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J K Sheehan

Publications and source records attributed to J K Sheehan.

At least 19 recordsLinked to original sources
PubMed1991

Mucus glycoproteins from cystic fibrotic sputum. Macromolecular properties and structural 'architecture'.

Mucus glycoproteins (mucins) were isolated from sputum of patients with cystic fibrosis (CF) after separation into sol and gel phases. The mucus gel was solubilized with gentle stirring in 6 M-guanidinium chloride supplemented with proteinase inhibitors, and purification of mucins was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/guanidinium chloride. Density-gradient centrifugation also revealed a heterogeneity of the macromolecules, the pattern of which varied between individuals, and mucins from the gel phase was pooled as 'heavy' and 'light' fractions. Gel chromatography on Sepharose CL-2B showed that the heavy fraction contained a larger proportion of smaller species than the 'light' fraction and that the gel phase mucins were much larger than those from the sol. An apparently homogeneous high-Mr mucin population from one individual contained approx. 70% (w/w) carbohydrate, the major sugars being N-acetylglucosamine (17.8%), N-acetylgalactosamine (6.7%), galactose (20.7%), fucose (13.2%) and sialic acid (11.4%). These mucins had an S020.w of 47 S, and an Mr of 15 x 10(6) -20 x 10(6), and rate-zonal centrifugation revealed a polydisperse size distribution [range (5-30) x 10(6)] with a weight-average Mr of 17 x 10(6). The whole mucins were visualized with electron microscopy as linear and apparently flexible threads, disperse in size. Reduction produced subunits which were included on Sepharose CL-2B, and subsequent trypsin digestion yielded high-Mr glycopeptides which were further retarded. The size distributions and fragmentation patterns of mucin from two other CF patients were the same, as studied by gel chromatography, rate-zonal centrifugation and electron microscopy. We conclude that CF mucins are heterogeneous in both size and buoyant density and that the various populations, though differing in buoyant density, share the same architecture and macromolecular properties and are, in this respect, similar to mucins from normal respiratory secretions [Thornton, Davies, Kraayenbrink, Richardson, Sheehan & Carlstedt (1990) Biochem. J. 265, 179-186] and human cervical mucus [Carlstedt & Sheehan (1989) SEB Symp. XLIII 289-316].

Adolescent↗
PubMed1991

Heterogeneity of mucus glycoproteins from cystic fibrotic sputum. Are there different families of mucins?

High-Mr mucin glycopeptides prepared from sputum of an individual with cystic fibrosis (CF) were studied by ion-exchange h.p.l.c. The glycopeptides were heterogeneous and a number of partially resolved populations were identified. Whole mucins from the gel phase were separated into four fractions by isopycnic density-gradient centrifugation in CsCl, and high-Mr glycopeptides from these fractions were examined by ion-exchange h.p.l.c. The acidic nature of the high-Mr glycopeptides increased with increasing buoyant density of the intact mucins, and a periodate-Schiff (PAS)-rich and an extremely high-iron diamine (HID)-reactive component were present in the lowest and highest density fractions respectively. The various glycopeptide populations were identified in different proportions in mucins from four other individuals with CF. CF sputum thus seems to contain distinct mucin populations containing different oligosaccharide clusters corresponding to these high-Mr glycopeptides.

Centrifugation, Density Gradient↗
PubMed1991

Mucins in cat airway secretions.

