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PubMed2006

Disulfide bonds determine growth hormone receptor folding, dimerisation and ligand binding.

The growth hormone receptor contains seven cysteine residues in its extracellular domain. The six in the growth hormone binding domain form disulfide bonds, and help the receptor to gain its correct three-dimensional structure. In this study we replaced the cysteine for serine and alanine residues and investigated their role in growth hormone receptor folding, dimerisation and signal transduction. Folding and growth hormone binding capacity of the wild-type growth hormone receptor require less than two minutes for completion. Although less efficient, all mutant receptors arrive at the cell surface as pre-formed dimers. Disulfide bond C38-C48 is important for efficient maturation. The middle disulfide-bond, C83-C94, is important for ligand binding. Removing disulfide bond C108-C122 has little effect without affecting signalling. When two or all disulfide bonds are changed, ligand binding and activation are blocked. Dimerisation is delayed when all disulfide bonds are destroyed.

Animals↗

PubMed2000

Direct identification of a novel disulfide bond linkage system of new isolated isomer (isomer V) in recombinantly produced h-IGF-I.

Insulin-like growth factor I (IGF-I or somatomedin C) is a serum polypeptide with three intramolecular disulfide bonds. In the course of synthesis by the recombinant DNA method, three disulfide bond isomers, all of which have Cys18-Cys61 with three combinations of two disulfide bonds formed by Cys6, Cys47, Cys48 and Cys52, were identified. Natural type, isomer II, was proved to have a Cys6-Cys48, Cys18-Cys61, Cys47-Cys52 disulfide bond system. Now, the fourth isomer, isomer V which doesn't have Cys18-Cys61 disulfide, has been isolated, and its novel disulfide bond linkage system was identified by a chemical synthetic method. The supposed conformation constrained in 3D structure for isomer V would be discussed for its biological activity.

3T3 Cells↗

PubMed1999

Increased acetylation of histones induced by diallyl disulfide and structurally related molecules.

In previous studies, diallyl disulfide induced differentiation in DS19 mouse erythroleukemic cells. A mechanism mediated by increased histone acetylation was investigated. Diallyl disulfide caused increased acetylation of H4 and H3 histones in DS19 cells and K562 human leukemic cells. Diallyl disulfide was more effective than diallyl monosulfide and diallyl sulfone. Acetylation was also induced in rat hepatoma and human breast cancer cells by diallyl disulfide or its metabolite, allyl mercaptan. Allyl mercaptan was a more potent inhibitor of histone deacetylase than diallyl disulfide. Differentiation in erythroleukemic cells by diallyl disulfide and allyl mercaptan may be mediated through induction of histone acetylation.

Acetylation↗

PubMed2001

Effects of diallyl disulfide and other donors of sulfane sulfur on the proliferation of human hepatoma cell line (HepG2).

Effects of potential sulfane sulfur precursors (diallyl disulfide, cystamine, 2-mercaptoethanol disulfide, thiosulfate, immunothiole and pyridoxal phosphate jointly with cystine) on [3H]-thymidine incorporation in human hepatoma (HepG2) cells were studied. Of the tested compounds, diallyl disulfide, cystamine and 2-mercaptoethanol disulfide were found to cause significant inhibition of HepG2 cells proliferation. Moreover, pyridoxal phosphate jointly with cystine suppressed [3H]-thymidine incorporation, but the differences between that system and control cells were insignificant. In the case of thiosulfate, no significant difference was observed. The present study shows that diallyl disulfide, found in garlic, is effective in inhibiting [3H]-thymidine incorporation in human hepatoma HepG2 cell cultures. Similar antiproliferative effects on HepG2 cells are shown by such systems being a source of sulfane sulfur as cystamine or 2-mercaptoethanol disulfide. Thus, it may be concluded that these donors of reactive sulfane sulfur may be responsible for inhibition of the proliferation of HepG2 cells. It is suggested that the observed antiproliferative properties of the investigated compounds are connected with the presence of the highly reactive sulfane sulfur.

