with hyaluronan, which might result in many different bound conformations of the polysaccharide.

Preliminary study pointing out a significant alteration in the biochemical composition of MUC2 in colorectal mucinous carcinoma.

OBJECTIVES: In this study, we characterized colonic MUC2 mucin from a mucinous carcinoma cell line and tried to find out carcinoma-associated alterations by comparing the results with those obtained from its benign phenotype previously. DESIGN AND METHODS: The molecular size distribution of the extracted molecules and their reactivity with two different MUC2 polypeptide antibodies indicated the presence of precursor and mature forms of the mucin in both cell lines. Isopycnic density gradient centrifugation gave good resolution of mature and precursor forms of MUC2 as assessed by agarose gel electrophoresis. Using this approach, we compared the different forms of MUC2 between benign and malign colonic cells. RESULTS: In the comparison, we detected some aberrant glycosylated MUC2 molecules in mucinous carcinoma cell line. Agarose gel electrophoretic analysis of the low-density fractions indicated that these molecules are more charged than precursors, however, they are smaller and/or less glycosylated than mature MUC2 molecules. CONCLUSION: The identification of unusual partially glycosylated forms of the major colonic mucin MUC2 is novel and unexpected. Implication of defective processes in the post translational modification/ processing of MUC2 opens a new field in the cancer mucin biology.

Characterization of mucins from cultured normal human tracheobronchial epithelial cells.

Early-passage normal human tracheobronchial epithelial (NHTBE) cells grown in air-liquid interface cultures in medium containing retinoids differentiate into a mucociliary epithelium over a 2- to 3-wk period and express increasing mRNA levels of the airway mucin genes MUC5AC and MUC5B as the cultures age; the levels of MUC2 mRNA were very low throughout the study. Using specific antibodies to MUC5AC and MUC5B mucins, we noted a gradual increase in these two mucins in the intracellular and apically secreted pools as a function of time. A low level of MUC2 mucin was detected, which did not change with time. The intracellular and apically secreted mucins isolated from day 14 and day 21 cultures by density gradient centrifugation were similar in density to those previously isolated from human respiratory mucus secretions. The sedimentation rate of the apically secreted mucins indicated that they were highly oligomerized, polydisperse macromolecules similar to those previously documented from in vivo secretions. In contrast, the cell-associated mucins from the cultured NHTBE cells were much smaller, possibly only monomers and dimers. Anion-exchange chromatography detected no differences in charge density between the reduced and carboxymethylated cell-associated and secreted forms of the MUC5AC and MUC5B mucins. The MUC5AC mucin was of similar charge density to its in vivo counterpart; however, MUC5B was more homogeneous than that found in vivo. Finally, evidence is presented for an intracellular NH(2)-terminal cleavage of the MUC5B mucins. These studies indicate that the mucins produced by cultured NHTBE cells are similar to those found in human airways, suggesting that this cell culture model is suited for studies of respiratory mucin biosynthesis, processing, and assembly.

Glycosaminoglycan conformation: do aqueous molecular dynamics simulations agree with x-ray fiber diffraction?

Glycosaminoglycan-protein interactions are biologically important and require an appreciation of glycan molecular shape in solution, which is presently unavailable. In previous studies we found strong similarity between aqueous molecular dynamics (MD) simulations and published x-ray diffraction refinements of hyaluronan. We have applied a similar approach here to chondroitin and dermatan, attempting to clarify some of the issues raised by the x-ray diffraction literature relating to chondroitin and dermatan sulfate. We predict that chondroitin has the same beta(1-->4) linkage conformation as hyaluronan, and that their average beta(1-->3) conformations differ. This is explained by changes in hydrogen-bonding across this linkage, resulting from its axial hydroxyl, causing a different sampling of left-handed helices in chondroitin (2.5- to 3.5-fold) as compared with hyaluronan (3.0- to 4.0-fold). Few right-handed helices, which lack intramolecular hydrogen-bonds, were sampled during our MD simulations. Thus, we propose that the 8-fold helix observed in chondroitin-6-sulfate, represented in the literature as an 8(3) helix (right-handed), though it has never been refined, is more likely to be 8(5) (left-handed) helix. Molecular dynamics simulations implied that (4)C(1) and (2)S(O), but not (1)C(4), forms of iduronate could be used in refinements of dermatan x-ray fiber diffraction patterns. Current models of 8-fold dermatan sulfate chains containing (4)C(1) iduronate refine to right-handed helices, which possess no intramolecular hydrogen-bonds. However, MD simulations predict that models containing (2)S(O) iduronate could provide better (8(5) helix) starting structures for refinement. Thus, the 8-fold dermatan sulfate refinement (8(3) helix) could be in error.

Physical characterization of the MUC5AC mucin: a highly oligomeric glycoprotein whether isolated from cell culture or in vivo from respiratory mucous secretions.