Mucous secretions were obtained from cat tracheas that had received [3H]glucose and [35S]sulphate to radiolabel mucus glycoproteins biosynthetically. Samples were collected under resting ('basal') conditions as well as after pilocarpine stimulation and were separated into gel and sol phases by centrifugation. Macromolecules were partially purified by using gel chromatography on Sepharose CL-4B, and the species that were eluted with the void volume were then separated into two major populations with isopycnic density-gradient centrifugation in CsCl. The major component from the gel phase of pilocarpine-induced secretions had a buoyant density typical of mucins and was observed as linear and apparently flexible chains by electron microscopy. Reduction of disulphide bonds gave subunits that could be further cleaved by trypsin digestion into components of approximately the same size as the high-Mr glycopeptides obtained from other mucins after this treatment. In contrast, the dominant species in the gel phase of the 'basal' secretion had a significantly higher buoyant density than expected for mucins and was largely unaffected by reduction, as studied by gel chromatography. The macromolecules were fragmented by trypsin, suggesting that they contain a polypeptide backbone. This more dense component also predominated in the sol phase both from the 'basal' secretions and from the pilocarpine-released secretions. Digestion with DNAase, chondroitin ABC lyase or heparan sulphate lyase had no effect, which shows that this component is not DNA, a dermatan sulphate/chondroitin sulphate or a heparan sulphate proteoglycan. In contrast, endo-beta-galactosidase and keratanase caused some fragmentation, suggesting that the molecules contain some linkages of the poly-(N-acetyl-lactosamine) type, although the degradation was not as extensive as expected for keratan sulphate. Treatment with alkaline borohydride resulted in extensive fragmentation of the high-Mr glycopeptides from both components, indicating that the glycans were oligosaccharides that were probably O-linked. The monosaccharide compositions of both components were consistent with that expected for mucins. The data are in keeping with the major component from the pilocarpine-stimulated gel secretions being a mucus glycoprotein and the more dense component being a mucin-like molecule, possibly related to the keratanase-sensitive material isolated from canine trachea by Varsano, Basbaum, Forsberg, Borson, Caughey & Nadel [(1987) Exp. Lung Res. 13, 157-184].

Animals↗
PubMed1991

Evidence for shared epitopes within the 'naked' protein domains of human mucus glycoproteins. A study performed by using polyclonal antibodies and electron microscopy.

Polyclonal antibodies were raised in rabbits towards reduced subunits of human cervical mucus glycoproteins. The reduced subunits almost completely inhibited the antiserum, whereas the intact mucins and the heavily glycosylated fragments obtained after digestion of reduced subunits with trypsin (T-domains) caused only partial inhibition. Periodate oxidation of intact mucins, reduced subunits and T-domains caused no effect on the antibody response, and fragments obtained by more extensive proteolysis of the reduced subunits (P-domains) showed no inhibitory activity. By using electron microscopy, antibodies from T-domain-adsorbed antisera were revealed as bound to cervical mucin reduced subunits, either directly or with colloidal gold-Protein A. Binding sites (100-150 nm apart) were observed at the ends and at internal positions of the reduced subunits. We conclude that the antibodies do not recognize carbohydrate structures but are directed to two kinds of protein epitopes, one shared by whole mucins, reduced subunits and T-domains, and the other specific to the reduced subunit fragment. The latter epitopes are 'cryptic' and are probably shielded within folded protein domains stabilized by disulphide bonds. Human bronchial, cervical, gastric and salivary mucus glycoproteins share some of these cryptic epitopes.

Antibodies↗
PubMed1991

Immunochemical analysis of high molecular-weight human salivary mucins (MG1) using monoclonal antibodies.

Using four Mabs with different specificities for salivary mucins, an ELISA has been developed in which human whole saliva, glandular salivas, salivary protein fractions and purified, high molecular-weight, mucin fractions (MG1) isolated from human submandibular and sublingual glandular tissues have been immunochemically analysed. All four Mabs reacted with MG1s. Three of them reacted with the purified, low molecular-weight salivary mucins (MG2). None was reactive with parotid saliva. MG1 preparations isolated from submandibular and sublingual glandular tissues of one and the same individual displayed different patterns of reactivity with these Mabs, indicating that they differ immunochemically. Analysis of the MG1s in salivas derived from individual salivary glands showed differences in immunochemical composition. These results indicate that the MG1 fraction in human whole saliva consists of several immunochemically different species.

ABO Blood-Group System↗
PubMed1991

Mucin structure. The structure and heterogeneity of respiratory mucus glycoproteins.

