scholarly journals Identification of MUC5B, MUC5AC and small amounts of MUC2 mucins in cystic fibrosis airway secretions

1999 ◽  
Vol 344 (2) ◽  
pp. 321-330 ◽  
Author(s):  
Julia R. DAVIES ◽  
Naila SVITACHEVA ◽  
Louise LANNEFORS ◽  
Ragnhild KORNFÄLT ◽  
Ingemar CARLSTEDT
Keyword(s):  
Cystic Fibrosis ◽  
High Speed ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Insoluble Residue ◽  
Guanidinium Chloride ◽  
Charge Densities ◽  
Cystic Fibrosis Airway ◽  
Gel Phase ◽  
Two Populations

To investigate the genetic identities of the mucins secreted in cystic fibrosis (CF) airways, sputum was collected from five individuals. Samples were separated into gel and sol phases by high-speed centrifugation and the gel phase was extracted in 6 M guanidinium chloride. The ‘insoluble’ residue remaining after extraction of the gel phase was brought into solution by reduction/alkylation. Density-gradient centrifugation in CsCl revealed polydisperse distributions of sialic acid-containing mucins in the gel phase, insoluble residue and sol phase fractions and the degree of variation between the different individuals was low. Antibodies recognizing MUC5AC and MUC5B identified these mucins in each of the fractions. MUC2, however, was present only as a component of the insoluble residue from the gel which accounted for less than 4% by mass of the total mucins. MUC5B and MUC5AC from the gel phase were large oligomeric species composed of disulphide-bond linked subunits and MUC5B was present as two populations with different charge densities which are likely to correspond to MUC5B ‘glycoforms’. The sol phase contained, in addition to MUC5AC and MUC5B, mainly smaller mucins which did not react with the antisera and which were probably degraded. MUC5AC appeared to be enriched in the sol, suggesting that this mucin may be more susceptible to proteolytic degradation than MUC5B. The mucins present in sputum remained broadly similar during acute exacerbation and following antibiotic treatment, although the relative amount of an acidic MUC5B glycoform was decreased during infection.

scholarly journals Macromolecular organization of saliva: identification of ‘insoluble’ MUC5B assemblies and non-mucin proteins in the gel phase

2000 ◽  
Vol 351 (2) ◽  
pp. 421-428 ◽  
Author(s):  
Claes WICKSTRÖM ◽  
Cecilia CHRISTERSSON ◽  
Julia R. DAVIES ◽  
Ingemar CARLSTEDT
Keyword(s):  
Monoclonal Antibody ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Secretory Component ◽  
Significant Fraction ◽  
Guanidinium Chloride ◽  
Whole Saliva ◽  
Gel Phase ◽  
Macromolecular Organization

Stimulated human submandibular/sublingual (HSMSL) and whole saliva were separated into sol and gel phases and mucins were isolated by density-gradient centrifugation in CsCl/4M guanidinium chloride. MUC5B and MUC7 were identified using anti-peptide antisera raised against sequences within the MUC5B and MUC7 apoproteins respectively. MUC7 was found mainly in the sol phase of both HSMSL and whole saliva, but some MUC7 was consistently present in the gel phase, suggesting that this mucin may interact with the salivary gel matrix. In HSMSL saliva, MUC5B was found in the gel phase; however, most of the material was ‘insoluble’in guanidinium chloride and was only brought into solution by reduction. In whole saliva, the MUC5B mucin was present both in the sol and gel phases although some material was again ‘insoluble’. Rate-zonal centrifugation of whole saliva showed that MUC5B mucins in the sol phase were smaller than those in the gel phase, suggesting differences in oligomerization and/or degradation. Antibodies against IgA, secretory component, lysozyme and lactoferrin were used to study the distribution of non-gel-forming proteins in the different phases of saliva. The majority of these proteins was found in the sol phase of both HSMSL and whole saliva. However, a significant fraction was present in the gel phase of whole saliva, suggesting a post-secretory interaction with the salivary gel matrix. A monoclonal antibody against a parotid salivary agglutinin was used to show that this protein is present mainly in the gel phase of both whole saliva and parotid secretion.