We have isolated the high-M(r) mucins from growth medium of the early stage of an HT-29 cell culture by gel chromatography and isopycnic density gradient centrifugation. The mucins (buoyant density 1.34-1.44 g/ml) were reactive with an anti-peptide antiserum (MAN-5ACI) raised against a sequence from within the MUC5AC mucin. Similar antisera raised against the MUC2 and MUC5B mucins were not reactive. The MUC5AC reduced-mucin subunits exhibited a homogeneous charge distribution on anion-exchange chromatography, but appeared as two bands, one major and one more minor, after agarose gel electrophoresis. The unreduced mucins had an average M(r) in excess of 40 MDa and were visualized in the electron microscope as large, fine filamentous threads (many microns in length) that after reduction were greatly reduced in size (number average length 570 nm). Agarose gel electrophoresis of unreduced MUC5AC mucins identified a major band just entering the gel with evidence of a 'ladder' of faster-migrating minor bands. Partial reduction of the mucins increased the proportion of the faster bands and at least 16 could be discriminated. M(r) measurements showed that these bands differed by single monomer units. The mucins behaved as very stiff extended structures in solution and this characteristic might explain the poor separation of different-sized oligomers in sedimentation-rate experiments. The cell-culture mucin preparation had similar characteristics of charge and buoyant density to MUC5AC mucins from respiratory secretions in vivo. In addition the MUC5AC mucin from respiratory tract secretions exhibited similar behaviour, reduced and unreduced on agarose gel electrophoresis, indicating that the mucin has a similar molecular phenotype in vivo and in vitro.

Identification in vitreous and molecular cloning of opticin, a novel member of the family of leucine-rich repeat proteins of the extracellular matrix.

A prominent 45-kDa component was identified by protein staining following SDS-polyacrylamide gel electrophoresis of a 4 M guanidine hydrochloride extract from bovine vitreous collagen fibrils. Peptide sequences obtained from this component were used as a basis for the cloning (from human retinal cDNA) and sequencing of a novel member of the leucine-rich repeat extracellular matrix protein family that we have named opticin. Opticin mRNA was found by reverse transcription polymerase chain reaction in ligament and skin as well as in retina. An open reading frame containing 332 amino acids was identified, the first 19 amino acids representing a signal peptide. The deduced amino acid sequence of the mature protein encodes a 35-kDa protein with a calculated isoelectric point of 5.4. The central domain of this protein consists of six B-type leucine-rich repeats. This domain is flanked by cysteine clusters including a C-terminal two-cysteine cluster containing an additional leucine-rich repeat. The N-terminal region contains a cluster of potential O-glycosylation sites, and analysis of bovine vitreous opticin demonstrated the presence of sialylated O-linked oligosaccharides substituting the core protein. Opticin shows highest protein sequence identity to epiphycan (42%) and osteoglycin (35%) and belongs to Class III of the leucine-rich repeat extracellular matrix protein family.

A comparison of post-operative morbidity following outpatient dental care under general anaesthesia in paediatric patients with and without disabilities.

An investigation was undertaken of morbidity after outpatient general anaesthesia for comprehensive dental care in a group of paediatric patients over a 1-year period. Data were collated from the patient's hospital notes and from the response to a questionnaire sent to parents/carers. Clinical data were obtained for 55 cases (age range 3-17 years) for whom parents/carers had returned questionnaires. There were 27 intellectually and/or physically impaired patients, the other 28 being anxious or phobic. After discharge, 44% of all parents/carers reported symptoms post-operatively in their child, the prevalence being similar in both groups. The symptoms were nausea/vomiting (20%), unexpected drowsiness (13%) and the need for pain relief at home (13%). Dental procedures were routine restorations (42%), or a combination of restoration, extractions and preventive care for the remainder. Only four patients had extractions only. One teenager had to be admitted for persistent nausea and vomiting, despite prophylactic measures. In conclusion, post-operative morbidity appears to be low after outpatient general anaesthesia for dental procedures, and is no greater in patients with disabilities.

A study of the intracellular and secreted forms of the MUC2 mucin from the PC/AA intestinal cell line.

In this study we present data on the entire population of MUC2 molecules secreted from and within the cell layer of an intestinal cell line. The molecular size distribution of the extracted molecules and their reactivity with two different MUC2 polypeptide antibodies indicated the presence of precursor and mature forms of the mucin. Oligomerized forms of the mucin were found in both the cell layer and medium; however, precursor forms were confined to the cell layer. Isopycnic density gradient centrifugation gave good resolution of mature and precursor forms of MUC2 as assessed by agarose gel electrophoresis. Three different populations of MUC2 were identified: one at low density (>1.3 g/ml) containing the N-glycosylated, non-O-glycosylated polypeptide; a second at intermediate density (1.3-1.35 g/ml) which may represent partially O-glycosylated intermediates; and a third at high density (1.36-1.48 g/ml) containing the mature MUC2 mucins. Rate-zonal centrifugation and agarose electrophoretic analysis of the low-density fraction indicated that the N-glycosylated MUC2 polypeptide was present as putative monomer and dimer/oligomer species. The combination of isopycnic density gradient centrifugation with agarose electrophoresis provides a new and simple approach that allows us to follow the MUC2 gene product from polypeptide through to the mature glycosylated mucin.

