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PubMed1994

Chaperone-like activity of protein disulfide isomerase in the refolding of a protein with no disulfide bonds.

D-Glyceraldehyde-3-phosphate dehydrogenase (GAP-DH) is a protein containing no disulfide bonds; the guanidine HCl-denatured enzyme shows only a limited extent of refolding and reactivation upon dilution, and the enzyme is particularly prone to aggregation during the dilution process. With increasing GAPDH concentration, reactivation decreases and aggregation increases. The presence of protein disulfide isomerase in the dilution mixture markedly increases reactivation of GAPDH and at the same time prevents the aggregation of GAPDH as shown by light-scattering measurements. It is suggested that upon dilution, denatured GAPDH is faced with two competing processes of correct folding and assembly to yield the native enzyme and non-productive association of the partially refolded species to form aggregates. Independent of the isomerase activity as no disulfide bond is present in GAPDH, protein disulfide isomerase assists the refolding of GAPDH to its active state by suppressing aggregation in a way closely similar to the action of chaperones.

Disulfides↗
PubMed2005

Engineering disulfide bonds of the novel human beta-defensins hBD-27 and hBD-28: differences in disulfide formation and biological activity among human beta-defensins.

Human beta-defensins comprise a large number of peptides that play a functional role in the innate and adaptive immune system. Recently, clusters of new beta-defensin genes with predominant expression in testicular tissue have been discovered on different chromosomes by bioinformatics. beta-Defensins share a common pattern of three disulfides that are essential for their biological effects. Here we report for the first time the chemical synthesis of the new fully disulfide-bonded beta-defensins hBD-27 and hBD-28, and compare the results with synthetic procedures to obtain the known hBD-2 and hBD-3. While hBD-27 was readily converted into a product with the desired disulfide pattern by oxidative folding, hBD-28 required a selective protective group strategy to introduce the three disulfide bonds. The established synthetic processes were applied to the synthesis of hBD-2, which, like hBD-27, was accessible by oxidative folding, whereas hBD-3 required a selective strategy comparable to hBD-28. Experimental work demonstrated that trityl, acetamidomethyl, and t-butyl are superior to other protection strategies. However, the suitable pairwise arrangement of the protective groups can be different, as shown here for hBD-3 and hBD-28. Determination of the minimum inhibitory concentration against different bacteria revealed that hBD-27, in contrast to other beta-defensins tested, has virtually no antimicrobial activity. Compared to the other peptides tested, hBD-27 showed almost no cytotoxic activity, measured by hemoglobin release of erythrocytes. This might be due to the low positive net charge, which is significantly higher for hBD-2, hBD-3, and hBD-28.

Amino Acid Sequence↗
PubMed2000

The cell adhesion molecule retina cognin is a cell surface protein disulfide isomerase that uses disulfide exchange activity to modulate cell adhesion.

The retina cell adhesion molecule, R-cognin, shares cDNA sequence with protein disulfide isomerase (PDI) but has a different molecular size and subcellular location. We asked whether R-cognin originated from a unique PDI gene transcript or was a product of posttranscriptional processing. The 3'-terminal partial cDNA clone for R-cognin was extended by both 5' RACE and by PCR from sequence near the 5' end of the PDI-translated region. The cDNA sequence was compared to those of chicken, bovine, and human PDI. The R-cognin cDNA sequence was identical to that of chicken PDI and differed by less than 10% from mammalian PDI proteins. The role of the disulfide exchange activity characteristic of both proteins was studied by assessing the cell-aggregation-enhancing ability and tissue specificity of R-cognin and recombinant human PDI and its derivatives. Chicken and normal human PDI proteins showed tissue- and developmental-specific enhancement of cell aggregation identical to R-cognin, and this activity was blocked by inactivation of the -WCGHC- motifs which function in disulfide exchange. Dependence of retina cell aggregation on disulfide exchange activity was shown by blocking that activity with the inhibitor, DTNB, or with a recombinant human PDI with the -WCGHC- motif cysteines mutated. The results suggest that one -WCGHC- motif in R-cognin is sufficient and that the more C-terminal motif is most active. We conclude that R-cognin is a tissue-specific protein product of the standard PDI chicken gene. The -WCGHC- motif in mature R-cognin is necessary, but not sufficient, for cell adhesion.