scholarly journals Mucus glycoproteins from cystic fibrotic sputum. Macromolecular properties and structural ‘architecture’

1991 ◽  
Vol 276 (3) ◽  
pp. 667-675 ◽  
Author(s):  
D J Thornton ◽  
J K Sheehan ◽  
H Lindgren ◽  
I Carlstedt
Keyword(s):  
Electron Microscopy ◽  
Density Gradient ◽  
Proteinase Inhibitors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Gel Phase ◽  
Mucus Glycoproteins

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].

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

1991 ◽  
Vol 276 (3) ◽  
pp. 677-682 ◽  
Author(s):  
D J Thornton ◽  
J K Sheehan ◽  
I Carlstedt
Keyword(s):  
Cystic Fibrosis ◽  
Ion Exchange ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Reactive Component ◽  
Density Fractions ◽  
Gel Phase ◽  
Acidic Nature ◽  
Mucus Glycoproteins

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.

scholarly journals Interactions between different corneal proteoglycans

1978 ◽  
Vol 173 (3) ◽  
pp. 935-939 ◽  
Author(s):  
P Speziale ◽  
M S Speziale ◽  
L Galligani ◽  
C Balduini
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Original Sample ◽  
Chemical Analyses ◽  
Guanidinium Chloride ◽  
Keratan Sulphate ◽  
Cscl Density Gradient ◽  
Two Peaks ◽  
Fraction Density

Proteoglycans were extracted from bovine cornea with 4M-guanidinium chloride and purified by CsCl-density-gradient centrifugation. Under associative conditions two fractions were found: one capable of forming assemblies of high molecular weight and another lacking this property. The heavier fraction (density 1.59 g/ml) was eluted as a single retarded peak from Sepharose 2B, but on DEAE-Sephadex chromatography, gave two peaks: the first (eluted with 0.75 M-NaCl) contained mainly proteochondroitin sulphate and the second (eluted with 1.25 M-NaCl) mainly proteokeratan sulphate. Each of these proteoglycans was more retarded on Sepharose 2B than was the original sample from density-gradient centrifugation. Re-aggregation was obtained by recombination of the two fractions. The lighter fraction (density 1.44 g/ml), containing predominantly keratan sulphate chains, was eluted from DEAE-Sephadex as a single peak with 1.25 M-NaCl and was retarded on Sepharose 2B: this fraction was not able to form aggregates with proteochondroitin sulphate. Chemical analyses of the carbohydrate and protein moieties of the proteoglycans from DEAE-Sephadex confirmed that, in the cornea, different subunits are present with characteristic aggregation properties and hydrodynamic volumes.

scholarly journals MUC5B is a major gel-forming, oligomeric mucin from human salivary gland, respiratory tract and endocervix: identification of glycoforms and C-terminal cleavage

1998 ◽  
Vol 334 (3) ◽  
pp. 685-693 ◽  
Author(s):  
Claes WICKSTRÖM ◽  
Julia R. DAVIES ◽  
Gitte V. ERIKSEN ◽  
Enno C. I. VEERMAN ◽  
Ingemar CARLSTEDT
Keyword(s):  
Respiratory Tract ◽  
Gradient Centrifugation ◽  
Guanidinium Chloride ◽  
Charge Densities ◽  
Terminal Domain ◽  
Disulphide Bonds ◽  
Whole Saliva ◽  
Submucosal Glands ◽  
Before And After ◽  
Ion Exchange Hplc