Allyl Compounds↗

PubMed2002

[Escherichia coli disulfide-forming related proteins: structures, functions and their application in gene engineering for expressing heterologous proteins in Escherichia coli].

The formation of disulfide bonds in secreted proteins of E. coli is a synergetic process depending on a series of Dsb proteins containing DsbA, DsbB, DsbC, DsbD, DsbE and DsbG. DsbA functions as an oxidant to form a disulfide bond between two -SH- in vivo and DsbB reactivates DsbA by reoxidizing it. Both DsbC and DsbG, two periplasmic proteins with isomerase activity, can correct mis-paired disulfide bonds introduced by DsbA although they recognize different substrates. DsbD, an inner membrane protein, plays a role in reducing DsbC and DsbG in vivo. It is regarded that DsbE has the similar function with DsbD. All DsbA, DsbC and DsbG have chaperone activity besides involving in the formation of disulfide bonds. Furthermore, their chaperone activity can promote the formation of protein disulfide bonds. There are a few reports dealing with soluble expression of heterologous proteins containing disulfide bonds assisted by DsbA and DsbC in E. coli. So far there has been no reports about the soluble expression of heterologous proteins promoted by DsbG. Our experiments first demonstrated that both DsbC and DsbG can improve the expression of single chain antibodies as soluble and functional forms in E. coli, and DsbG has additive effects with DsbC.

Bacterial Proteins↗

PubMed1992

The structure of hepadnaviral core antigens. Identification of free thiols and determination of the disulfide bonding pattern.

A set of wild-type and mutant human, woodchuck, and duck hepatitis viral core proteins have been prepared and used to study the free thiol groups and the disulfide bonding pattern present within the core particle. Human (HBcAg) and woodchuck (WHcAg) core proteins contain 4 cysteine residues, whereas duck (DHcAg) core protein contains a single cysteine residue. Each of the cysteines of HBcAg has been eliminated, either singly or in combinations, by a two-step mutagenesis procedure. All of the proteins were shown to have very similar physical and immunochemical properties. All assemble into essentially identical core particle structures. Therefore disulfide bonds are not essential for core particle formation. No intra-chain disulfide bonds occur. Cys107 is a free thiol buried within the particle structure, whereas Cys48 is present partly as a free sulfhydryl which is exposed at the surface of the particle. Cys61 is always and Cys48 is partly involved in interchain disulfide bonds with the identical residues of another monomer, whereas Cys183 is always involved in a disulfide bond with the Cys183 of a different monomer. WHcAg has the same pattern of bonding, whereas DHcAg lacks any disulfide bonds, and the single free sulfhydryl, Cys153 which is equivalent to Cys107 of HBcAg, is buried.

Amino Acid Sequence↗

PubMed1990

Role of disulfide bond formation in the folding of human chorionic gonadotropin beta subunit into an alpha beta dimer assembly-competent form.

The malignant trophoblastic cell line JAR was used as a model system to study protein folding in intact cells. We have used this model previously to identify conformational intermediates in the production of an assembly-competent form of the human chorionic gonadotropin beta subunit (Ruddon, R. W., Krzesicki, R. F., Norton, S. E., Beebe, J. S., Peters, B. P., and Perini, F. (1987) J. Biol. Chem. 262, 12533-12540). The earliest biosynthetic precursor of the human chorionic gonadotropin beta subunit detectable in JAR cells pulse labeled for 2 min is p beta 1, a form that lacks half of the six intrachain disulfide bonds observed in the fully processed dimer form of beta and that does not combine with the alpha subunit. p beta 1 is rapidly (t1/2 approximately 4 min) converted into p beta 2, which has a full complement of intrachain disulfide bonds and does combine with the alpha subunit. In this study, we have identified the three late forming disulfide bonds involved in the transition of p beta 1 into the assembly-compete form, p beta 2. The last three disulfide bonds to form are those between cysteines 9 and 90, 23 and 72, and 93 and 100. These were identified in JAR cell lysates that had been pulse labeled with [35S]cysteine for 2 or 5 min followed by trapping of the cysteine thiols with iodoacetic acid before immunopurification of the beta subunit forms. Immunopurified p beta 1 was treated with trypsin under nonreducing conditions to liberate [35S]cysteine-containing peptides from the disulfide-linked beta core polypeptide. These tryptic peptides were then separated by high performance liquid chromatography and sequenced to determine the location of the carboxymethyl-[35S]cysteine residues. The three late forming disulfide bonds are most likely the ones involved in stabilizing the conformation of the beta subunit that is required for combination with alpha to form the biologically functional alpha beta heterodimer.