Amino Acid Sequence↗
PubMed1992

Determination of disulfide bond arrangement in bombyxin-IV, an insulin superfamily peptide from the silkworm, Bombyx mori, by combination of thermolysin digestion of natural peptide and selective synthesis of disulfide bond isomers.

The mode of disulfide linkages in bombyxin-IV, an insulin superfamily peptide consisting of A- and B-chains, was determined as A6-A11, A7-B10, and A20-B22. An intermolecular bond of A20-B22 was identified by sequencing and mass spectrometric analysis of the fragments generated by thermolysin digestion of natural bombyxin-IV. The mode of the remaining two bridges was determined by chemical and selective synthesis of three possible disulfide bond isomers of bombyxin-IV. A- and B-chains were synthesized by solid-phase method, and three disulfide bonds were bridged stepwise and in a fully controlled manner. Retention time on reversed-phase high-performance liquid chromatography (HPLC), thermolysin digests, and biological activity of the synthetic [A6-A11, A7-B10, A20-B22-cystine]-bombyxin-IV revealed that it was identical with the natural bombyxin-IV. Two other isomers with respect to disulfide bond arrangement, [A6-A7, A11-B10, A20-B22-cystine]- and [A6-B10, A7-A11, A20-B22-cystine]-bombyxin-IVs, were distinguishable from the natural one by use of HPLC, thermolysin digestion, and bioassay.

Amino Acid Sequence↗
PubMed1981

Partial purification of biologically active, low molecular weight, human antihemophilic factor free of Von Willebrand factor. II. Further purification with thiol-disulfide interchange chromatography and additional evidence for disulfide bonds susceptible to limited reduction.

Thiol-disulfide interchange chromatography was used in the preparation of partially purified (approx. 17 000-fold) low molecular weight, Mr approximately or equal to 115 000, human antihemophilic factor essentially free of Von Willebrand factor. This antihemophilic factor was prepared from fresh plasma which had undergone limited reduction with 1 mM dithiothreitol and was subsequently reacted with 2,2'-dipyridyl disulfide, a sulfhydryl reagent which readily undergoes disulfide exchange. Exchange of protein-2-pyridyl mixed disulfide with thiopropyl-Sepharose resulted in the chromatographic adsorption of approx. 96% of the coagulant activity, of which approx. 20% subsequently eluted with 1.0 mM dithiothreitol. After reductive displacement from the thiopropyl-Sepharose the antihemophilic factor could be S-alkylated with iodo-[1-14]acetamide. The ratio of coagulant activity to Von Willebrand factor-antigen activity was greater than 30 000 : 1. In contrast, reduced antihemophilic factor was alkylated with iodoacetamide prior to chromatography as a control, and showed no exchange with the thiopropyl-Sepharose, eluting quantitatively in the breakthrough volume. These studies reinforce our previous results (Harris, R.B., Newman, J. and Johnson, A.J. (1981) Biochim. Biophys. Acta 668, 456-470) that partial reduction with dithiothreitol exposes critical sulfhydryl groups which, when alkylated, maintains the antihemophilic factor in a low molecular weight form without inactivating procoagulant activity.

Chromatography↗
PubMed2006

Exploring synonymous codon usage preferences of disulfide-bonded and non-disulfide bonded cysteines in the E. coli genome.

