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 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 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 The effect of guanidinium chloride on the behaviour of human cervical-mucus glycoproteins. Evidence for unfolding regions of ordered structure in 6M-guanidinium chloride

1984 ◽  
Vol 221 (2) ◽  
pp. 499-504 ◽  
Author(s):  
J K Sheehan ◽  
I Carlstedt
Keyword(s):  
Frictional Coefficient ◽  
Correlation Spectroscopy ◽  
Cervical Mucus ◽  
Random Coil ◽  
Radius Of Gyration ◽  
Ordered Structure ◽  
Guanidinium Chloride ◽  
Photon Correlation ◽  
The Individual ◽  
Mucus Glycoproteins

The macromolecular properties of cervical-mucus glycoproteins (mucins) were studied as a function of the concentration of guanidinium chloride with conventional light-scattering, photon-correlation spectroscopy and sedimentation-velocity centrifugation. No evidence for an association of the mucins in 0.2M-NaCl as compared with 6M-guanidinium chloride was found at mucin concentrations below approx. 0.5 mg/ml. However, an increase in the frictional coefficient and in the radius of gyration occurred with increasing concentrations of guanidinium chloride, in particular between 4 M and 6 M, suggesting an expansion of the individual macromolecule. The change in the particle-scattering function is consistent with a transition from a ‘stiff’ random coil in 0.2 M-NaCl into a more flexible one in 6 M-guanidinium chloride. We suggest that the mucins contain regions of ‘ordered’ structure which can undergo a reversible ‘unfolding’ analogous to the behaviour of a conventional globular protein exposed to a denaturing solvent. Such regions might carry sites for specific interactions between mucins and/or be decisive for their conformation and macromolecular properties in physiological solvents.

scholarly journals Purification and partial characterization of a tumour-metastasis-associated high-Mr glycoprotein from rat 13762NF mammary adenocarcinoma cells

1987 ◽  
Vol 242 (3) ◽  
pp. 779-787 ◽  
Author(s):  
P A Steck ◽  
S M North ◽  
G L Nicolson
Keyword(s):  
Cellular Localization ◽  
Proteinase Inhibitors ◽  
Cell Clone ◽  
Gradient Centrifugation ◽  
Mammary Adenocarcinoma ◽  
Metastatic Tumour ◽  
Guanidinium Chloride ◽  
Tumour Metastasis ◽  
Adenocarcinoma Cells ◽  
Cscl Density Gradient

The expression of a high-Mr sialogalactoprotein (gp580) on rat 13762NF mammary adenocarcinoma cells was identified and correlated with spontaneous metastatic potential to colonize lung [Steck & Nicolson (1983) Exp. Cell Res. 147, 255-267]. Using a highly metastatic tumour-cell clone, MTLn3, we isolated and characterized gp580 from cells growing in vitro and in vivo in the mammary fat-pads of Fischer 344 rats. The glycoprotein was extracted with 4 M-guanidinium chloride/4% Zwittergent 3-12 solution in the presence of proteinase inhibitors. The extracts were then subjected to dissociative CsCl-density-gradient centrifugation, gel filtration on Sepharose CL-2B columns and ion-exchange chromatography on DEAE-Sephacel. The isolated glycoprotein possessed low electrophoretic mobility in SDS/polyacrylamide gels, and after desialylation bound 125I-labelled peanut agglutinin. Electrophoresis of gp580 in polyacrylamide-gradient gels resulted in a diffuse but homogeneous migrating band of Mr approx. 55,000. After removal of carbohydrate, gp580 was demonstrated to have a protein core of Mr approx. 150,000. The gp580 had a high density (1.430 g/ml) on isopycnic centrifugation in 4 M-guanidinium chloride and was resistant to most proteinases and other degradative enzymes, suggesting a mucin-like structure. Amino acid and carbohydrate analyses revealed that gp580 has high contents of serine, threonine, glutamic acid, aspartic acid, glucosamine and galactosamine; several acidic and neutral oligosaccharides were obtained from alkaline-borohydride digests. Cellular localization studies suggested that gp580 is associated mainly with the cell-surface and extracellular-matrix fractions of MTLn3 cells.

