A structurally driven analysis of thiol reactivity in mammalian albumins

Understanding the structural basis of protein redox activity is still an open question. Hence, by using a structural genomics approach, different albumins have been chosen to correlate protein structural features with the corresponding reaction rates of thiol exchange between albumin and disulfide DTNB. Predicted structures of rat, porcine, and bovine albumins have been compared with the experimentally derived human albumin. High structural similarity among these four albumins can be observed, in spite of their markedly different reactivity with DTNB. Sequence alignments offered preliminary hints on the contributions of sequence‐specific local environments modulating albumin reactivity. Molecular dynamics simulations performed on experimental and predicted albumin structures reveal that thiolation rates are influenced by hydrogen bonding pattern and stability of the acceptor C34 sulphur atom with donor groups of nearby residues. Atom depth evolution of albumin C34 thiol groups has been monitored during Molecular Dynamic trajectories. The most reactive albumins appeared also the ones presenting the C34 sulphur atom on the protein surface with the highest accessibility. High C34 sulphur atom reactivity in rat and porcine albumins seems to be determined by the presence of additional positively charged amino acid residues favoring both the C34 S^? form and the approach of DTNB. © 2010 Wiley Periodicals, Inc. Biopolymers 95:278–285, 2011.     

Initial steps in assembly of microfibrils. Formation of disulfide-cross-linked multimers containing fibrillin-1

Fibrillins are the major constituents of extracellular microfibrils. How fibrillin molecules assemble into microfibrils is not known. Sequential extractions and pulse-chase labeling of organ cultures of embryonic chick aortae revealed rapid formation of disulfide-cross-linked aggregates containing fibrillin-1. These results demonstrated that intermolecular disulfide bond formation is an initial step in the assembly process. To identify free cysteine residues available for intermolecular cross-linking, small recombinant peptides of fibrillin-1 harboring candidate cysteine residues were analyzed. Results revealed that the first four cysteine residues in the unique N terminus form intramolecular disulfide bonds. One cysteine residue (Cys(204)) in the first hybrid domain of fibrillin-1 was found to occur as a free thiol and is therefore a good candidate for intermolecular disulfide bonding in initial steps of the assembly process. Furthermore, evidence indicated that the comparable cysteine residue in fibrillin-2 (Cys(233)) also occurs as a free thiol. These free cysteine residues in fibrillins are readily available for intermolecular disulfide bond formation, as determined by reaction with Ellman's reagent. In addition to these major results, the cleavage site of the fibrillin-1 signal peptide and the N-terminal sequence of monomeric authentic fibrillin-1 from conditioned fibroblast medium were determined.     

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.     

Highly conserved cysteines of mouse core 2 beta1,6-N-acetylglucosaminyltransferase I form a network of disulfide bonds and include a thiol that affects enzyme activity

Core 2 beta1,6-N-acetylglucosaminyltransferase I (C2GnT-I) plays a pivotal role in the biosynthesis of mucin-type O-glycans that serve as ligands in cell adhesion. To elucidate the three-dimensional structure of the enzyme for use in computer-aided design of therapeutically relevant enzyme inhibitors, we investigated the participation of cysteine residues in disulfide linkages in a purified murine recombinant enzyme. The pattern of free and disulfide-bonded Cys residues was determined by liquid chromatography/electrospray ionization tandem mass spectrometry in the absence and presence of dithiothreitol. Of nine highly conserved Cys residues, under both conditions, one (Cys217) is a free thiol, and eight are engaged in disulfide bonds, with pairs formed between Cys59-Cys413, Cys100-Cys172, Cys151-Cys199, and Cys372-Cys381. The only non-conserved residue within the beta1,6-N-acetylglucosaminyltransferase family, Cys235, is also a free thiol in the presence of dithiothreitol; however, in the absence of reductant, Cys235 forms an intermolecular disulfide linkage. Biochemical studies performed with thiolreactive agents demonstrated that at least one free cysteine affects enzyme activity and is proximal to the UDP-GlcNAc binding site. A Cys217 --> Ser mutant enzyme was insensitive to thiol reactants and displayed kinetic properties virtually identical to those of the wild-type enzyme, thereby showing that Cys217, although not required for activity per se, represents the only thiol that causes enzyme inactivation when modified. Based on the pattern of free and disulfide-linked Cys residues, and a method of fold recognition/threading and homology modeling, we have computed a three-dimensional model for this enzyme that was refined using the T4 bacteriophage beta-glucosyltransferase fold.     