Respiratory mucus glycoproteins purified from both "normal" respiratory secretions and sputa of patients with a variety of hypersecretory conditions are high Mr linear molecules adopting a random coil configuration in solution. Studies on their polydispersity show them to have an Mr in the range 3 to 32 x 10(6) and a distribution of length from 200 nm to beyond 10 microns. These macromolecules are fragmented by reduction of intermolecular disulfide bonds into subunits, with Mr approximately 2 x 10(6) and length from 200 to 600 nm. Reduction not only cleaves the mucin molecule but opens, presumably by breaking intramolecular disulfide bonds, cryptic "naked" protein regions. Trypsin digestion of subunits yields high Mr glycopeptides (Mr, 300 to 500,000), presumably by cleavage of the peptide core within the unfolded "naked" protein domains. Respiratory mucus glycoproteins from infected sputum samples are usually heterogeneous in CsCl density gradients, in contrast to those from "normal" tracheobronchial secretions. The former are characterized by the presence of a number of different mucin species, and the basis for the separation of these mucins appears to be the variable presence of sialic acid and sulfate moieties in the oligosaccharide clusters. This heterogeneity may reflect a difference in cellular origin of the mucins and also may be clinically significant.

Glycoproteins↗
PubMed1990

Electron microscopy of cervical-mucus glycoproteins and fragments therefrom. The use of colloidal gold to make visible 'naked' protein regions.

Subunits of human cervical-mucus glycoproteins obtained by reductive cleavage of whole mucins and high-Mr glycopeptides (T-domains) obtained after their trypsin digestion were studied with electron microscopy after spreading the macromolecules in a monolayer of benzyldimethylalkylammonium chloride. Subunits were observed as linear and apparently flexible particles, with number- and weight-average lengths of 390 nm and 460 nm respectively. T-domains randomly distributed on the grid have number- and weight-average lengths of 90 nm and 103 nm respectively, whereas when aligned (possibly stretched by flow) they were longer, with number-average and weight-average lengths of 150 nm and 170 nm respectively. Subunits complexed with gold appeared as segmented structures, with a distribution of inter-gold distances similar to the length distribution for the relaxed T-domains. The whole mucins had few binding sites for gold, suggesting that reduction exposes hydrophobic protein-rich regions with high affinity for gold. Most T-domains had a binding site at one end, indicating the presence of a residual protruding naked peptide region. We conclude that mucins are assembled from subunits joined end-to-end, and that each subunit consists of alternating oligosaccharide 'clusters' (approx. 100 nm) and naked peptide regions which have (after reduction) a high affinity for colloidal gold.

Cervix Mucus↗
PubMed1990

Mucus glycoproteins from 'normal' human tracheobronchial secretion.

Mucous secretions were collected from tracheas of patients undergoing minor surgery under general anaesthesia with tracheal intubation, and mucus glycoproteins were isolated by using isopycnic density-gradient centrifugation in CsCl/guanidinium chloride. 'Whole' mucins were excluded from a Sepharose CL-2B gel, whereas subunits obtained after reduction were included. Trypsin digestion of subunits afforded high-Mr glycopeptides (T-domains), which were further included in the gel. The latter fragments are heterogeneous and comprise two or three populations, as indicated by gel chromatography and ion-exchange h.p.l.c. Rate-zonal centrifugation showed that the 'whole' mucins are polydisperse in size, with a weight-average Mr of (14-16) x 10(6). The macromolecules were observed by electron microscopy, as linear and apparently flexible thread-like structures. Subunits and T-domains had weight-average contour lengths of 490 nm and 160 nm respectively. It is concluded that mucus glycoproteins are present in secretions from the healthy lower respiratory tract. The 'whole' tracheal mucins are assembled from subunits, which in turn can be fragmented into high-Mr glycopeptides corresponding to the oligosaccharide domains typically found in mucus glycoproteins. The size and macromolecular architecture of the tracheal mucins is thus similar to that observed for mucins from human cervical mucus, chronic bronchitic sputum and pig stomach, providing yet another example of this general design of these macromolecules, i.e. subunits assembled end-to-end into very large linear and flexible macromolecules.

Bronchi↗
PubMed1990

Mucinophilic and chemotactic properties of Pseudomonas aeruginosa in relation to pulmonary colonization in cystic fibrosis.