High-quality data about protein structures and their gene sequences are essential to the understanding of the relationship between protein folding and protein coding sequences. Firstly we constructed the EcoPDB database, which is a high-quality database of Escherichia coli genes and their corresponding PDB structures. Based on EcoPDB, we presented a novel approach based on information theory to investigate the correlation between cysteine synonymous codon usages and local amino acids flanking cysteines, the correlation between cysteine synonymous codon usages and synonymous codon usages of local amino acids flanking cysteines, as well as the correlation between cysteine synonymous codon usages and the disulfide bonding states of cysteines in the E. coli genome. The results indicate that the nearest neighboring residues and their synonymous codons of the C-terminus have the greatest influence on the usages of the synonymous codons of cysteines and the usage of the synonymous codons has a specific correlation with the disulfide bond formation of cysteines in proteins. The correlations may result from the regulation mechanism of protein structures at gene sequence level and reflect the biological function restriction that cysteines pair to form disulfide bonds. The results may also be helpful in identifying residues that are important for synonymous codon selection of cysteines to introduce disulfide bridges in protein engineering and molecular biology. The approach presented in this paper can also be utilized as a complementary computational method and be applicable to analyse the synonymous codon usages in other model organisms.

Amino Acids↗
PubMed2000

Propensities for the formation of individual disulfide bonds in hen lysozyme and in the size and stability of disulfide-associated submolecular structures.

Hen lysozyme single-disulfide variants were constructed to characterize the structures associated with the formation of individual native disulfide bonds. Circular dichroism spectra and the effective concentration of protein thiol groups showed that the propensity for structure formation was relatively high for Cys-6-Cys-127 and Cys-30-Cys-115 disulfides. The urea concentration dependence of individual effective concentrations showed that the apparent sizes of the structures were 14-50% of the whole molecule. The intrinsic stability of each submolecular structure in a reduced form of protein, obtained by subtracting the entropic contribution of cross-linking, was highest for Cys-64-Cys-80 and lowest for Cys-76-Cys-94 disulfide bonds.

Animals↗
PubMed1999

2,2'-Bispyridyl disulfide rapidly induces intramolecular disulfide bonds in peptides.

A linear peptide containing two reduced cysteine residues can be rapidly converted to its oxidized cyclic form containing an intramolecular disulfide bond by adding an excess of 2,2'-bispyridyl disulfide (2,2'-dipyridyl disulfide or 2,2'-dithiodipyridine) to conventional buffer solutions. The reactants and products are easily separated by reverse-phase chromatography. This reaction will find wide application in forming intramolecular disulfide bonds because of its selectivity for free sulfhydryl groups, quickness, safety, and applicability under acidic conditions.

2,2'-Dipyridyl↗
PubMed1993

Disulfide bonds in recombinant human platelet-derived growth factor BB dimer: characterization of intermolecular and intramolecular disulfide linkages.

Interchain cystines of PDGF-BB dimer were characterized by Edman reaction and by SDS-PAGE analysis on the protein which was chemically cleaved at Trp-40. It was found that Cys-43 has a key role in dimer formation, asymmetrically cross-linked to a cysteine residue of another identical subunit. The remaining cystines participate in the intramolecular disulfide linkages. Pepsin digestion of PDGF-BB dimer generated several small peptides and one ubiquitous Cys-containing peptide. Sequence analyses of several Cys-containing peptides indicated the existence of three intramolecular disulfide linkages including Cys-16--Cys-60, Cys-49--Cys-97, and Cys-53--Cys-99. Two interchain disulfide bonds of Cys-43--Cys-52 between two subunits were deduced from the partial reduction and alkylation of PDGF-BB. This study provides chemically determined disulfide linkages of PDGF-BB.

Acetylation↗
PubMed1988

Reoxidation of the class I disulfides of the rat adipocyte insulin receptor is dependent upon the presence of insulin: the class I disulfide of the insulin receptor is extracellular.