Mucins from human whole saliva, as well as from respiratory- and cervical-tract secretions, were subjected to density-gradient centrifugation in CsCl/0.5 M guanidinium chloride. A polydisperse population of MUC5B mucins was demonstrated in all samples using anti-peptide antisera (LUM5B-2, LUM5B-3 and LUM5B-4) raised against sequences within the MUC5B mucin. The sequences recognized by the LUM5B-2 and LUM5B-3 antisera are located within the domains flanking the highly glycosylated regions of MUC5B, and reduction increased the reactivity with these antibodies, suggesting that the epitopes are partially shielded and that these regions are folded and stabilized by disulphide bonds. Rate-zonal centrifugation before and after reduction showed MUC5B to be a large oligomeric mucin composed of disulphide-linked subunits. In saliva and respiratory-tract secretions, populations of MUC5B mucins with different charge densities were identified by ion-exchange HPLC, suggesting the presence of MUC5B ‘glycoforms ’. In trachea, the F2 monoclonal antibody against the sulpho-Lewis C structure reacted preferentially with the later-to-be-eluted populations. An antibody (LUM5B-4) recognizing a sequence in the C-terminal domain of MUC5B identified, after reduction, the mucin subunits as well as smaller fragments, suggesting that some of the MUC5B mucins are cleaved within the C-terminal domain. Immunohistochemistry revealed that MUC5B is produced by cells dispersed throughout the human submandibular and sublingual glands, in the airway submucosal glands as well as the goblet cells, and in the epithelium and glands of the endocervix. The F2 antibody stained a subpopulation of the MUC5B-producing cells in the airway submucosal glands, suggesting that different cells may produce different glycoforms of MUC5B in this tissue.

scholarly journals Characterization and N-terminal sequence of human platelet proteoglycan

1988 ◽  
Vol 255 (3) ◽  
pp. 1007-1013 ◽  
Author(s):  
J P Périn ◽  
F Bonnet ◽  
P Maillet ◽  
P Jollès
Keyword(s):  
Core Protein ◽  
Proteinase Inhibitors ◽  
Human Platelet ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Exchange Chromatography ◽  
Terminal Sequence ◽  
Guanidinium Chloride ◽  
Cscl Density Gradient ◽  
Yolk Sac Tumour

Human platelet proteoglycan (P.PG) was prepared from a 4 M-guanidinium chloride platelet extract in the presence of proteinase inhibitors. The purification procedure included CsCl-density-gradient centrifugation, DEAE-Sepharose CL-6B ion-exchange chromatography and f.p.l.c. on a Mono Q HR 5/5 column. P.PG was recovered as a polydisperse molecule, but the protein core appeared to be at least 90% homogeneous. This observation could be due to partial proteolysis of the core protein during extraction. The N-terminal sequence of the human P.PG core protein was determined up to residue 66 and was shown to be highly homologous to the propeptide of an embryonic rat yolk-sac tumour proteoglycan (PG19); the significance of this homology is discussed.

scholarly journals Mucus glycoproteins from ‘normal’ human tracheobronchial secretion

1990 ◽  
Vol 265 (1) ◽  
pp. 179-186 ◽  
Author(s):  
D J Thornton ◽  
J R Davies ◽  
M Kraayenbrink ◽  
P S Richardson ◽  
J K Sheehan ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Lower Respiratory Tract ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Minor Surgery ◽  
Normal Human ◽  
Macromolecular Architecture ◽  
Mucus Glycoproteins

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.

scholarly journals Isolation and characterization of human cervical-mucus glycoproteins

1983 ◽  
Vol 211 (1) ◽  
pp. 13-22 ◽  
Author(s):  
I Carlstedt ◽  
H Lindgren ◽  
J K Sheehan ◽  
U Ulmsten ◽  
L Wingerup
Keyword(s):  
Density Gradient ◽  
Proteinase Inhibitors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Disc Electrophoresis ◽  
Guanidinium Chloride ◽  
Molecular Weights ◽  
Isolation And Characterization ◽  
Mucus Glycoproteins