Representative isolates of nonmucoid Pseudomonas aeruginosa were studied to investigate the hypothesis that mucinophilic and chemotactic properties in this species act as potential factors in the initial stages of pulmonary colonization in patients with cystic fibrosis (CF). Transmission electron microscopy with a surfactant monolayer technique was used in a novel manner to demonstrate the adhesion of all 10 P. aeruginosa strains examined to porcine gastric mucin and tracheobronchial mucin from a patient with CF. Control experiments showed that Escherichia coli K-12 and single representatives of Proteus mirabilis and Klebsiella aerogenes did not bind to these mucins. The Adler capillary technique, used to measure bacterial chemotactic response, showed that purified CF mucin acted as a chemoattractant for most P. aeruginosa strains, with the exception of the nonmotile mutant M2Fla- and the nonchemotactic mutant WR-5. The ability of the major sugar and amino acid components of mucin to act as chemoattractants was investigated. The degree of chemotaxis was strain specific; optimum chemotaxis was observed toward serine, alanine, glycine, proline, and threonine. No strain showed chemotaxis to N-acetylneuraminic acid, but all strains showed a strain-dependent chemotactic response to the sugars L-fucose, D-galactose, N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine. These results provide new information on the mucinophilic and chemotactic properties of nonmucoid P. aeruginosa and support the hypothesis that these properties could play a role in the initial stages of pulmonary colonization in patients with CF.

Amino Acids↗
PubMed1989

Quantitation of mucus glycoproteins blotted onto nitrocellulose membranes.

A sensitive assay for mucus glycoproteins (mucins) and fragments thereof is presented. The macromolecules are blotted onto nitrocellulose membranes and visualized using a periodate-Schiff (PAS) reaction and the color yield quantitated with an image analysis system used as a reflectance densitometer. At least 50 ng of the macromolecules was detected. "Whole" mucins and subunits were assayed on 0.2-micron pore size nitrocellulose membranes whereas immobilization of the high-molecular-weight mucin glycopeptides (Mr 300-500,000) required pretreatment of membranes with poly-L-lysine. Binding of the glycopeptides to the polylysine-treated membranes was found to decrease with increasing salt concentration suggesting an electrostatic interaction. The data obtained with this method and a solution PAS assay are in good agreement but the former is more sensitive and can be performed on samples dissolved in chaotropic solvents.

Collodion↗
PubMed1989

Structure and macromolecular properties of cervical mucus glycoproteins.

The endocervical canal is filled with a mucus gel, the properties of which vary during the ovulatory cycle. At mid-cycle the amount of mucus increases, mainly owing to an oestrogen-induced increased hydration of the gel, mucus becomes less visco-elastic and the penetration of the spermatozoa is facilitated. In contrast, under the influence of progesterone during the luteal phase, mucus turns into a less hydrated, highly visco-elastic structure which acts as a barrier to sperm. The mucus gel is formed by very large and structurally complex glycoproteins perfected by evolution to tease and disunite the scientists engaged in unravelling their secrets. The macromolecules are referred to as the mucus glycoproteins or the mucins. Hydrodynamic studies show that cervical mucus glycoproteins (Mr 10-15 x 10(6] behave as random coils, which occupy large spheroidal domains in dilute solution. The predicted 'linear' structure is supported by evidence obtained with electron microscopy. By this technique, the macromolecules are visualized as 'threads' with a skewed and polydisperse distribution of contour lengths (number-average length, 1.5 microns; range 0.5-5 microns). The macromolecules may be cleaved into subunits (Mr 2-3 x 10(6] by reduction of disulphine bonds and these fragments can be divided into large glycopeptides (T-domains; Mr 300,000-400,000) by trypsin. Most of the carbohydrate, which accounts for approximately 80% by weight of the macromolecule and occurs as a heterogeneous population of oligosaccharides, is enriched within the T-domains. The high-Mr glycopeptides thus correspond to long (of the order 100 nm) stretches of protein covered with 100-300 oligosaccharides which protect the core from proteolysis. These regions of the macromolecule are referred to as oligosaccharide 'clusters' and subunits of cervical mucins contain, on average, 3-5 of these 'clusters'. Each 'cluster' is flanked by stretches of protein which are less substituted with carbohydrate and, consequently, more sensitive to proteolysis. There is evidence that these parts of the core, referred to as the 'naked' regins, are folded and stabilized by disulphide bonds. Cervical mucus glycoproteins may thus be viewed as a linear array of oligosaccharide-rich 'clusters' alternating with structures reminiscent of a globular protein. Little is known about how the mucus glycoproteins interact to form the gel. The classical 'Odeblad concept' postulates that the mucins form bundles ('micelles') which are then interconnected in a hormone-dependent way. In contrast, light-scattering studies suggest that cervical mucus is an entangled net-work of long and flexible macromolecules.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbohydrate Sequence↗
PubMed1988

Only trace amounts of fatty acids are found in pure mucus glycoproteins.