Four free cysteine residues found in human IgG1 of healthy donors

Modifications with different thiol reagents demonstrated that 28 of 32 cysteine residues of human IgG1 are involved in the formation of disulfide bonds, and four cysteines remain free. So IgG1 is a protein possessing both free SH-groups and disulfide bonds. Only one of the four SH-groups is accessible for silver or mercury ions and hydrophobic reagents, whereas the remaining three SH-groups are masked and can be revealed only after deep denaturation of the protein. Detection of the masked cysteine residues was shown to depend on the kinetics of intramolecular changes occurring during denaturation of the protein and on the method of the assay of the SH-groups.     

Oxidation of cysteine and homocysteine by bovine albumin

The autooxidation of cysteine and homocysteine to their disulfide forms was determined by measuring the time course of thiol groups disappearance. We found the oxidative chemistry of cysteine and homocysteine to be quite different. In the absence of added Cu(II), cysteine autooxidized at a slower rate than homocysteine, though in its presence cysteine oxidation was much faster, homocysteine being found to be a poor responder to copper catalysis. Albumin speeded up the spontaneous oxidation of both aminothiols, the reaction being faster with cysteine than with homocysteine. The copper content of different albumins was found to be highly variable, ranging from 12.75 to 0.64 microg Cu(II)/g albumin. We propose that copper bound to albumin possesses redox cycling activity to perform cysteine oxidation since: (i) copper elimination by copper chelators markedly reduces oxidation; and (ii) a positive correlation exists between the albumin copper content and the oxidation reaction rate.     

Identification of plasma proteins containing sulfite-reactive disulfide bonds

Plasma protein S-sulfonate compounds (RS-SO-3) have previously been shown to form, presumably by sulfitolysis of disulfide bonds, as a result of exposure to sulfite. In the investigations reported here, we identify two proteins in rabbit plasma, namely albumin and plasma fibronectin, which contain reactive sites for S-sulfonate formation. Separation and identification of these proteins following in vitro and in vivo exposure to sulfite was accomplished primarily by column chromatographic and electrophoretic techniques. In addition, the structure of presumed S-sulfonate groups was confirmed by the identification of cysteinyl-S-sulfonate residues in protein hydrolysates generated by enzymatic digestion. The molar ratio of RS-SO-3 in both albumin and plasma fibronectin was less than one. Data from our experiments suggest that the mixed disulfide site of non- mercaptalbumin is the reactive site for S-sulfonate formation. The site(s) of formation within the plasma fibronectin molecule was not investigated. The possible physiological significance of disulfide sulfitolysis of albumin and plasma fibronectin is discussed.     

S-linked protein homocysteinylation: identifying targets based on structural, physicochemical and protein–protein interactions of homocysteinylated proteins

An elevated level of homocysteine, a thiol-containing amino acid is associated with a wide spectrum of disease conditions. A majority (>80 %) of the circulating homocysteine exist in protein-bound form. Homocysteine can bind to free cysteine residues in the protein or could cleave accessible cysteine disulfide bonds via thiol disulfide exchange reaction. Binding of homocysteine to proteins could potentially alter the structure and/or function of the protein. To date only 21 proteins have been experimentally shown to bind homocysteine. In this study we attempted to identify other proteins that could potentially bind to homocysteine based on the criteria that such proteins will have significant 3D structural homology with the proteins that have been experimentally validated and have solvent accessible cysteine residues either with high dihedral strain energy (for cysteine–cysteine disulfide bonds) or low pKa (for free cysteine residues). This analysis led us to the identification of 78 such proteins of which 68 proteins had 154 solvent accessible disulfide cysteine pairs with high dihedral strain energy and 10 proteins had free cysteine residues with low pKa that could potentially bind to homocysteine. Further, protein–protein interaction network was built to identify the interacting partners of these putative homocysteine binding proteins. We found that the 21 experimentally validated proteins had 174 interacting partners while the 78 proteins identified in our analysis had 445 first interacting partners. These proteins are mainly involved in biological activities such as complement and coagulation pathway, focal adhesion, ECM-receptor, ErbB signalling and cancer pathways, etc. paralleling the disease-specific attributes associated with hyperhomocysteinemia.     

