The disulphide bonds of human and rabbit gamma-globulins.
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ABSTRACT:
SUBMITTER: Cecil R
PROVIDER: S-EPMC1264677 | biostudies-other | 1965 Nov
REPOSITORIES: biostudies-other
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Project description:Disulphide bonds are stabilizing crosslinks in proteins and serve to enhance their thermal stability. In proteins that are small and rich in disulphide bonds, they could be the major determining factor for the choice of conformational state since their constraints on appropriate backbone conformation can be substantial. Such crosslinks and their positional conservation could itself enable protein family and functional association. Despite the importance of the field, there is no comprehensive database on disulphide crosslinks that is available to the public. Herein we provide information on disulphides in DSDBASE2.0, an updated and significantly expanded database that is freely available, fully annotated and manually curated database on native and modelled disulphides. The web interface also provides several useful computational tools that have been specifically developed for proteins containing disulphide crosslinks. The modelling of disulphide crosslinks is performed using stereochemical criteria, coded within our Modelling of Disulphides in Proteins (MODIP) algorithm. The inclusion of modelled disulphides potentially enhances the loop database substantially, thereby permitting the recognition of compatible polypeptide segments that could serve as templates for immediate modelling. The DSDBASE2.0 database has been updated to include 153,944 PDB entries, 216,096 native and 20,153,850 modelled disulphide bond segments from PDB January 2021 release. The current database also provides a resource to user-friendly search for multiple disulphide bond containing loops, along with annotation of their function using GO and subcellular localization of the query. Furthermore, it is possible to obtain the three-dimensional models of disulphide-rich small proteins using an independent algorithm, RANMOD, that generates and examines random, but allowed backbone conformations of the polypeptide. DSDBASE2.0 still remains the largest open-access repository that organizes all disulphide bonds of proteins on a single platform. The database can be accessed from http://caps.ncbs.res.in/dsdbase2.
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Project description:Most women with ovarian cancer are diagnosed at an advanced stage and there are few therapeutic options. Recently, monoclonal antibody therapies have had limited success, thus more effective antibodies are needed to improve long-term survival. In this report, we prepared polyclonal rabbit anti-ovarian cancer antibody (Poly Ab) by immunizing rabbits with the human ovarian cancer cell line SKOV3. The Poly Ab bound to SKOV3 and inhibited the cancer cells proliferation. Western blot analysis was conducted, which indicated that Poly Ab inhibited cancer cells through apoptosis involving the caspase signaling pathway including caspase-3 and caspase-9. Finally, compared with the control antibody, administration of Poly Ab reached 64% and 72% tumor inhibition in the subcutaneous and intraperitoneal xenograft mouse model, respectively. Our findings suggest that Poly Ab is an effective agent for apoptosis induction and may be useful as a safe anticancer agent for ovarian cancer therapy.
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Project description:Six peptides containing eight half-cystine residues were isolated in good yield, after either oxidation or reduction and carboxymethylation of fragment C-1, which contains the N-terminal half of the heavy chain of rabbit immunoglobulin G. The sequences of five of these peptides had been reported previously (Cebra, Steiner & Porter, 1968b; Wilkinson, 1969) and that of the sixth was established. Other peptides containing half-cystine residues were isolated in much lower yield and are presumed to be derived from minor sequence variants. The cystine-containing peptides from enzymic digests of whole immunoglobulin G and Fc fraction were studied by several techniques and the results obtained enable us to put forward a scheme of the arrangement of the inter- and intra-chain disulphide bonds.