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 Electron microscopy of cervical, gastric and bronchial mucus glycoproteins

1986 ◽  
Vol 239 (1) ◽  
pp. 147-153 ◽  
Author(s):  
J K Sheehan ◽  
K Oates ◽  
I Carlstedt
Keyword(s):  
Electron Microscopy ◽  
Proteinase Inhibitors ◽  
Gradient Centrifugation ◽  
Uranyl Acetate ◽  
Gastric Mucus ◽  
Bronchial Mucus ◽  
Mucus Gel ◽  
Macromolecular Architecture ◽  
Mucus Glycoproteins ◽  
Low Shear

Mucus glycoproteins (mucins) were obtained from human cervical and pig gastric mucus as well as from chronic-bronchitic sputum after low-shear extraction. The mucus gel was solubilized in guanidinium chloride supplemented with proteinase inhibitors, and the macromolecules were purified by using isopycnic density-gradient centrifugation. The macromolecules were spread in monolayers of benzyldimethylalkyl-ammonium chloride and studied with electron microscopy after staining with uranyl acetate and/or shadowing with platinum/carbon. The mucins appeared as flexible linear threads with lengths varying from approx. 200 nm to about 400 nm. No regularly branched or star-shaped structures were observed. The macromolecular architecture of cervical, respiratory and gastric mucins is thus similar.

scholarly journals Purification and partial characterization of the major cell-associated heparan sulphate proteoglycan of rat liver

1991 ◽  
Vol 273 (2) ◽  
pp. 415-422 ◽  
Author(s):  
M Lyon ◽  
J T Gallagher
Keyword(s):  
Molecular Mass ◽  
Proteinase Inhibitors ◽  
Gradient Centrifugation ◽  
Heparan Sulphate ◽  
Exchange Chromatography ◽  
Guanidinium Chloride ◽  
Apparent Homogeneity ◽  
Core Proteins ◽  
Sds Page ◽  
Triton X

Heparan sulphate proteoglycans were solubilized from whole rat livers by homogenization and dissociative extraction with 4 M-guanidinium chloride containing Triton X-100 and proteinase inhibitors. The extract was subjected to trichloroacetic acid precipitation and the proteoglycan remained soluble. This was then purified to apparent homogeneity by a combination of (a) DEAE-Sephacel chromatography, (b) digestion with chondroitinase ABC followed by f.p.l.c. Mono Q ion-exchange chromatography, and (c) density-gradient centrifugation in CsCl and 4 M-guanidinium chloride. Approx. 1.5 mg of proteoglycan was obtained from 30 livers with an estimated recovery of 25%. The purified proteoglycan was eluted from Sepharose CL6B as an apparently single polydisperse population with a Kav. of 0.19 and displayed a molecular mass of greater than or equal to 200 kDa (relative to protein standards) by SDS/PAGE. Its heparan sulphate chains were eluted with a Kav. of 0.44 and have an estimated molecular mass of 25 kDa. Digestion of the proteoglycan with a combination of heparinases yielded core proteins of 77, 49 and 44 kDa. Deglycosylation using trifluoromethanesulphonic acid, though slightly decreasing the sizes, gave an identical pattern of core proteins. Electrophoretic detergent blotting demonstrated that all of the core proteins were hydrophobic and are probably integral plasma membrane molecules. The peptide maps generated by V8 proteinase digestion of the two major core proteins (77 and 49 kDa) were very similar, suggesting that these two core proteins are structurally related.

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 dermatan sulphate proteoglycan from human uterine cervix

1983 ◽  
Vol 209 (2) ◽  
pp. 497-503 ◽  
Author(s):  
N Uldbjerg ◽  
A Malmström ◽  
G Ekman ◽  
J Sheehan ◽  
U Ulmsten ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Uterine Cervix ◽  
Proteinase Inhibitors ◽  
Gradient Centrifugation ◽  
Average Molecular Weight ◽  
Deae Cellulose ◽  
Sedimentation Equilibrium ◽  
Dermatan Sulphate ◽  
Guanidinium Chloride ◽  
Isolation And Characterization

Proteoglycans were extracted from human uterine cervix with 4 M-guanidinium chloride in the presence of proteinase inhibitors. They were purified by density-gradient centrifugation in 4 M-guanidinium chloride/CsCl (starting density 1.32 g/ml) followed by DEAE-cellulose and Sepharose chromatography. Only one polydisperse proteoglycan was found. s020,w was 2.1S and the weight-average molecular weight was 73 000 (sedimentation-equilibrium centrifugation) to 110 500 (light-scattering). The core protein was monodisperse, with an apparent molecular weight of 47 000. The proteoglycan contained about 30% protein and probably two or three glycosaminoglycan side chains per molecule. High contents of aspartate, glutamate and leucine were found. The glycan moiety of the proteoglycan was exclusively dermatan sulphate, with a co-polymeric structure with approximately equal quantities of iduronic acid- and glucuronic acid-containing disaccharides.

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