The presence of noncovalently associated lipids and covalently bound fatty acids was investigated in preparations of mucus glycoproteins obtained by using density-gradient centrifugation in CsCl/guanidinium chloride. No phospholipids, glycolipids, cholesterol, or triglycerides could be detected. However, small amounts of extractable fatty acids were consistently found, the sum of which ranged from 0.3 to 0.9 micrograms/mg of glycoprotein. The amount of fatty acid released after subsequent treatment with KOH ranged from 0 to 27 ng/mg of glycoprotein. We conclude that density-gradient centrifugation in CsCl/guanidinium chloride is very efficient in removing noncovalently associated lipids from mucus glycoproteins and that covalently bound fatty acids are probably not present in the macromolecules.

Animals↗
PubMed1987

A study of the interaction between cartilage proteoglycan and link protein.

The interaction between proteoglycan and link protein extracted from bovine articular cartilage (15-18-month-old animals) was investigated in 0.5 M-guanidinium chloride. The proteoglycans, radiolabelled as the aggregate (A1 fraction), were fractionated by two 'dissociative' density-gradient centrifugations (A1D1D1) followed by a rate-zonal centrifugation (S1) to yield an A1D1D1S1 preparation. At least 65% of these proteoglycans were able to bind to hyaluronate, but only 52% were able to bind to link protein as assessed by chromatography on Sepharose CL-2B. Over 80% of the [3H]link-protein preparation, radiolabelled as the aggregate, was able to interact with proteoglycan as assessed by chromatography on Sepharose CL-4B. Equilibrium-boundary-centrifugation studies performed at low link-protein concentrations (2.42 x 10(-9) M-5.93 x 10(-8) M) were analysed by Scatchard-type plots and indicated a Kd of 1.5 x 10(-8) M and a stoichiometry, n = 0.56, i.e. approx. 56% of those proteoglycans capable of binding to link protein had a strong site for link protein if a 1:1 stoichiometry were assumed. However, experiments performed at higher link-protein concentrations (3.5 x 10(-7) M and 8 x 10(-7) M) yielded stoichiometry values which were link-protein-concentration-dependent. Non-equilibrium binding studies using chromatography on Sepharose CL-2B and rate-zonal centrifugation yielded apparent stoichiometries between 0.6 and 7.5 link-protein molecules per proteoglycan monomer as a function of increasing link-protein concentration. It was concluded that a proportion of the proteoglycan molecules had a strong site for binding a single link protein (Kd 1.5 x 10(-8) M) and that at high link-protein concentrations a weaker, open-ended, process of link-protein self-association nucleated upon the strong link-protein-proteoglycan complex occurred. Hyaluronate oligosaccharides appeared to abolish a proportion of this self-association (as observed by Bonnet, Dunham & Hardingham [(1985) Biochem. J. 228, 77-85] in a study of link-protein-hyaluronate-oligosaccharide interactions) so as to leave a link protein:proteoglycan stoichiometry of 2. It is not clear whether this second link-protein molecule binds directly to the proteoglycan or to the first link protein.

Animals↗
PubMed1987

The detection of substructures within proteoglycan molecules. Electron-microscopic immuno-localization with the use of Protein A-gold.

