Further evidence for a flexible and highly expanded spheroidal model for mucus glycoproteins in solution.
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ABSTRACT:
SUBMITTER: Harding SE
PROVIDER: S-EPMC1154172 | biostudies-other | 1983 Mar
REPOSITORIES: biostudies-other
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Project description:The major structural components of protective mucus hydrogels on mucosal surfaces are the secreted polymeric gel-forming mucins. The very high molecular weight and extensive O-glycosylation of gel-forming mucins, which are key to their viscoelastic properties, create problems when studying mucins using conventional biochemical/structural techniques. Thus, key structural information, such as the secondary structure of the various mucin subdomains, and glycosylation patterns along individual molecules, remains to be elucidated. Here, we utilized Raman spectroscopy, Raman optical activity (ROA), circular dichroism (CD), and tip-enhanced Raman spectroscopy (TERS) to study the structure of the secreted polymeric gel-forming mucin MUC5B. ROA indicated that the protein backbone of MUC5B is dominated by unordered conformation, which was found to originate from the heavily glycosylated central mucin domain by isolation of MUC5B O-glycan-rich regions. In sharp contrast, recombinant proteins of the N-terminal region of MUC5B (D1-D2-D'-D3 domains, NT5B), C-terminal region of MUC5B (D4-B-C-CK domains, CT5B) and the Cys-domain (within the central mucin domain of MUC5B) were found to be dominated by the ?-sheet. Using these findings, we employed TERS, which combines the chemical specificity of Raman spectroscopy with the spatial resolution of atomic force microscopy to study the secondary structure along 90 nm of an individual MUC5B molecule. Interestingly, the molecule was found to contain a large amount of ?-helix/unordered structures and many signatures of glycosylation, pointing to a highly O-glycosylated region on the mucin.
| S-EPMC5218386 | biostudies-literature
Project description: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.
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Project description:Evidence is presented from three experimental systems to support the allosteric model of Walker et al. (1975) (Biochem. J. 147, 425-433) which explains the substrate-concentration-dependent transition observed in the RNAase (ribonuclease)-catalysed hydrolysis of 2':3'-cyclic CMP (cytidine 2':3'-cyclic monophosphate). 1. Kinetic studies of the initial rate of hydrolysis of 2':3'-cyclic CMP show that the midpoint of the transition shifts to lower concentrations of 2':3'-cyclic CMP in the presence of the substrate analogues 3'-CMP, 5'-CMP, 3'-AMP, 3'-UMP and Pi; 2'-CMP and 2'-UMP do not cause such a shift. 2. Trypsin-digestion studies show that a conformational change in RNAase to a form less susceptible to tryptic inactivation is induced in the presence of the substrate analogues 3'-CMP, 5'-CMP, 3'-AMP, and 3'-UMP. 2'-CMP, 2'-AMP and 2'-UMP do not induce this conformational change. 3. Equilibrium-dialysis experiments demonstrate the multiple binding of molecules of 3'-CMP, 3'-AMP and 5'-AMP to a molecule of RNAase. 2'-CMP binds the ratio 1:1 over the analogue concentration range studied.
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Project description: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 X 10(6) and 5.9 X 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%).
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Project description: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.
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Project description:The depolymerization of bovine cervical glycoprotein resulting from cleavage of disulphide bonds. Pronase digestion and both procedures sequentially was assessed by using gel filtration. Cleavage of disulphide bonds followed by Pronase digestion produced more extensive depolymerization than did either treatment alone, and gel filtration of the products resulted in two major peaks of glycosylated material on Sepharose CL-2B and Sepharose 4B. The glycopolypeptides in both peaks had similar sugar and sulphate compositions, but they migrated to different extents on gel electrophoresis. Electrophoretic studies indicated that both glycopolypeptides were derived from the same glycoprotein molecule and not from a mixture of two similar glycoproteins. Pronase digestion of glycoproteins in which the disulphide bonds had been labelled with iodo-[1-14C]acetamide revealed that most of the cysteine residues were situated in regions susceptible to Pronase. The results show the presence of two types of structural regions in bovine cervical glycoprotein, namely 'naked' peptide or non-glycosylated regions and glycopolypeptide subunit regions in which glycopolypeptides of two different sizes predominate. Comparison of the cervical glycoproteins isolated from mucus secreted during oestrus and pregnancy, by the methods outlined above, did not reveal any structural differences in the glycoproteins to explain the different physical properties of the mucus secreted under these conditions.
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Project description: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].
| S-EPMC1151057 | biostudies-other
Project description: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.
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