Amino Acid Sequence↗

PubMed1991

Disulfide bonding controls the processing of retroviral envelope glycoproteins.

The mitogenic membrane glycoprotein (gp55) encoded by Friend erythroleukemia virus is inefficiently processed from the rough endoplasmic reticulum (RER) and only 3-5% reaches plasma membranes. Because this processed component (gp55P) contains larger and more complex oligosaccharides, it can be separated from RER gp55. In nonreducing conditions, gp55P is a unique disulfide-bonded dimer, whereas RER gp55 consists of monomers and dimers with diverse intrachain and interchain disulfide bonds. This suggests that gp55 folds heterogeneously and that only one homodimer is competent for export from the RER. Pulse-chase analyses of gp55 components labeled with radioactive amino acids indicated that formation of diverse disulfide-bonded components occurred within minutes of polypeptide synthesis and that malfolded components did not later isomerize to generate dimers competent for export from the RER. Chemical studies suggested that all 12 cysteines of gp55 were oxidized within 5 min after synthesis of the protein. In contrast, the envelope glycoprotein precursor (gPr90) encoded by a replication-competent murine leukemia virus folds more homogeneously, and it is then processed and cleaved to form an extracellular glycoprotein gp70 plus a transmembrane protein p15E. The fully processed glycoprotein contains an unoxidized cysteine sulfhydryl that isomerizes reversibly with a disulfide bond that links gp70 to p15E. Consequently, only a proportion of gp70 and p15E is disulfide-bonded, and dissociation occurs when the environment becomes even slightly reducing. The gp55 glycoprotein appears to be an extreme example of protein malfolding associated with imprecise and irreversible disulfide bonding. We discuss evidence that folding inefficiencies are common for retroviral proteins that have newly evolving pathogenic functions.

Blotting, Western↗

PubMedDate not supplied

Monitoring protein cleavage and concurrent disulfide bond assignment using thermospray LC/MS.

The arrangement of the disulfide bridges of Cucurbita maxima trypsin inhibitor, CMTI I, has been confirmed by enzymatic and chemical cleavages of the native protein and analysis of the resulting disulfide-bridged fragments using thermospray liquid chromatography/mass spectrometry. Although the disulfide bridges of CMTI I have recently been assigned from the x-ray crystallographic structure, direct chemical analysis of the S-S bonds using classical techniques proved difficult. The CMTI I molecule is extremely resistant to enzymatic digestion, and only one site of the peptide chain (Met-8) can be used efficiently for chemical cleavage. A series of degradative conditions were employed in the studies reported here. The progress of protein modification was monitored directly by high-performance liquid chromatography/thermospray mass spectrometry. The disulfide pairings could be deduced directly from the mass spectra of the peptides produced by the fragmentation processes and resolved by high-performance liquid chromatography. In two instances, fragments involving a disulfide bond were isolated and further analyzed, and these confirmed the mass spectral assignments. The disulfide bridges identified, 3-20, 10-22 and 16-28, correspond to those of the x-ray structure and are consistent with those assigned for two other closely related trypsin inhibitors.

Amino Acid Sequence↗

PubMed1990

Isolation and characterization of disulfide-bonded peptides from the three globular domains of aggregating cartilage proteoglycan.