Mechanism of bovine serum albumin aggregation during ultrafiltration

Protein fouling is a critical problem for ultrafiltration. In this study, we adopted bovine serum albumin (BSA) as a model protein and polysulfone membrane as a typical ultrafiltration membrane. We then investigated the factors of the protein denaturation and aggregation, such as stirring shear stress and intermolecular exchange of disulfide during ultrafiltration, and discussed the BSA fouling mechanism. Fourier transform-infrared analysis revealed that magnetic stirring did not cause any difference in the secondary structural change of BSA gel-like deposits on the ultrafiltration membrane. BSA aggregates were collected from BSA gel-like deposits on the ultrafiltration membrane by centrifugation. Polyacrylamide gel electrophoresis in SDS analysis of BSA aggregates proved that the major binding of the BSA aggregates involved intermolecular disulfhydryl binding and that capping the free thiol group in BSA molecules with cysteine induced a remarkable decrease in the amount of the BSA aggregates during ultrafiltration. We concluded that one of the main factors in the BSA aggregation during ultrafiltration is the intermolecular exchange of disulfide through cysteinyl residue. We also found that the BSA aggregation caused a decrease in alpha-helix from 66% to 50% and an increase in beta-sheet from 20% to 36%, which was presumably because the cysteine residues associated with the intermolecular disulfide bonds had been located in alpha-helices. Copyright John Wiley & Sons, Inc.     

Thiol-disulfide interchange between cystine and N-2-mercaptoethyl-1, 3-diaminopropane as a potential treatment for cystinuria

The radioprotective compound WR2721 is a thiophosphate, which, when administered orally, is activated at the acid pH of the stomach to its free thiol (MDP). The free thiol is a mucolytic compound which acts via the reduction of disulfide bonds of mucin molecules. An equimolar mixture of MDP and cysteine, in urine at pH 6.0 and 37 degrees C, when oxidized by molecular oxygen, preferentially forms the soluble mixed disulfide between MDP and cysteine. The disulfide cystine will undergo thiol-disulfide interchange with MDP; as a result, cystine crystals are effectively dissolved. Moreover, in the presence of catalytic amounts of free thiol, the disulfide of MDP will undergo thiol-disulfide interchange with cystine to dissolve cystine crystals. The mixed disulfide of MDP with cysteine is soluble in urine at pH 6.0 and 37 degrees C to at least 100 mg/ml. Chromatographic procedures which permit the analysis of MDP and its mixed disulfide derivatives as MDP-sulfonic acid are described. By these procedures, it was demonstrated that 20% of a single oral dose of WR2721 was excreted as MDP derivatives in the urine of normal volunteers. These procedures will permit the evaluation of WR2721 in the treatment of cystinuria.     

Thiol dependent isomerization of bovine albumin

Albumin isomerizes to the aged form in the presence of cysteine at pH 8.9 and low ionic strength. Albumins with a high fatty acid and Cu(II) content do not produce isomers, and recover this capacity after an acid expansion. Isomers have the free thiol group fully oxidized (non-mercaptalbumin) and have been isolated for the first time from aged albumins by anion and cation exchange chromatography. Isomers have a higher susceptibility to limited tryptic digestion and show a decrease in the fluorescence of bound dansylamide, a typical marker of site I. Aging in the presence of phenylarsine oxide, which complexes vicinal thiols, impairs the formation of isomers and increases the free -SH groups of albumin.     

Analysis of free sulfhydryl groups and disulfide bonds in Na+,K+-ATPase

The content of free SH groups and disulfide bonds in the purified pig kidney Na+,K+-ATPase was determined by ammetric titration with silver nitrate. In the native enzyme, most of the free SH groups are masked due to their location in the polypeptide chain regions poorly accessible to SH reagents. Denaturation with 5% SDS and 8 M urea makes these regions accessible thus revealing 22 free SH groups/mol of the protein. After complete blocking of free SH groups with silver ions, 8 SH groups/mol of the protein are being released upon sulfitolysis which indicates the presence of four disulfide bonds in the enzyme. At least one disulfide bridge is located in the alpha-subunit whereas the beta-subunit contains three disulfide bonds.     

Dissociation of bovine cytochrome c1 subcomplex and the status of cysteine residues in the subunits

Purified bovine heart two-band cytochrome c1 subcomplex was dissociated by treatment with p-chloromercuribenzoic acid (pCMB) into its heme subunit and a colorless subunit called hinge protein, which is essential for the formation of cytochrome c1-c complex. The subcomplex was found by titration to react with 4 mol of pCMB per mol of cytochrome c1. The contents of mercury of the dissociated heme subunit and the hinge protein were 3 and 1 mol per mol of polypeptide, respectively. These results, together with the sequence analysis, indicated that the three cysteine residues in cytochrome c1 heme subunit not involved in heme-binding existed in free thiol form. One of the five cysteine residues in the hinge protein was in free form and four in two disulfide bonds. The dissociated hinge protein was digested with staphylococcal protease and the cysteine-containing peptides were separated by reversed-phase high-performance liquid chromatography (HPLC). The content of mercury and the result of performic acid oxidation of cystine peptides revealed that Cys-30 existed in free thiol form and two disulfide bridges were formed between Cys-24 and Cys-68 and between Cys-40 and Cys-54. The conformation of the hinge protein was predicted to be composed largely of either two-alpha-helical or four-alpha-helical conformation with the amino (N)-terminal 20 residues being in a random structure.     