Mucus glycoproteins (mucins) were extracted from human cervical pregnancy mucus by 6 M-guanidinium chloride in the presence of proteinase inhibitors. Purification was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/ guanidinium chloride gradients. The purified macromolecules represented approx. 85% of the total and were devoid of nucleic acids and proteins, as judged by analytical density-gradient centrifugation, disc electrophoresis and u.v. spectroscopy. Sedimentation-velocity centrifugation revealed a single unimodal peak with S20,W 50.1S in 0.2M-NaCl and 37.0S in 6 M-guanidinium chloride. Molecular weights obtained by light-scattering were 9.7 × 10(6) and 5.9 × 10(6) in 0.2M-NaCl and 6 M-guanidinium chloride respectively. The chemical analyses were typical of those of epithelial mucins. The macromolecules contained approx. 20% (w/w) of protein, and 65% (w/w) was accounted for as carbohydrate. Serine and threonine constituted 32 mol/100 mol and proline 10 mol/100 mol of the amino acids. The major sugars found were N-acetylglucosamine (12.8%), N-acetylgalactosamine (9.7%), galactose (18.7%), sialic acid (15.0%) and fucose (7.5%).

scholarly journals Differences in the rates of aggregation of proteoglycans from human articular cartilage and chondrosarcoma

1983 ◽  
Vol 215 (3) ◽  
pp. 705-708 ◽  
Author(s):  
M T Bayliss ◽  
G D Ridgway ◽  
S Y Ali
Keyword(s):  
Articular Cartilage ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Equilibrium Density ◽  
Gel Chromatography ◽  
Human Articular Cartilage ◽  
Guanidinium Chloride ◽  
Adult Human ◽  
Comparison Of The Results

Pieces of adult human articular cartilage and chondrosarcoma were incubated in the presence of [35S]sulphate. After continuous or pulse-change incorporation of radioactivity, proteoglycans were extracted with 4.0 M-guanidinium chloride, purified by equilibrium density-gradient centrifugation and fractionated by gel chromatography. A comparison of the results suggests that the formation of stable aggregates occurs at a lower rate in articular cartilage than in chondrosarcoma.

scholarly journals Biosynthesis of the MUC2 mucin: evidence for a slow assembly of fully glycosylated units

1996 ◽  
Vol 315 (3) ◽  
pp. 1055-1060 ◽  
Author(s):  
John K. SHEEHAN ◽  
David J. THORNTON ◽  
Marj HOWARD ◽  
Ingemar CARLSTEDT ◽  
Anthony P. CORFIELD ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Molecular Mass Distribution ◽  
Cell Layer ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Guanidinium Chloride ◽  
Peptide Antibody ◽  
Agarose Electrophoresis ◽  
Average Size ◽  
Entire Distribution

The human colonic cell line PC/AA was grown to near confluency over 24 days and labelled with [14C]proline and [3H]glucose over the last 48 h in culture. The cell layer was extracted with 6 M guanidinium chloride and the mature fully glycosylated mucins were isolated at a density of 1.45 g/ml by using density-gradient centrifugation in CsCl/4 M guanidinium chloride. These mucins were identified as MUC2 with an anti-peptide antibody. The macromolecules were fragmented by reduction into two distinct populations of MUC2 subunits as assessed by agarose electrophoresis. The MUC2 mucin was polydisperse in length, ranging from 500 nm to many microns and its molecular-mass distribution, assessed by rate-zonal centrifugation, ranged from 5×106 to 40×106 Da. However, the metabolically labelled MUC2 mucins, though found throughout the whole distribution, were of much smaller average size. Since the entire distribution is not uniformly radiolabelled over 48 h, the formation of the largest species must be preceded by glycosylation and occur slowly, over several days, via the assembly of fully glycosylated units which are likely to be at least dimers [Asker, Baeckstrom, Axelsson, Carlstedt, and Hansson (1995) Biochem. J. 308, 873–880].

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