The aggregating cartilage proteoglycan core protein contains two globular domains near the N terminus (G1 and G2) and one near the C terminus (G3). The G1-G3 domains contain 10, 8, and 10 cysteine residues, respectively. The disulfide assignments of the G1 domain have previously been deduced (Neame, P. J., Christner, J. E., and Baker, J. R. (1987) J. Biol. Chem. 262, 17768-17778) as Cys1-Cys2, Cys3-Cys6, Cys4-Cys5, Cys7-Cys10, and Cys8-Cys9, in which the numbers cited after the half-cystine residues are their relative positions from the N terminus. Here we describe a method for the isolation of disulfide-bonded peptides from tryptic digests of bovine nasal cartilage monomer. Sequence analysis of these peptides has allowed us to confirm the pairings previously determined for the G1 domain and to assign a disulfide pattern for the G2 domain of Cys11-Cys14, Cys12-Cys13, Cys15-Cys18, and Cys16-Cys17, in which the Cys15-Cys18 pairing was deduced indirectly. Similarly, for the G3 domain, a pattern of Cys19-Cys20, Cys21-Cys24, Cys22-Cys23, Cys25-Cys27, and Cys26-Cys28 was assigned, in which the Cys22-Cys23 pair was deduced indirectly. The G2 domain therefore contains disulfide bonding which is characteristic of the tandem repeat structures found in the G1 domain and link protein, and the G3 domain contains the three disulfide linkages previously assigned to the family of C-type animal lectins. The method described here, which combines anion-exchange, cation-exchange, and reversed-phase chromatography, should have broad application to the isolation of disulfide-bonded peptides from other heavily glycosylated proteins and proteoglycans.

Aggrecans↗

PubMed1990

The disulfide structure of mouse lysosome-associated membrane protein 1.

The disulfide structure of mouse lysosome-associated membrane protein 1 has been determined by reverse-phase isolation and sequence analysis of the cysteine-containing tryptic fragments of the reduced and non-reduced deglycosylated protein. Half-cystines were distinguished (a) by their localization within tryptic or chymotryptic peptides that formed reverse-phase peaks unique to the reduced digests and (b) by their 3H-carboxymethylation only after reduction of the protein. The disulfide arrangement of the cysteines was assigned after isolation of disulfide-linked peptide pairs. Each pair chromatographed as a peak present in the nonreduced (but not the corresponding reduced) tryptic digest. NH2-terminal sequencing as well as reduction, alkylation, and rechromatography of the disulfide-linked fragments led to the following assignment of disulfide bonds: Cys11 and Cys50, Cys125 and Cys161, Cys198 and Cys235, and Cys303 and Cys340. This structure creates four 36-38-residue loops that are symmetrically placed within the two halves of the protein's intraluminal domain. The loops formed by the Cys11-Cys50 and Cys198-Cys235 bridges are homologous, and the Cys125-Cys161 and Cys303-cys340 loops form a second set of homologous domains. The conservation of cysteine residues among lysosome-associated membrane proteins 1 and 2 suggests that this disulfide arrangement is common to both members of this family of lysosomal membrane glycoproteins.

Amino Acid Sequence↗

PubMed1989

Structure of human lysosomal membrane glycoprotein 1. Assignment of disulfide bonds and visualization of its domain arrangement.

The amino acid sequence of one of the major lysosomal membrane glycoproteins, lysosome-associated membrane protein 1 (lamp-1), was deduced from its cDNA sequence (Fukuda, M., Viitala, J., Matteson, J., and Carlsson, S. R. (1988) J. Biol. Chem. 263, 18920-18928). This amino acid sequence suggests that lamp-1 contains a hinge-like structure and could form disulfide bridges that are observed in the immunoglobulin superfamily. To test this possibility, we have determined the positions of the disulfide bridges by isolating and sequencing cystine-containing peptides which contain disulfide bridges. The results indicate that disulfide arrangement of lamp-1 is different from that of immunoglobulins. Each molecule contains, in total, four loops formed by disulfide bonds, and each loop contains 36-39 amino acid residues. However, none of the disulfide bonds connects two domains that are separated by a hinge-like structure. The results indicate that the hinge region has no ordered structure, and the relative positions of the two domains can be altered in space. Examination of the ultrastructure of lamp-1 by electron microscopy showed that the hinge-like structure actually functions as a hinge. These results indicate that the lamp-1 molecule represents a novel family of glycoproteins with unique structural properties.

