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

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 X 10(6] were degraded into 'subunits' (Mr approx. 2 X 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.     

Macromolecular architecture and hydrodynamic properties of human cervical mucins

Cervical mucus glycoproteins (mucins) were extracted by using slow stirring in 6M-guanidinium chloride supplemented with proteinase inhibitors. Subsequent purification was achieved by isopycnic density-gradient centrifugation in CsCl/guanidinium chloride. The whole mucins (Mr 10 X 10(6) - 15 X 10(6)) were degraded into subunits (Mr 2 X 10(6) - 3 X 10(6)) by reduction. Trypsin digestion of subunits afforded glycopeptides (T-domains) with Mr 0.4 X 10(6). The relationship between the intrinsic viscosity and Mr for the whole mucins and the fragments suggests that cervical mucins are linear flexible macromolecules. This view is supported by hydrodynamic data.     

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

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.     

A study of equilibrium binding of link protein to hyaluronate.

Link protein was extracted from bovine femoral-head cartilage, radiolabelled while in the proteoglycan-aggregate stage, and then purified by density-gradient centrifugation and gel chromatography. The purity of the preparation was assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and two species with approx. mol.wts. 45000 and 48000 were observed. Sedimentation-velocity experiments were performed in 0.5 M-guanidinium chloride/5 mM-phosphate, pH 7.4, and yielded an SO20, w of 4.75S. The proportion of link protein unable to interact with hyaluronate was determined by chromatography on Sepharose CL-4B. The binding of link protein to high-molecular-weight hyaluronate was studied by frontal-gel chromatography on Sepharose CL-4B in 0.5 M-guanidinium chloride/5 mM-phosphate/0.1% bovine serum albumin, pH 7.4. Experiments were performed at 10, 17 and 25 degrees C and the results were treated as described by Scatchard [(1949) Ann. N.Y. Acad. Sci. 51, 660-672]. Dissociation constants of approx. (1-4) X 10(-8) M were obtained. The length of hyaluronate occupied per link-protein molecule was determined to be six to seven disaccharides.     

New isolation procedure and further biochemical characterization of glycoproteins IIb and IIIa from human platelet plasma membrane.

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.     

Isolation and characterization of dermatan sulphate proteoglycan from human uterine cervix.

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.     

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.     

Interactions between different corneal proteoglycans.

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.     

The composition and proposed subunit structure of egg-white beta-ovomucin. The isolation of an unreduced soluble ovomucin.

1. New preparations of reduced carboxymethylated beta-ovomucin (S-carboxymethyl-beta-ovomucin) were homogeneous by sedimentation analysis, analytical sedimentation to equilibrium in CsCl gradients, and disc electrophoresis in sodium dodecyl sulphate. 2. Degradation of S-carboxymethyl-beta-ovomucin with either CNBr or trypsin indicated the presence of a subunit (approx. mol. wt. 112300). 3. Electron microscopy showed that S-carboxymethyl-beta-ovomucin consisted of chains of globular units (approx. mol. wt. 103 000). IN 6M-guanidinium chloride S-carboxymethyl-beta-ovomucin existed mainly as an aggregate (mol. wt. 720 000). 4. S-Carboxymethyl-beta-ovomucin contained ester sulphate (4.24%, W/W) and carbohydrate (60%, W/W), which consisted of large amounts of galactose (22%, W/W), galactosamine (8.9%, W/W) and sialic acid (10.6%, W/W). 5. An unreduced soluble fibrous component (component SGH) extracted from crude ovomucin precipitate with 5M-guanidinium chloride contained beta-ovomucin (approx. 70%, W/W). By using the Scheraga-Mandelkern equation the molecular weight of component SGH was calculated to be 11.5 times 10(6).     

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.     

Physicochemical characterization of the ribosomal RNA species of the Mollusca. Molecular weight, integrity and secondary-structure features of the RNA of the large and small ribosomal subunits.