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Project description:Goblet-cell mucin (mucin 1) was isolated and purified from human small-intestinal scrapings. After application of mucin 1 to DEAE-Bio-Gel (A) columns, most of the glycoprotein (76-94% of hexoses) was eluted in the first peak (designated mucin 2). Minor amounts of acidic glycoproteins were eluted with 0.2m- and 0.4m-NaCl in later peaks. Analyses of mucin 1 and mucin 2 revealed mucin 2 to be a monodisperse highly glycosylated glycoprotein containing 6.3% by wt. of protein, N-acetylgalactosamine, N-acetylglucosamine, galactose and fucose. Mucin 1 was similar in composition, but was polydisperse and contained more protein (12.3% by wt.) as well as N-acetylneuraminic acid. Analytical CsCl-gradient ultracentrifugation showed both mucin 1 and mucin 2 to have a major component with an average buoyant density of 1.47000g/ml. Mucin 1 also contained a slightly less-dense minor glycoprotein component. After exhaustive reduction and alkylation mucin 1 retained its major component, but partly dissociated into two lighter glycoprotein components. Mucin 2, in contrast, did not change its density distribution after reduction. Band ultracentrifugation in (2)H(2)O-containing iso-osmotic buffers showed that mucin 1 contained a major fast-sedimenting component (s(o)=37+/-2S), and a minor amount of a slower-sedimenting component. After reduction there was an increased quantity of the latter component, for which an s(o) value of 14.5S was calculated. In contrast, mucin 2 was unaltered by reduction (s(o)=33+/-2S). These findings indicate that the major component of goblet-cell mucin (mucin 2) does not dissociate after S-S-bond reduction, and thus does not apparently rely for its polymeric structure on the association of subunits through covalent disulphide bonds. However, the effects of reduction on mucin 1 suggest that in the native mucin intramolecular disulphide bonds in the minor glycoproteins may stabilize their structure, permitting secondary non-covalent interactions to develop with the major dense mucin (mucin 2) protein.
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Project description:The importance of disulphide bond in mediating viral peptide entry into host cells is well known. In the present work, we elucidate the role of disulphide (SS) bond in partitioning mechanism of membrane-active Hepatitis A Virus-2B (HAV-2B) peptide, which harbours three cysteine residues promoting formation of multiple SS-bonded states. The inclusion of SS-bond not only results in a compact conformation but also induces distorted α-helical hairpin geometry in comparison to SS-free state. Owing to these, the hydrophobic residues get buried, restricting the insertion of SS-bonded HAV-2B peptide into lipid packing defects and thus the partitioning of the peptide is completely or partly abolished. In this way, the disulphide bond can potentially regulate the partitioning of HAV-2B peptide such that the membrane remodelling effects of this viral peptide are significantly reduced. The current findings may have potential implications in drug designing, targeting the HAV-2B protein by promoting disulphide bond formation within its membrane-active region.
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Project description:Cancer-associated fibroblasts (CAFs) constitute a major component of the tumor microenvironment. CAFs regulated the growth and development, invasion and metastasis of primary tumors, as well as response to treatment. Recent studies indicated that monoclonal antibody therapies had limited success, thus more effective polyclonal antibodies (Poly Abs) is urgently needed. Poly Abs is a possible alternative because they target multiple antigens simultaneously. In this report, we prepared Poly Abs by immunizing rabbits with the bFGF-activated fibroblasts. The Poly Abs inhibited the cancer cells proliferation as revealed by MTT analysis. The Poly Abs induced apoptosis as indicated by flow cytometric analysis, and microscopic observation of apoptotic changes in morphology. Compared with the control IgG, Poly Abs significantly inhibited tumor cells migration as indicated by wound healing and transwell analysis in vitro, and lung metastasis analysis in vivo. Serial intravenous injections of Poly Abs inhibited tumor growth in mice bearing murine CT26 colon carcinoma. Ki67 analysis indicated that Poly Abs significantly inhibited tumor cells proliferation, as compared to control Ig G treatments. Our findings suggested that Poly Abs was an effective agent for apoptosis induction, migration and metastasis inhibition. The Poly Abs may be useful as a safe anticancer agent for cancer immunotherapy in the future.
