The macromolecular structure of human cervical-mucus glycoproteins. Studies on fragments obtained after reduction of disulphide bridges and after subsequent trypsin digestion.
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ABSTRACT:
SUBMITTER: Carlstedt I
PROVIDER: S-EPMC1152144 | biostudies-other | 1983 Aug
REPOSITORIES: biostudies-other
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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:Pig small-intestinal mucus glycoprotein, of molecular weight 1.72 X 10(6), is cleaved by Pronase digestion into glycoprotein subunits of molecular weight 4.5 X 10(5). Of the protein component of the native glycoprotein 29% by weight was lost on Pronase digestion, with no loss of carbohydrate. The non-glycosylated region of the protein that was lost with proteolytic digestion had a broad spectrum of amino acid residues, in contrast with the glycosylated region of the protein, which was resistant to proteolysis and was rich in serine, threonine and proline residues. Reduction with 0.2M-mercaptoethanol dissociated the Pronase-digested glycoprotein subunits into smaller glycoprotein subunits of molecular weight 2.7 X 10(5). On reduction, the native glycoprotein was dissociated into subunits of molecular weight 2.4 X 10(5), a similar size to those obtained from reduction of the Pronase-digested glycoprotein. On reductive dissociation of the native glycoprotein, in addition to glycoprotein subunits, protein was also released principally as a component of 90000 molecular weight. This protein was separated quantitatively from the reduced glycoprotein in amounts compatible with one 90000-mol.wt. protein molecule per 1.72 X 10(6)-mol.wt. native glycoprotein molecule. No 90000-mol.wt. protein was released on reduction of the isolated Pronase-digested glycoprotein. Pig small-intestinal mucus glycoprotein is therefore a covalent polymer of glycoprotein subunits joined by disulphide bridges. This polymeric structure differs in important respects from that previously shown for gastric mucus, in particular with respect to the size and number of component subunits per native molecule.
| S-EPMC1162883 | biostudies-other
Project description:Trypsin is the most used enzyme in proteomics. Nevertheless, proteases with complementary cleavage specificity have been applied in special circumstances. In this work, we analyzed the characteristics of five protease alternatives to trypsin for protein identification and sequence coverage when applied to S. pombe whole cell lysates. The specificity of the protease heavily impacted the number of proteins identified. Proteases with higher specificity led to the identification of more proteins than proteases with lower specificity. However, AspN, GluC, chymotrypsin, and proteinase K largely benefited from being paired with trypsin in sequential digestion, as had been shown by us for elastase before. In the most extreme case, predigesting with trypsin improves the number of identified proteins for proteinase K by 731%. Trypsin predigestion also improved the protein identifications of other proteases, AspN (+62%), GluC (+80%), and chymotrypsin (+21%). Interestingly, the sequential digest with trypsin and AspN yielded even a higher number of protein identifications than digesting with trypsin alone.
| S-EPMC7377536 | biostudies-literature
Project description:1. A new method is described for ;fingerprinting' cysteic acid peptides derived from the disulphide bridges of proteins. Cystine peptides are separated by paper electrophoresis and oxidized on paper by performic acid vapour. Electrophoresis at right angles to the first direction produces parallel groups of cysteic acid peptides lying off a diagonal. This ;fingerprint' reveals the way in which the cysteic acid peptides were originally joined in the protein. 2. The method allows a very easy selective purification of cysteic acid peptides. 3. By applying this method to bovine chymotrypsinogen A, we found that the half-cystine residues were linked 1-122, 42-58, 136-201, 168-182 and 191-220.
| S-EPMC1270086 | biostudies-other
Project description:The construction of a trypsin column for rapid and efficient protein digestion in proteomics is described. Electrospun and alcohol-dispersed polymer nanofibers were used for the fabrication of highly stable trypsin coatings, which were prepared by a two-step process of covalent attachment and enzyme cross-linking. In a comparative study with the trypsin coatings on as-spun and nondispersed nanofibers, it has been observed that a simple step of alcohol dispersion improved not only the enzyme loading but also the performance of protein digestion. In-column digestion of enolase was successfully performed in less than 20 min. By applying the alcohol dispersion of polymer nanofibers, the bypass of samples was reduced by filling up the column with well-dispersed nanofibers, and subsequently, interactions between the protein and the trypsin coatings were improved, yielding more complete and reproducible digestions. Regardless of alcohol dispersion or not, trypsin coatings showed better digestion performance and improved performance stability under recycled uses than covalently attached trypsin, in-solution digestion, and commercial trypsin beads. The combination of highly stable trypsin coatings and alcohol dispersion of polymer nanofibers has opened up a new potential to develop a trypsin column for online and automated protein digestion.