1. The rRNA species of the Cephalopoda Octopus vulgaris and Loligo vulgaris were found to have unexpectedly high sedimentation coefficients and molecular weights. In 0.1 M-NaCl the L-rRNA (RNA from large ribosomal subunit) has the same s20 value as the L-rRNA of the mammals (30.7S), whereas the S-rRNA (RNA from small ribosomal subunit) sediments at a faster rate (20.1S) than the S-rRNA of both the mammals and the fungi (Neurospora crassa) (17.5S). The molecular weights of the L-rRNA were determined by gel electrophoresis in formamide and found to be 1.66 X 10(6) (Octupus) and 1.89 X 10(6) (Loligo); the mol.wt. of the S-rRNA of both species is 0.96 X 10(6), i.e. much larger than that of the mammals (0.65 X 10(6)) and almost coincident with that of the '23S' RNA of the prokaryotes. 2. By contrast, the less evolved Gastropoda and Lamellibranchiata (Murex trunculus and Macrocallista chione) have S-rRNA and L-rRNA species with mol.wts. of 0.65 X 10(6) and approx. 1.40 X 10(6).3. All the mature L-rRNA molecules of the cephalopoda are composed of two unequal fragments held together by regions of hydrogen-bonding having a similar, low, thermal stability in the two species; the molecular weights of the two fragments composing the L-rRNA are estimated to be 0.96 X 10(6) and 0.88 X 10(6) (Loligo) and 0.96 X 10(6) and 0.65 X 10(6) (Octupus). THe S-rRNA of both species is a continuous chain with exactly the same molecular weight (0.96 X 10(6)) as the heavier of the two fragments of the L-rRNA. 4. The secondary-structure features of the L-rRNA and S-rRNA species of the Caphalopoda were investigated by thermal 'melting' analysis in 4.0 M-guanidinium chloride; 60-70% of the residues are estimated to form short, independently 'melting' bihelical segments not more than 10 base-pairs in length. 5. Bases are unevenly distributed between non-helical and bihelical portions of the rRNA molecules, G and C residues being preferentially concentrated in bihelical comains. 6. The secondary-structure regions of the L-rRNA species of Octopus and Loligo are heterogenous, including two discrete fractions of independently 'melting' species that give rise to biphasic 'melting' profiles: a fraction consisting of shorter (G + C)-poorer segments (60-68% G + C, not more than 5 base-pairs in length) and a fraction consisting of longer (G + C)-richer segments (80-88% G + C, 5-10 base-pairs in length). No evidence for heterogeneity has been detected in the S-rRNa.     

Hydrodynamic properties of human cervical-mucus glycoproteins in 6M-guanidinium chloride.

Cervical mucins and fragments thereof were studied by sedimentation-velocity, rotatory viscometry and laser light-scattering performed as photon-correlation spectroscopy as well as low-angle total-intensity measurements. The Mr of the whole mucins is 10 X 10(6)-15 X 10(6), whereas fragments obtained after reduction of disulphide bonds ('subunits') have Mr 2.1 X 10(6)-2.9 X 10(6), depending on the method used. Subsequent trypsin digestion of subunits afforded glycopeptides with Mr approx. 0.4 X 10(6). The high frictional ratio for the whole mucins is interpreted as a large degree of expansion. The Stokes radius calculated from the diffusion coefficient is approx. 110nm for the whole mucins, which is in agreement with that estimated from the radius of gyration (130nm) by using the concept of the equivalent hydrodynamic sphere. The ratio of the concentration-dependence parameter for the reciprocal sedimentation coefficient (Ks) to the intrinsic viscosity ( [eta] ) for the whole mucins is 1.42, suggesting that the individual macromolecule occupies a spheroidal domain in solution. The relationship between [eta] and Mr for whole mucins, subunits and T-domains suggests that they are linear flexible macromolecules behaving as somewhat 'stiff' random coils. This conclusion is supported by the relationships between the sedimentation coefficients, the diffusion coefficients and the Mr. The hydrodynamic behaviour of the mucins is thus close to that expected for coiling macromolecules entrapping a lot of solvent, which is consistent with the postulated polymeric structure.     

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.     

Nature of sulphated macromolecules in mouse Reichert''s membrane. Evidence for tyrosine O-sulphate in basement-membrane proteins.

Seven different sulphated macromolecules were detected in 6 M-guanidinium chloride extracts of metabolically [35S]sulphate-labelled mouse Reichert's membrane and were partially separated. Polypeptide bands of apparent Mr 50 000, 150 000 (tentatively identified as entactin) and 170 000 contained essentially tyrosine O-sulphate as the labelled component. Most of the radioactive sulphate was incorporated into three different proteoglycans, which could be separated by chromatography and density-gradient centrifugation before and after enzymic degradation. Enzymic analysis of glycosaminoglycans and of protein cores by immunoassays identified these components as low-density and high-density forms of heparan sulphate proteoglycan and a high-density form of chondroitin sulphate or dermatan sulphate proteoglycan.     

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

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.     

Purification and characterization of goblet-cell mucin of high Mr from the small intestine of sheep.

Crude soluble mucus from sheep small intestine was freed of nearly all the nucleic acid contaminants by precipitation with protamine sulphate and treatment with nucleases. After removal of non-covalently bound proteins by equilibrium density-gradient centrifugation in CsCl, a high-Mr glycoprotein was isolated by repeated h.p.l.c. from the partially purified mucin. The high degree of purity of the high-Mr mucin was borne out by (a) the observation of a single boundary on analytical ultracentrifugation in the presence of 5M-guanidinium chloride and (b) the observation of apparent monodispersity on sedimentation-equilibrium analysis. The Mr of the highly purified mucin, determined by sedimentation equilibrium, was 5.0 (+/- 0.1) X 10(6) and was concentration-independent. Finally, only goblet cells and the mucus blanket lining the intestinal epithelial cells were immunofluorescent when guinea-pig anti-(highly purified mucin) serum was used in an indirect immunofluorescence assay. The above antiserum reacted with apparently equal strength with goblet cells and with free mucin in abomasum, caecum and colon. The chemical composition of the glycoprotein was 66% carbohydrate and 34% protein, 45% of the latter being composed of valine and threonine. The glycoprotein migrated anodally on immunoelectrophoresis and contained 7.1% (w/w) sulphate. Neutral hexoses accounted for nearly half of the total carbohydrate content, followed by galactosamine and glucosamine. Whereas fucose and sialic acid were present in only small amounts, uronic acid was not detectable in the highly purified mucus glycoprotein.     