Elements of the quaternary structure of the native and dithiothreitol- (DTT) reduced rat adipocyte insulin receptor have been elucidated by vectorial probing and subunit cross-linking. The charged reducing agents glutathione and beta-mercaptoethylamine were used to reduce the class I disulfides of the receptor in intact adipocytes, demonstrating the extracellular location of the disulfide directly. This interpretation was confirmed by use of DTT as a reducing agent and the nonpermeant sulfhydryl blocking reagent Thiolyte MQ to prevent the reoxidation of the class I sulfhydryl groups which occurred when they were not blocked. It was found that the above reoxidation of the receptor is dependent on the concentration of insulin in the nanomolar range, not occurring measurably at 4 degrees C in its absence. Cross-linking studies with ethylene glycol bis(succinimidyl succinate) demonstrated that the alpha subunits could not be cross-linked to each other after reduction of the class I disulfides, suggesting that the interaction between the receptor heterodimers may be due primarily to the disulfide bonds.

Adipose Tissue↗
PubMed2006

Mechanistic implications of the structure of the mixed-disulfide intermediate of the disulfide oxidoreductase, 2-ketopropyl-coenzyme M oxidoreductase/carboxylase.

The structure of the mixed, enzyme-cofactor disulfide intermediate of ketopropyl-coenzyme M oxidoreductase/carboxylase has been determined by X-ray diffraction methods. Ketopropyl-coenzyme M oxidoreductase/carboxylase belongs to a family of pyridine nucleotide-containing flavin-dependent disulfide oxidoreductases, which couple the transfer of hydride derived from the NADPH to the reduction of protein cysteine disulfide. Ketopropyl-coenzyme M oxidoreductase/carboxylase, a unique member of this enzyme class, catalyzes thioether bond cleavage of the substrate, 2-ketopropyl-coenzyme M, and carboxylation of what is thought to be an enzyme-stabilized enolacetone intermediate. The mixed disulfide of 2-ketopropyl-coenzyme M oxidoreductase/carboxylase was captured through crystallization of the enzyme with the physiological products of the reaction, acetoacetate, coenzyme M, and NADP, and reduction of the crystals with dithiothreitol just prior to data collection. Density in the active-site environment consistent with acetone, the product of reductive decarboxylation of acetoacetate, was revealed in this structure in addition to a well-defined hydrophobic pocket or channel that could be involved in the access for carbon dioxide. The analysis of this structure and that of a coenzyme-M-bound form provides insights into the stabilization of intermediates, substrate carboxylation, and product release.

Acetoacetates↗
PubMed2001

First examples of oxidizing secondary alcohols to ketones in the presence of the disulfide functional group: synthesis of novel diketone disulfides.

The disulfide functionality is present in a number of organic compounds of interest in the fields of both chemistry and biology. Because the disulfide group is known to be highly susceptible to further oxidation by a wide range of agents, performing a chemoselective oxidation without further oxidizing the disulfide moiety poses a synthetic challenge. Reported herein are the first examples of such a chemoselective oxidation in which a series of novel secondary alcohol disulfides 2a-f have been converted to the corresponding symmetrical diketones 3a-f utilizing a modified Swern oxidation.

Alcohols↗
PubMed1977

Formation of the four isomers of hen egg white lysozyme containing three negative disulfide bonds and one open disulfide bond.

Reduced partially carboxymethylated hen egg white lysozyme (mucopeptide N-acetylmuramoylhydrolase; EC 3.2.1.17) (approximately 0.8 mol of [1-(14)C]carboxymethyl groups) was air oxidized at pH 8.0 and 37 degrees in the presence of 1.5 mM 2-mercaptoethanol for 36 hr. Gel filtration of this product gave the lower (native) and higher hydrodynamic volume forms, both containing radioactivity (approximately 35 and 65%, respectively). Ion exchange chromatography of the lower hydrodynamic volume forms yielded renatured lysozyme, two major radioactive samples (LH(C) and LH(D)) eluting at the positions of monocarboxymethylated lysozyme, and two minor radioactive samples eluting at the positions of dicarboxymethylated lysozyme. Sample LH(C) (approximately 23% of the radioactivity) was essentially homogeneous with respect to electrophoretic mobility, exhibited approximately 39% of the enzymic activity of lysozyme, and contained 0.95 mol of [(14)C]carboxymethyl groups. Sample LH(D) (approximately 8% of the radioactivity) was also enzymically active and contained approximately 0.5 mol of [(14)C]carboxymethyl groups; this low value is apparently due to contamination of noncarboxymethylated species. The radioactive tryptic peptides from samples LH(C) and LH(D) were characterized. The results indicated that all eight isomers, containing three presumably native disulfide bonds and one free and one carboxymethylated sulfhydryl group, are formed on air oxidation of reduced partially carboxymethylated lysozyme. Since in each of these isomers the formation of one of the four native disulfide bonds is permanently blocked, it would follow that no one of the four disulfide bonds of native lysozyme is obligatory in the formation of the other three native disulfide bonds.