| S-EPMC2950708 | biostudies-literature
Project description:Evidence is presented for the disulphide bridges in bovine Factor X. The protein was degraded by chemical and enzymic means, and all 12 disulphide bridges were isolated in separate peptides except for bridges nos. 6/7 in the light chain. All the disulphide bridges were found to be in positions corresponding to those found in other homologous domains. This report is the first verification of an epidermal-growth-factor-homologous domain having the same disulphide-bonding pattern as that found in mouse epidermal growth factor.
| S-EPMC1148212 | biostudies-other
Project description:BACKGROUND: Phospholipase C (PLC) is an enzyme that plays pivotal role in a number of signaling cascades. These are active in the plasma membrane and triggers cellular responses by catalyzing the hydrolysis of membrane phospholipids and thereby generating the secondary messengers. Phosphatidylinositol-PLC (PI-PLC) specifically interacts with phosphoinositide and/or phosphoinositol and catalyzes specific cleavage of sn-3- phosphodiester bond. Several isoforms of PLC are known to form and function as dimer but very little is known about the molecular basis of the dimerization and its importance in the lipid interaction. PRINCIPAL FINDINGS: We herein report that, the disruption of disulphide bond of a novel PI-specific PLC of C. reinhardtii (CrPLC) can modulate its interaction affinity with a set of phospholipids and also the stability of its dimer. CrPLC was found to form a mixture of higher oligomeric states with monomer and dimer as major species. Dimer adduct of CrPLC disappeared in the presence of DTT, which suggested the involvement of disulphide bond(s) in CrPLC oligomerization. Dimer-monomer equilibrium studies with the isolated fractions of CrPLC monomer and dimer supported the involvement of covalent forces in the dimerization of CrPLC. A disulphide bridge was found to be responsible for the dimerization and Cys7 seems to be involved in the formation of the disulphide bond. This crucial disulphide bond also modulated the lipid affinity of CrPLC. Oligomers of CrPLC were also captured in in vivo condition. CrPLC was mainly found to be localized in the plasma membrane of the cell. The cell surface localization of CrPLC may have significant implication in the downstream regulatory function of CrPLC. SIGNIFICANCE: This study helps in establishing the role of CrPLC (or similar proteins) in the quaternary structure of the molecule its affinities during lipid interactions.
| S-EPMC3380823 | biostudies-literature
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 β-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 α-helix/unordered structures and many signatures of glycosylation, pointing to a highly O-glycosylated region on the mucin.
| S-EPMC5218386 | biostudies-literature
Project description:The five cystine peptides of chymotrypsinogen B have been isolated as pairs of cysteic acid peptides by the diagonal electrophoretic technique of Brown & Hartley (1963, 1966). The sequences of these peptides have been shown to be very similar to those of chymotrypsinogen A, and indicate that both zymogens have the same pattern of disulphide bridges. The determination of the sequence of residues 14-21 in chymotrypsinogen B has shown that residue 18 differs in the chymotrypsinogens from the corresponding residue in trypsinogen. Eight differences between chymotrypsinogens A and B are found in sequences accounting for 72 of the 245 residues.
| S-EPMC1198434 | biostudies-other
Project description:The arrangement of the disulphide bridges of the major component of the light chains of immunoglobulins (kappa-chains) has been studied in the Bence-Jones proteins. Three disulphide bridges have been found. An interchain bridge at the C-terminus has been shown to occur in the dimers of all the proteins studied and was characterized by symmetrical peptides. In the monomer form, the C-terminal half-cystine of the corresponding peptides was linked to a lone half-cystine residue. A second common disulphide-bridge peptide in which a single amino acid difference could be related to the Inv factors of the individual proteins was found in Bence-Jones proteins and in the kappa-chains of normal and abnormal immunoglobulins. Peptides characteristic of a third disulphide bridge studied in three specimens were found to have differences in some residues, but also striking similarities. A methionine peptide has also been characterized in two specimens as a by-product of the technique employed. It is suggested that a general manner of folding may be a common feature of the heterogeneous population of kappa-chains: one bridge which folds an invariable stretch of the chain, another bridge which folds a stretch that varies from protein to protein, and a bridge at the C-terminus which is the interchain link.
| S-EPMC1270114 | biostudies-other