Mucin gene expression during differentiation of human airway epithelia in vitro. Muc4 and muc5b are strongly induced.

Mucus hypersecretion is characteristic of chronic airway diseases. However, regulatory mechanisms are poorly understood. Human airway epithelial cells grown on permeable supports at the air-liquid interface (ALI) develop a mucociliary morphology resembling that found in vivo. Such cultures provide a model for studying secretory cell lineage, differentiation, and function, and may provide insight regarding events leading to mucus hypersecretion. The mucin gene expression profile of well-differentiated human airway epithelial cells in culture has not yet been established. We compared expression of all the currently described mucin genes in poorly differentiated (conventional cultures on plastic) and well-differentiated (ALI) human nasal and bronchial epithelial cells. Differentiation-dependent upregulation of MUC3, MUC5AC, MUC5B, and MUC6 messenger RNA (mRNA) was demonstrated using reverse transcriptase-polymerase chain reaction (RT-PCR). Northern blot analysis showed a similar increase for MUC4 and demonstrated that induction of MUC4 and MUC5B expression depended on retinoic acid. MUC1, MUC2, MUC7, and MUC8 mRNAs were also detected by RT-PCR, but these genes did not appear to be strongly regulated as a function of differentiation. Mucin gene expression was similar in bronchial and nasal cells. Thus, mucociliary differentiation of human airway epithelia in vitro entails upregulation of several mucin genes.

Physical characterization of a low-charge glycoform of the MUC5B mucin comprising the gel-phase of an asthmatic respiratory mucous plug.

We have previously noted that sequential extraction of an asthmatic mucous exudate with 6 M guanidinium chloride yielded a fraction of the mucins that were most resistant to solubilization and of high Mr [Sheehan, Richardson, Fung, Howard and Thornton (1995) Am. J. Respir. Cell Mol. Biol. 13, 748-756]. Here we show that this mucin fraction is dominated (at least 96% of the total) by the low-charge glycoform of the MUC5B gene product. Seen in the electron microscope the mucins appeared mainly as compact 'island' structures composed of linear threads often emanating from globular 'nodes' rather than the discrete linear threads more typical of mucins that we have previously described. The effect of reducing agents was as expected for other gel-forming mucins, i.e. reduced subunits or monomers of Mr 3x10(6)) were produced within 15 min of treatment. Kinetic experiments on the cleavage of the intact mucins with the proteinase trypsin indicated two clear regimes of fragmentation. An initial rapid cleavage generated mucins ranging from Mr=4x10(6) to 30x10(6) that in the electron microscope appeared as polydisperse threads (500-3000 nm in length), similar to normal and other respiratory mucins that we have previously characterized. A subsequent slower fragmentation over many hours yielded a major fragment of Mr 3x10(6) and length 200-600 nm, very similar in size and Mr to the subunits obtained by reduction. The results suggest that the MUC5B mucin is assembled, first into polydisperse linear threads, which are then linked together via a protein-mediated process. This might involve part of the mucin polypeptide or an as yet unidentified protein(s). The high proteinase susceptibility of the linkage suggests that it might be a point of control for mucin size and thus mucus rheology.

Salivary mucin MG1 is comprised almost entirely of different glycosylated forms of the MUC5B gene product.

The MG1 population of mucins was isolated from human whole salivas by gel chromatography followed by isopycnic density gradient centrifugation. The reduced and alkylated MG1 mucins, separated by anion exchange chromatography, were of similar size (radius of gyration 55-64 nm) and molecular weight (2.5-2.9 x 10(6) Da). Two differently-charged populations of MG1 subunits were observed which showed different reactivity with monoclonal antibodies to glycan epitopes. Monosaccharide and amino acid compositional analyses indicated that the MG1 subunits had similar glycan structures on the same polypeptide. An antiserum recognizing the MUC5B mucin was reactive across the entire distribution, whereas antisera raised against the MUC2 and MUC5AC mucins showed no reactivity. Western blots of agarose gel electrophoresis of fractions across the anion exchange distribution indicated that the polypeptide underlying the mucins was the product of the MUC5B gene. Amino acid analysis and peptide mapping performed on the fragments produced by trypsin digestion of the two MG1 populations yielded data similar to that obtained for MUC5B mucin subunits prepared from respiratory mucus (Thornton et al., 1997) and confirmed that the MUC5B gene product was the predominant mucin polypeptide present. Isolation of the MG1 mucins from the secretions of the individual salivary glands (palatal, sublingual, and submandibular) indicate that the palatal gland is the source of the highly charged population of the MUC5B mucin.