Amino Acid Sequence↗
PubMed2003

Not every disulfide lasts forever: disulfide bond formation as a redox switch.

Cellular compartments differ dramatically in their redox potentials. This translates directly into variations in the extent of disulfide bond formation within proteins, depending on their cellular localization. It has long been assumed that proteins that are present in the reducing environment of the cytosol do not possess disulfide bonds. The recent discovery of a number of cytosolic proteins that use specific and reversible disulfide bond formation as a functional switch suggests that this view needs to be revised. Oxidative stress-induced disulfide bond formation appears to be the main strategy to adjust the protein activity of the oxidative stress transcription factors Yap1 and OxyR, the molecular chaperone Hsp33, and the anti-sigma factor RsrA. This elegant and rapid regulation allows the cells to respond quickly to environmental changes that manifest themselves in the accumulation of reactive oxygen species.

Animals↗
PubMed1983

Purification and characterization of a Bacillus megaterium disulfide reductase specific for disulfides containing pantethine 4',4"-diphosphate.

An NADH-linked disulfide reductase specific for disulfides containing pantethine 4',4"-diphosphate moieties was purified 23,000-fold to homogeneity from spores of Bacillus megaterium. The enzyme had a native molecular weight of 122,000 with two apparently identical subunits, contained one molecule of flavin adenine dinucleotide per subunit, and was inhibited by the vicinal dithiol reagent arsenite. The enzyme was active only on disulfides containing pantethine 4',4"-diphosphate moieties, including pantethine 4',4"-diphosphate, oxidized coenzyme A, and coenzyme A in disulfide linkage to acyl carrier protein. However, the Km values for pantethine 4',4"-diphosphate and oxidized coenzyme A were 0.65 and 7.4 mM, respectively. The enzyme was at a low level in log-phase cells but increased up to 10-fold early in the stationary phase and had a similar specific activity in both the mother cell and the forespore compartment; the enzyme activity fell only slowly during spore germination and outgrowth. The enzyme was not detected in several eucaryotic sources and was present in at most a low level in a number of gram-negative bacteria. Surprisingly, the specific activity of this enzyme varied more than 200-fold in extracts from different Bacillus species, with values in B. subtilis being 5- to 6-fold lower and values in B. cereus and B. sphaericus being 8- and 35-fold higher, respectively, than the maximum value in B. megaterium. However, the high specific activity in B. sphaericus did not represent more enzyme protein than in B. megaterium. The possible function of this newly discovered enzyme is discussed.

Bacillus↗
PubMed2002

A structural model of pestivirus E(rns) based on disulfide bond connectivity and homology modeling reveals an extremely rare vicinal disulfide.

E(rns) is a pestivirus envelope glycoprotein and is the only known viral surface protein with RNase activity. E(rns) is a disulfide-linked homodimer of 100 kDa; it is found on the surface of pestivirus-infected cells and is secreted into the medium. In this study, the disulfide arrangement of the nine cysteines present in the mature dimer was established by analysis of the proteolytically cleaved protein. Fragments were obtained after digestion with multiple proteolytic enzymes and subsequently analyzed by liquid chromatography-electrospray ionization mass spectrometry. The analysis demonstrates which cysteine is involved in dimerization and reveals an extremely rare vicinal disulfide bridge of unknown function. With the assistance of the disulfide arrangement, a three-dimensional model was built by homology modeling based on the alignment with members of the Rh/T2/S RNase family. Compared to these other RNase family members, E(rns) shows an N-terminal truncation, a large insertion of a cystine-rich region, and a C-terminal extension responsible for membrane translocation. The homology to mammalian RNase 6 supports a possible role of E(rns) in B-cell depletion.