Role of thiols, pH and cathepsin D in the lysosomal catabolism of serum albumin.

Attempts were made to assess the role of thiols and to determine the cathepsins involved in the degradation of serum albumin in mouse liver and kidney lysosomes. Unlike cysteine or beta-mercaptoethanol, reduced glutathione (GSH) did not stimulate the degradation of formaldehyde-treated albumin in liver lysosomes, suggesting that the tripeptide did not penetrate the membrane. However, GSH was a much more effective stimulant of proteolysis in kidney lysosomes than was cysteine at low concentrations, and the effect was saturable at 1-2 mM concentrations. Thiols did not stimulate proteolysis in lysosomes when the disulphide bonds of albumin were reduced and alkylated, suggesting that the stimulatory effects were solely due to disulphide-bond reduction in protein substrates. Results obtained with thiols and iodoacetamide suggested that albumins denatured by disulphide-bond reduction and alkylation, disulphide-bond reduction without alkylation, or by treatment with 8 M-urea, were all degraded primarily by cathepsin D in lysosomes, but formaldehyde-denatured albumin was attacked by thiol proteinases. These findings correlated well with studies on the degradation of these proteins by rat liver lysosome (tritosome) extracts. Studies with the proteinase inhibitors leupeptin and pepstatin and the stimulatory effects of thiols in these extracts suggested that formaldehyde-denatured albumin was degraded primarily by the thiol proteinases, but that native albumin or albumins denatured by disulphide-bond reduction or by treatment with 8 M-urea were attacked by cathepsin D. Denaturation of serum albumin by any of the methods used caused a shift in the pH optimum of albumin catabolism by tritosome extracts or by purified cathepsin D from approx. 3-4 to 5-6. These results were discussed in terms of a possible mechanism for the catabolic aspect of serum albumin turnover.     

Structural studies on metal-serum albumin. IV. The interaction of Zn(II), Cd(II) and Hg(II) with HSA and BSA.

There have been no detailed and reliable studies on the environment and configuration of Zn(II), Cd(II) and Hg(II) in the metal centers of human serum albumin and bovine serum albumin to date. In this paper the authentic evidence for the involvement of the cystinyl sulfur atoms in the ligation to the zinc group ions has been obtained from the X-ray photoelectron spectra. The belief that each of the zinc group ions possesses several similar binding sites in human- and bovine serum albumin and is bound to the deprotonated thiol group (-RS-) of the cysteinyl residues to form tetrahedral and linear metal centers has been further confirmed by the treatment of ligand to metal charge transfer data with Jorgensen's method. According to these results, we have inferred that these binding sites may be located at the seventeen disulfide bridges, most likely at the seven pairs of adjacent disulfide bridges between positions 75 and 567, in the serum albumins.     

Functional rescue of the nephrogenic diabetes insipidus-causing vasopressin V2 receptor mutants G185C and R202C by a second site suppressor mutation

Mutations in the gene of the G protein-coupled vasopressin V2 receptor (V2 receptor) cause X-linked nephrogenic diabetes insipidus (NDI). Most of the missense mutations on the extracellular face of the receptor introduce additional cysteine residues. Several groups have proposed that these residues might disrupt the conserved disulfide bond of the V2 receptor. To test this hypothesis, we first calculated a structure model of the extracellular receptor domains. The model suggests that the additional cysteine residues may form a second disulfide bond with the free, nonconserved extracellular cysteine residue Cys-195 rather than impairing the conserved bond. To address this question experimentally, we used the NDI-causing mutant receptors G185C and R202C. Their Cys-195 residues were replaced by alanine to eliminate the hypothetical second disulfide bonds. This second site mutation led to functional rescue of both NDI-causing mutant receptors, strongly suggesting that the second disulfide bonds are indeed formed. Furthermore we show that residue Cys-195, which is sensitive to "additional cysteine" mutations, is not conserved among the V2 receptors of other species and that the presence of an uneven number of extracellular cysteine residues, as in the human V2 receptor, is rare among class I G protein-coupled receptors.     