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Project description:Carboxymethylation using 14C- or 3H-labelled iodoacetic acid has been used to identify the cysteine residues in bovine rhodopsin involved in the formation of the two intramolecular disulphide bridges. Iodo[2-14C]acetic acid was used to modify 5.8-5.9 residues of cysteine under non-reducing conditions. After dialysis and reduction of disulphide bridges by 2-mercaptoethanol, iodo[2-3H]acetic acid was employed to covalently modify 3.3-3.6 residues of cysteine. Peptide purification and sequencing has unambiguously shown that cysteine residues 322 and 323 are only carboxymethylated after reduction of disulphide bridges. Indirect evidence presented, now coupled with the earlier finding [Findlay & Pappin (1986) Biochem. J. 238, 625-642] suggests that the other disulphide bridge is formed between cysteine residues 110 and 187. A comparison is made of all the sequences of mammalian rhodopsins and colour pigments and attention is drawn to the fact that whereas Cys-322 and Cys-323 are conserved only in three rhodopsins (bovine, ovine and human), the residues corresponding to Cys-110 and Cys-187 are found in all the visual proteins (from rods as well as human cones).
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Project description:Many extracellular globular proteins have evolved to possess disulphide bonds in their native conformations, which aids in thermodynamic stabilisation. However, disulphide bond breakage by heating leads to irreversible protein denaturation through disulphide-thiol exchange reactions. In this study, we demonstrate that methanethiosulphonate (MTS) specifically suppresses the heat-induced disulphide-thiol exchange reaction, thus improving the heat-resistance of proteins. In the presence of MTS, small globular proteins that contain disulphides can spontaneously refold from heat-denatured states, maintaining wild-type disulphide pairing. Because the disulphide-thiol exchange reaction is triggered by the generation of catalytic amounts of perthiol or thiol, rapid and specific perthiol/thiol protection by MTS reagents prevents irreversible denaturation. Combining MTS reagents with another additive that suppresses chemical modifications, glycinamide, further enhanced protein stabilisation. In the presence of these additives, reliable remnant activities were observed even after autoclaving. However, immunoglobulin G and biotin-binding protein, which are both composed of tetrameric quaternary structures, failed to refold from heat-denatured states, presumably due to chaperon requirements. Elucidation of the chemical modifications involved in irreversible thermoinactivation is useful for the development of preservation buffers with optimum constitutions for specific proteins. In addition, the impact of disulphide bond breakage on the thermoinactivation of proteins can be evaluated using MTS reagents.
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Project description:Nek2 is a dimeric serine/ threonine protein kinase that belongs to the family of NIMA-related kinases (Neks). Its N-terminal catalytic domain and its C-terminal regulatory region are bridged by a leucine zipper, which plays an important role in the activation of Nek2's catalytic activity. Unusual conformational dynamics on the intermediary/slow timescale has thwarted all attempts so far to determine the structure of the Nek2 leucine zipper by means of X-ray crystallography and Nuclear Magnetic Resonance (NMR). Disulfide engineering, the strategic placement of non-native disulfide bonds into flexible regions flanking the coiled coil, was used to modulate the conformational exchange dynamics of this important dimerization domain. The resulting reduction in exchange rate leads to substantial improvements of important features in NMR spectra, such as line width, coherence transfer leakage and relaxation. These effects were comprehensively analyzed for the wild type protein, two single disulfide bond-bearing mutants and another double disulfide bonds-carrying mutant. Furthermore, exchange kinetics were measured across a wide temperature range, allowing for a detailed analysis of activation energy (ΔG‡) and maximal rate constant (k'ex). For one mutant carrying a disulfide bond at its C-terminus, a full backbone NMR assignment could be obtained for both conformers, demonstrating the benefits of the disulfide engineering. Our study demonstrates the first successful application of 'kinetic' disulfide bonds for the purpose of controlling the adverse effects of protein dynamics. Firstly, this provides a promising, robust platform for the full structural and functional investigation of the Nek2 leucine zipper in the future. Secondly, this work broadens the toolbox of protein engineering by disulfide bonds through the addition of a kinetic option in addition to the well-established thermodynamic uses of disulfide bonds.
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