Amino Acid Sequence↗
PubMed1985

Augmentation of in vitro antibody response by disulfide compounds. I. Comparison between intermolecular and intramolecular disulfides.

The following disulfide compounds: 2-hydroxyethyldisulfide (2-MEox), 2-aminoethyldisulfide (cystamine) and oxidized dithiothreitol (DTTox) were found to augment the in vitro antibody response to sheep erythrocytes in murine lymphocytes as effectively as their reduced forms when they were added to the culture medium. We, however, found out that the mode of action of DTTox was greatly different from that of 2-MEox or cystamine. 2-MEox and cystamine showed augmenting effects on the proliferative response to lipopolysaccharide and on the uptake of (35S)-cystine by the lymphocytes. In contrast to these intermolecular disulfides, DTTox, an intramolecular disulfide compound, was found to be inactive in these systems. 2-MEox and cystamine, but not DTTox, were reduced to thiol forms by the intact lymphocytes or by the cell homogenate. Thus, it is likely that DTTox did not behave as the reduced form in the lymphocyte culture in contrast to 2-MEox and cystamine, suggesting that the disulfide form itself plays an important role in augmenting effects of DTTox.

Animals↗
PubMed1989

The arrangement of disulfide loops in human alpha 2-HS glycoprotein. Similarity to the disulfide bridge structures of cystatins and kininogens.

The complete disulfide loop structure of human alpha 2-HS glycoprotein has been elucidated. alpha 2-HS glycoprotein isolated from human plasma was found to be a two-chain protein composed of a heavy and a light chain. The heavy chain comprises the A-chain of alpha 2-HS glycoprotein (Yoshioka, Y., Gejyo, F., Marti, T., Rickli, E. E., Bürgi, W., Offner, G. D., Troxler, R. F., and Schmid, K. (1986) J. Biol. Chem. 261, 1665-1676) and part of the connecting peptide which has been predicted from the corresponding cDNA sequence (Lee, C. C., Bowman, B. H., and Yang, F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4403-4407), whereas the light chain corresponds to the beta-chain of alpha 2-HS glycoprotein (Gejyo, F., Chang, J. L., Bürgi, W., Schmid, K., Offner, G. D., Troxler, R. F., Van Halbeek, H., Dorland, L., Gerwig, G. J., Vliegenthart, J. F. G. (1983) J. Biol. Chem. 258, 4966-4971). Twelve half-cystine residues are present in the alpha 2-HS glycoprotein molecule, and 11 of them are positioned in the heavy chain and a single one in the light chain of the molecule; they form six disulfide bridges. The first and the last half-cystine residues of the amino acid sequence of alpha 2-HS glycoprotein are engaged in the formation of a loop spanning the extreme NH2- and COOH-terminal portions of the molecule, thereby connecting the heavy and light chains. The other 10 half-cystines residues are linked consecutively in the heavy chain and form five loops which span 4-19 amino acid residues. Among them are two pairs of loops which are characterized by mutual sequence homology. The particular arrangement of disulfide loops in alpha 2-HS glycoprotein is similar to the patterns of linearly arranged and tandemly repeated disulfide loops of cysteine proteinase inhibitors, i.e. the cystatins and the kininogens. It is concluded that alpha 2-HS glycoprotein represents a structural prototype of a novel family among the cystatin superfamily, characterized by the presence of two cystatin-like building blocks. Extensive similarity among the NH2-terminal sequences of alpha 2-HS glycoprotein and human histidine-rich glycoprotein suggest that the latter protein is another candidate protein of this new family.

Amino Acid Sequence↗