Proteoglycan monomers from pig laryngeal cartilage were examined by electron microscopy with benzyldimethylalkylammonium chloride as the spreading agent. The proteoglycans appeared as extended molecules with a beaded structure, representing the chondroitin sulphate chains collapsed around the protein core. Often a fine filamentous tail was present at one end. Substructures within proteoglycan molecules were localized by incubation with specific antibodies followed by Protein A-gold (diameter 4 nm). After the use of an anti-(binding region) serum the Protein A-gold (typically one to three particles) bound at the extreme end of the filamentous region. A small proportion of the labelled molecules (10-15%) showed the presence of gold particles at both ends. A monoclonal antibody specific for a keratan sulphate epitope (MZ15) localized a keratan sulphate-rich region at one end of the proteoglycan, but gold particles were not observed along the extended part of the protein core. This distribution was not changed by prior chondroitin AC lyase digestion of the proteoglycan. Localization with a different monoclonal antibody to keratan sulphate (5-D-4) caused a change in the spreading behaviour of a proportion (approx. 20%) of the proteoglycan monomers that lost their beaded structure and appeared with the chondroitin sulphate chains projecting from the protein core. In these molecules the Protein A-gold localized antibody (5-D-4) along the length of the protein core whereas in those molecules with a beaded appearance it labelled only at one end. Labelling with either of the monoclonal antibodies was specific, as it was inhibited by exogenously added keratan sulphate. The differential localization achieved may reflect structural differences within the proteoglycan population involving keratan sulphate and the protein core to which it is attached. The results showed that by this technique substructures within proteoglycan molecules can be identified by Protein A-gold labelling after the use of specific monoclonal or polyclonal antibodies.

Animals↗
PubMed1987

An enzyme-linked immunosorbent assay (ELISA) of denatured cartilage link protein.

Antiserum (MB007) was raised in rabbits to SDS-denatured cartilage link protein in order to develop an enzyme-linked immunosorbent assay (ELISA) to quantify link protein in cartilage extracts. The antibodies were characterized by using native and denatured link protein, either as the immobilised or the inhibiting antigen in the assay, and shown to bind more effectively to denatured link protein. At low concentrations, neither hyaluronate (0-30 micrograms/ml), proteoglycan (0-50 micrograms/ml) nor hyaluronate-binding region (0-3 micrograms/ml) competitively inhibited the link protein assay. However, at higher concentrations of proteoglycan (50 micrograms/ml-4 mg/ml) and hyaluronate-binding region (3-40 micrograms/ml) inhibition was observed. A more highly purified proteoglycan and a further purified hyaluronate-binding region preparation showed identical behaviour. The inhibition produced by proteoglycan and hyaluronate-binding region occurred at approximately equivalent molar concentrations (assuming Mr of 10(6) and 7 X 10(4), respectively). These results suggest that a significant proportion of these polyclonal antibodies recognize an epitope common to link protein and hyaluronate-binding region. However, the possibility that these effects are due to contamination with covalently bound link protein cannot be excluded. Trypsinated aggregates (0-10 micrograms/ml) produced no inhibition in the link protein ELISA, but as higher concentrations the inhibition was approximately 2000-fold lower than might have been expected from the link protein concentration present. Thus, the accessibility and/or binding of the antibodies to link protein was substantially decreased, illustrating masking of the link protein antigenic sites, as found by A. Ratcliffe and T.E. Hardingham (Biochem. J. 213 (1983) 371-378). These studies indicate that link protein in tissue extracts may be quantified in the concentration range 30-200 ng/ml and in the presence of hyaluronate, proteoglycan and hyaluronate-binding region, provided that both the immobilised and extracted link proteins are denatured.

Animals↗
PubMed1987

Size heterogeneity of human cervical mucus glycoproteins. Studies performed with rate-zonal centrifugation and laser light-scattering.

Mucus glycoproteins (mucins) from cervical pregnancy mucus were fractionated by using rate-zonal centrifugation in a gradient of guanidinium chloride. The distribution of the macromolecules, as assessed by using sialic acid determination, suggested the presence of three populations of different size. Individual fractions were subjected to laser light-scattering performed as total-intensity measurements as well as photon correlation spectroscopy. The results showed that points of inflexion were present in the distribution of both Mr and DT (translational diffusion coefficient) and that the three populations have Mr values of approx. 24 X 10(6), 16 X 10(6) and 6 X 10(6) respectively. The weight-average Mr for the whole distribution, as calculated from the values obtained for the individual fractions, was 13.6 X 10(6), which is in good agreement with that found for the unfractionated material (11.1 X 10(6]. Plots of log RG (radius of gyration) and log (1/DT) versus log Mr are in keeping with the macromolecules being linear flexible chains.

Centrifugation, Zonal↗