Amino Acid Sequence↗

PubMed1988

Characterization of disulfide-linked crystallins associated with human cataractous lens membranes.

In order to characterize possible disulfide-linked interactions between lens fiber cell membranes and crystallins, two-dimensional diagonal electrophoresis has been used in combination with Western blot analysis. When these blots were probed with monospecific antisera against alpha, beta and gamma crystallins, membrane from five individual normal lenses showed no disulfide-bonded components. Membrane from 13 individual cataractous human lenses showed no disulfide-bonded alpha crystallin, but did show significant amounts of disulfide-bonded beta crystallin in four out of the 13 lenses studied, and significant amounts of disulfide-bonded gamma crystallin in 10 out of the 13 lenses studied. Together, these studies demonstrate that intermolecular disulfide bonding of crystallins to purified fiber cell membranes is found only in cataractous lenses, and that the predominant polypeptide species involved in this interaction is gamma crystallin.

Cataract↗

PubMed1988

[Cytoarchitectonics of the tracheobronchial lymph nodes in rats exposed to carbon disulfide].

Cytoarchitectonics and rearrangement of cellular composition in functionally different zones of the rat tracheobronchial lymph nodes have been studied at a short effect of the industrial carbon disulfide vapour at a maximally admissible concentration (MAC) (1 mg/m3 and 10 mg/m3), as well as after the effect of MAC of carbon disulfide in the inhaled air for 4, 7, 14 days. The inhalation of the carbon disulfide vapours results in certain changes of the cytoarchitectonics of the lymph nodes studied in dependence on concentration and duration of the substance effect in the air inhaled. The effect of the MAC carbon disulfide, when the experiment lasts for 2 days, is accompanied with a decreased lymphocytopoiesis in all the structural components of the node and with a sharp intensification in the number of plasma cells of the medullary cords, that demonstrates local immunocytopoiesis. An opposite picture is noted, when carbon disulfide is applied in an elevated concentration (10 mg/m3)--plasmic reaction is sharply inhibited in the medullary cords, lymphocytopoiesis is noticeably increased, certain signs of allergic reaction are also seen. A prolonged effect of carbon disulfide vapours (up to 14 days) in MAC results in lymphocytopenia of the nodes, in increasing destructive processes and in decreasing cell proliferation. Differences in reaction of the structural components are revealed in the nodes studied at the toxic effect: in the cortical plateau and in the medullary cords suppression of the lymphocytopoietic activity is noted, and in the medullary cords--inhibition of immunocytopoiesis.

Administration, Inhalation↗

PubMed1986

In vivo formation and stability of engineered disulfide bonds in subtilisin.

Computer modeling suggested that a disulfide bond could be built into Bacillus amyloliquefaciens subtilisin between positions 22 (wild-type, Thr) and 87 (Ser) or between positions 24 (Ser) and 87 (Ser). Single cysteines were introduced into this cysteine-free protease at positions 22, 24, or 87 by site-directed mutagenesis of the cloned subtilisin gene. The corresponding double-cysteine mutants were constructed, and recombinant plasmids were expressed in Bacillus subtilis. Double-cysteine mutant enzymes were secreted as efficiently as wild-type, and disulfide bonds were formed quantitatively in vivo. These disulfide bonds were introduced approximately 24 A away from the catalytic site and had no detectable effect on either the specific activities or the pH optima of the mutant enzymes. The equilibrium constants for the reduction of the mutant disulfide bonds by dithiothreitol were determined to be 82 +/- 22 and 20 +/- 5 for Cys22/Cys87 and Cys24/Cys87, respectively. Studies of autoproteolytic inactivation of wild-type subtilisin support a relationship between autolytic stability and conformational stability of the protein. The stabilities of Cys24/Cys87 and wild-type enzymes to autolysis were essentially the same; however, Cys22/Cys87 was actually less stable to autolysis. Reduction of the disulfide cross-bridge lowered the autolytic stability of both double-cysteine mutants relative to their disulfide forms. This correlates with a lowered autolytic stability for the Cys22 and Cys87 single-cysteine mutants, and the fact that an intramolecular hydrogen bond between the hydroxyl groups of Thr22 and Ser87 is likely to be disrupted in the Cys22 and Cys87 single-cysteine mutant proteins.