Monoclonal antibody recognizing a core epitope on mucin.

Monoclonal antibody TH1 (IgM) was prepared by immunizing mice with deglycosylated (TFMSA-treated) cystic fibrosis mucin. TH1 reacted strongly with TFMSA treated cystic fibrosis mucin but not with the fully glycosylated mucin, indicating reactivity with a core mucin epitope. TH1 showed no reactivity with ovine mucin (98% of glycans as sialyl-Tn) but reacted strongly with desialylated ovine mucin, indicating the epitope for this mab was the Tn-antigen (O-linked GalNAc). However, TH1 showed no reactivity with Tn-positive red blood cells, and the binding of TH1 was not inhibited by GalNAc at 2.5 mg/ml, illustrating the importance of the peptide sequence to which the GalNAc is attached. TH1 stained the majority of cancers of the colon, lung, stomach, ovary, breast, and cervix, and the cellular distribution of this antigen in normal tissue suggested reactivity with immature mucin. This antibody appears to be a useful reagent for the detection of immature mucin.

The large chondroitin sulphate proteoglycan versican in mammalian vitreous.

Hyaluronan is a major component of the vitreous gel. Hyaluronan-binding macromolecules, including the aggregating proteoglycans, have been shown to perform an important role in maintaining the structural integrity of a number of tissues. However, there have not previously been any biochemical data to establish the presence of these types of macromolecules in vitreous. Bovine vitreous gel was solubilized (apart from a residual collagenous pellet) in 4 M guanidine hydrochloride and after dialysis into phosphate buffered saline analyzed by gel filtration chromatography with in-line measurement of refractive index and multi-angle laser light scattering. The concentration of hyaluronan in whole vitreous was found to be 0.57 mg/ml. The average molecular weight of the hyaluronan was found to be 170,000 (after isolation of the vitreous hyaluronan by isopycnic centrifugation in 0.5 M guanidine hydrochloride and papain digestion). Following Superose 12 gel filtration chromatography of the Streptomyces hyaluronan lyase digested vitreous extract, a pool of material was identified at or near the void volume of the column, and this material was shown to contain sulphated proteoglycans. Analysis of fractions following Superose 12 gel filtration chromatography by Western blotting showed that this pool of material contained the chondroitin sulphate proteoglycans versican and type IX collagen. Link protein was also identified in vitreous extracts by Western blotting. In whole vitreous, the concentration of versican was found to be 21.4+/-2.8 microg/ml and of link protein 0.62+/-0.07 microg/ml. Versican and link protein were thus present in approximately 1:1 molar ratios, but hyaluronan was present in a molar excess of 150 times. Therefore, aggregating proteoglycans are present in vitreous but, assuming that they bind to hyaluronan in-vivo, their overall density along the hyaluronan is much lower than that found in other tissues.

Dynamic exchange between stabilized conformations predicted for hyaluronan tetrasaccharides: comparison of molecular dynamics simulations with available NMR data.

Studies of the hyaluronan (HA) tetrasaccharides are important for understanding hydrogen-bonding in the HA polymer, as they are probably the smallest oligomers in which characteristics of the constituent monosaccharides and the polymer are simultaneously exhibited. Here we present extensive molecular dynamics simulations of the two tetrasaccharides of HA in dilute aqueous solution. These simulations have confirmed the existence of intramolecular hydrogen-bonds between the neighboring sugar residues of HA in solution, as proposed by Scott (1989). However, our simulations predict that these intramolecular hydrogen-bonds are not static as previously proposed, but are in constant dynamic exchange on the sub-nanosecond time-scale. This process results in discrete internal motion of the HA tetrasaccharides where they rapidly move between low energy conformations. Specific interactions between water and intramolecular hydrogen-bonds involving the hydroxymethyl group were found to result in differing conformations and dynamics for the two alternative tetrasaccharides of HA. This new observation suggests that this residue may play a key role in the entropy and stability of HA in solution, allowing it to stay soluble up to high concentration. The vicinal coupling constants3 J NHCH of the acetamido groups have been calculated from our aqueous simulations of HA. We found that high values of 3J NHCH approximately 8 Hz, as experimentally measured for HA, are consistent with mixtures of both trans and cis conformations, and thus3 J NHCH cannot be used to imply a purely trans conformation of the acetamido. The rapid exchange of intramolecular hydrogen-bonds indicates that although the structure is at any moment stabilized by these hydrogen-bonds, no one hydrogen-bond exists for an extended period of time. This could explain why NMR often fails to provide evidence for intramolecular hydrogen-bonds in HA and other aqueous carbohydrate structures.