Separation and characterization of two populations of aggregating proteoglycans from cartilage.

Intermediary gel immunoelectrophoresis was used to show that purified aggregating cartilage proteoglycans from 2-year-old steers contain two distinct populations of molecules and that only one of these is immunologically related to non-aggregating cartilage proteoglycans. The two types of aggregating proteoglycans were purified by density-gradient centrifugation in 3.5M-CsCl/4M-guanidinium chloride and separated by zonal rate centrifugation in sucrose gradients. The higher-buoyant-density faster-sedimenting proteoglycan represented 43% of the proteoglycans in the extract. It had a weight-average Mr of 3.5 X 10(6), did not contain a well-defined keratan sulphate-rich region, had a quantitatively dominant chondroitin sulphate-rich region and contained 5.9% protein and 23% hexosamine. The lower-buoyant-density, more slowly sedimenting, proteoglycan represented 15% of the proteoglycans in the extract. It had a weight-average Mr of 1.3 X 10(6), contained both the keratan sulphate-rich and the chondroitin sulphate-rich regions and contained 7.3% protein and 23% hexosamine. Each of the proteoglycan preparations showed only one band on agarose/polyacrylamide-gel electrophoresis. The larger proteoglycan had a lower mobility than the smaller. The distribution of chondroitin sulphate chains along the chondroitin sulphate-rich region was similar for the two types of proteoglycans. The somewhat larger chondroitin sulphate chains of the larger proteoglycan could not alone account for the larger size of the proteoglycan. Peptide patterns after trypsin digestion of the proteoglycans showed great similarities, although the presence of a few peptides not shared by both populations indicates that the core proteins are partially different.     

Isolation and biochemical characterization of the tryptic fragments of bovine nasal-cartilage proteoglycan monomer of high buoyant density.

Relatively homogeneous fractions of proteoglycan fragments were prepared from tryptic digests of the 4M-guanidinium chloride extract of bovine nasal cartilage. Glycosaminoglycan-containing fragments were separated from non-proteoglycan contaminants by ion-exchange chromatography and fractionated by equilibrium density-gradient centrifugation under dissociative conditions. The fractions of highest buoyant density were chromatographed on a column of Sepharose 4B, digested with chondroitinase ABC and chromatographed on a column of Sepharose 6B, yielding two distinct fractions: fraction B/6B-4 contained fragments from the chondroitin sulphate-bearing region of the proteoglycan monomer, and fraction B/6B-2 fragments from the keratan sulphate-rich region, most probably including a chondroitin sulphate-bearing monomer segment. By dansyl chloride analysis, fraction B/6B-2 had alanine and leucine as sole and fraction B/6B-4 had isoleucine and leucine as greatly predominant N-terminal amino acids, indicative of the relative homogeneity of these preparations of cartilage proteoglycan monomer fragments.     

Matrix proteins bound to associatively prepared proteoglycans from bovine cartilage.

Proteoglycans were extracted from bovine tracheal cartilage by high-speed homogenization, the use of dissociative solvents being avoided. The homogenate was fractionated by gel chromatography, sucrose-density-gradient centrifugation and ion-exchange chromatography. A previously unrecognized protein, cartilage matrix protein, was identified by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It cofractionated with the proteoglycans in all systems, indicating an interaction. The cartilage matrix protein-proteoglycan complex was dissociated by treatment with 4M-guanidinium chloride. The complex again formed when the guanidine was removed. The cartilage matrix protein has a mol.wt. of more than 200000. On reduction it yields subunits with a mol.wt. of approx. 60000.     

Purification and structural characterization of a cartilage matrix protein.

The cartilage matrix protein is a major non-collagenous protein in bovine cartilage. It was purified from a 5 M-guanidinium chloride extract of bovine tracheal cartilage by sequential CsCl-density-gradient centrifugation, gel chromatography in guanidinium chloride and differential precipitation. The molecular weight of the intact protein is 148 000, determined by sedimentation-equilibrium centrifugation. It was dissociated to three subunits of molecular weight 52 000 by reduction of disulphide bonds. The cartilage matrix protein was insoluble in low-salt solutions and behaved abnormally on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The content of cysteine was high, whereas the contents of aromatic amino acids were low. The carbohydrate content was 3.9% (w/w). Glycopeptides obtained after papain digestion were heterogenous on gel chromatography. Asparagine/aspartic acid was enriched in the purified glycopeptides, indicating the presence of N-glycosidic linkages to protein.