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 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 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 The macromolecular structure of human cervical-mucus glycoproteins. Studies on fragments obtained after reduction of disulphide bridges and after subsequent trypsin digestion

1983 ◽  
Vol 213 (2) ◽  
pp. 427-435 ◽  
Author(s):  
I Carlstedt ◽  
H Lindgren ◽  
J K Sheehan
Keyword(s):  
Proteinase Inhibitors ◽  
Gradient Centrifugation ◽  
Cervical Mucus ◽  
Trypsin Digestion ◽  
Radius Of Gyration ◽  
Guanidinium Chloride ◽  
Disulphide Bonds ◽  
Tentative Model ◽  
The Relationship ◽  
Mucus Glycoproteins

Human cervical-mucus glycoproteins (mucins) were extracted with 6 M-guanidinium chloride in the presence of proteinase inhibitors and purified by isopycnic density-gradient centrifugation. The whole mucins (Mr approx. 10 × 10(6] were degraded into ‘subunits’ (Mr approx. 2 × 10(6] by reduction of disulphide bonds. Trypsin digestion of the ‘subunits’ produced glycopeptides with Mr approx. 380000, which appear to be rod-like with a length of approx. 105 nm. The relationship between the radius of gyration and the Mr value obtained by light-scattering for whole mucins, ‘subunits’ and ‘domains’ suggest that cervical-mucus glycoproteins are linear flexible macromolecules composed of, on the average, four or five ‘domains’/subunit and four subunits/whole mucin macromolecule. The shape-dependent particle scattering function for the whole mucins and the ‘subunits’ are in accordance with that of a linear flexible chain. No evidence for a branched or a star-like structure was found. A tentative model for cervical mucins is proposed.

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 An interaction between lysozyme and mucus glycoproteins. Implications for density-gradient separations

1979 ◽  
Vol 181 (3) ◽  
pp. 717-724 ◽  
Author(s):  
J M Creeth ◽  
J L Bridge ◽  
J R Horton
Keyword(s):  
Sialic Acid ◽  
Ionic Strength ◽  
Density Gradient ◽  
Blood Group ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Ph Values ◽  
The Common ◽  
Mucus Glycoproteins

1. Some mucus glycoproteins form soluble complexes with lysozyme at neutral pH values. 2. The extent of complex-formation was determined, by an ultracentrifugal difference method, for a range of glycoproteins covering the common blood-group specificities. 3. Interaction was strongest with those glycoproteins of blood-group Lea specificity; these were also richest in sialic acid. 4. Interaction diminished with increase of ionic strength, and was not detectable at I 0.50; however, an asialoglycoprotein was found to retain some activity. The interaction is accordingly primarily, but probably not exclusively, coulombic in origin. 5. The buoyant density of lysozyme in CsCl, CsBr, CsI and Cs2SO4 was determined; the values in the last three salts are anomalously high. This finding accounts for the previously noted difficulty of separating free protein from glycoproteins by single-stage centrifugation in CsBr. 6. Conditions for effective separation of glycoproteins from secretions containing lysozyme by density-gradient centrifugation are reported.

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