Autolysis↗

PubMed1985

Disulfide spacer between methotrexate and poly(D-lysine). A probe for exploring the reductive process in endocytosis.

Poly(D-lysine) is taken up avidly by cultured cells through adsorptive endocytosis and can serve as a carrier to increase cellular uptake of other molecules. While direct conjugation of methotrexate to poly(D-lysine) yields a conjugate devoid of cytotoxic effects because poly(D-lysine) is not digested in lysosomes, the indirect conjugation using a triglycine spacer or a disulfide spacer strongly inhibits the growth of both the wild type and the methotrexate transport-defective Chinese hamster ovary cells. Cell treatment with 3 mM NH4Cl or 50 micrograms/ml leupeptin prevents the effect of conjugate with the triglycine spacer, but not of conjugate with the disulfide spacer. On the other hand, preincubation with 2-mercaptoethanol abolishes the effect of the drug-disulfide conjugate in the methotrexate transport-defective mutant, but not the effect of the drug-triglycine conjugate. The disulfide conjugate shows an identical cytotoxic effect in alpha-minimal essential medium and RPMI 1640 media, even though cells grown in the latter have only half the glutathione content as cells grown in the former medium. We conclude that the reductive process through which methotrexate is released from the disulfide spacer (a) occurs inside cells and not at the cell surface, (b) requires neither acid pH nor lysosomal enzymes, and (c) is not mediated by a glutathione-disulfide exchange reaction requiring high glutathione concentrations. Although the cellular compartment in which this reductive process occurs is not yet identified, there are reasons to assume that it is prelysosomal.

Animals↗

PubMed1979

Domain structure of fibronectin and its relation to function. Disulfides and sulfhydryl groups.

Hamster cell fibronectin is a glycoprotein consisting of two 230,000-dalton subunits in a disulfide-bonded dimer. The molecule is composed of domains which can be separated by partial proteolytic cleavage. The carbohydrates, disulfide bonds, and a single free sulfhydryl group per chain are distributed nonuniformly among these regions. All the interchain disulfides are within 10,000 daltons of the end of the molecule and are removed by mild proteolysis which also generates 200,000- and 25,000-dalton fragments which do not contain interchain disulfides. The 200,000-dalton fragment contains all or most of the carbohydrate side chains, and the free sulfhydryl group, but is relatively poor in cystine. The 25,000-dalton fragment is carbohydrate-free and cystine-rich but has no free sulfhydryl groups. There is heterogeneity in carbohydrate content among the monomeric chains of intact fibronectin and the 200,000-dalton fragments. The gelatin binding site of fibronectin is in the 200,000 fragment. Intact disulfide bonds are required for binding of fibronectin to cells and to gelatin and blockage of the free sulfhydryl groups prevents binding of fibronectin to cells, suggesting that intermolecular disulfide bonding may be important.

Amino Acids↗

PubMed1984

[Conformation analysis of a mixed hemoglobin-glutathione disulfide].

Theoretical conformational analysis was carried out for a mixed disulfide of hemoglobin with glutathione. The conformational mobility of the beta-subunit C-terminal fragment in methemoglobin and deoxyhemoglobin either with free or glutathione-blocked reactive SH-groups was examined. The most stable conformations of the mixed disulfide were delineated. Its spatial structure was shown to be dependent on the hemoglobin state prior to the S-S bond formation: a disulfide made with deoxyhemoglobin had more closely interwoven hemoglobin and glutathione chains than the methemoglobin-derived disulfide. However, in both cases disulfide formation brought about the alterations in the spatial structure of hemoglobin as well as glutathione. The changes in the hemoglobin biochemical properties accompanying the association with glutathione were rationalized in the frames of the mixed disulfide structural analysis.

Disulfides↗