Functional characterization of Mia40p, the central component of the disulfide relay system of the mitochondrial intermembrane space

Mia40p and Erv1p are components of a translocation pathway for the import of cysteine-rich proteins into the intermembrane space of mitochondria. We have characterized the redox behavior of Mia40p and reconstituted the disulfide transfer system of Mia40p by using recombinant functional C-terminal fragment of Mia40p, Mia40C, and Erv1p. Oxidized Mia40p contains three intramolecular disulfide bonds. One disulfide bond connects the first two cysteine residues in the CPC motif. The second and the third bonds belong to the twin CX(9)C motif and bridge the cysteine residues of two CX(9)C segments. In contrast to the stabilizing disulfide bonds of the twin CX(9)C motif, the first disulfide bond was easily accessible to reducing agents. Partially reduced Mia40C generated by opening of this bond as well as fully reduced Mia40C were oxidized by Erv1p in vitro. In the course of this reaction, mixed disulfides of Mia40C and Erv1p were formed. Reoxidation of fully reduced Mia40C required the presence of the first two cysteine residues in Mia40C. However, efficient reoxidation of a Mia40C variant containing only the cysteine residues of the twin CX(9)C motif was observed when in addition to Erv1p low amounts of wild type Mia40C were present. In the reconstituted system the thiol oxidase Erv1p was sufficient to transfer disulfide bonds to Mia40C, which then could oxidize the variant of Mia40C. In summary, we reconstituted a disulfide relay system consisting of Mia40C and Erv1p.     

The sulfhydryl groups of the thiol-dependent cytolytic toxin from Bacillus alvei evidence for one essential sulfhydryl group

Alveolysin, an extracellular protein toxin (M_r ? 63,000) excreted by Bacillus alvei and purified to homogeneity was shown to contain four cysteine residues. All thiol groups of the hemolytically active toxin preparation were free as found by direct titration by 5,5′-dithiobis (2-nitrobenzoic acid) and confirmed by the absence of disulfide bond. Toxin alkylation with tosyl lysine chloromethyl ketone resulted in the complete loss of hemolytic activity and the disappearance of only one thiol group with no modification of histidine residues. These results support the conclusion that one essential thiol group is implicated in the membrane-disrupting activity of alveolysin.     

Studies in serum support rapid formation of disulfide bond between unpaired cysteine residues in the VH domain of an immunoglobulin G1 molecule

Recombinant monoclonal antibodies undergo extensive posttranslational modifications. In this article, we characterize major modifications, separated by cation exchange chromatography, on an immunoglobulin G1 (IgG1) monoclonal antibody (mAb). We found that N-terminal cyclization of glutamine residues to pyroglutamate on the light and heavy chains are the major isoforms resolved during cation exchange chromatography. However, using CEX, we also separated and identified isoforms with unpaired cysteine residues in the V_H domain of the molecule (Cys22-Cys96). Omalizumab, a therapeutic anti-IgE antibody, has unpaired cysteine residues in the V_H domain between Cys22 and Cys96, and the Fab fragment, containing the unpaired cysteine residues, is reported to have reduced potency. Dynamic interchain disulfide rearrangement, with slow kinetics, was recently reported to take place in serum for an IgG2 molecule and resulted in predictable mature isoforms. Analytical evaluation of our mAb, after recovery from serum, revealed that the unpaired intrachain cysteine residues (Cys22-Cys96) reformed their disulfide bond. The significance of this study is that correct pairing occurred rapidly, and we speculate that thiol molecules such as cysteine, homocysteine, and glutathione in serum provide an environment, outside the endoplasmic reticulum, for correct linkage.     

Evidence for intramolecular disulfide bond shuffling in the folding of mutant human lysozyme

Our previous results using the Saccharomyces cerevisiae secretion system suggest that intramolecular exchange of disulfide bonds occurs in the folding pathway of human lysozyme in vivo (Taniyama, Y., Yamamoto, Y., Kuroki, R., and Kikuchi, M. (1990) J. Biol. Chem. 265, 7570-7575). Here we report on the results of introducing an artificial disulfide bond in mutants with 2 cysteine residues substituting for Ala83 and Asp91. The mutant (C83/91) protein was not detected in the culture medium of the yeast, probably because of incorrect folding. Thereupon, 2 cysteine residues Cys77 and Cys95 were replaced with Ala in the mutant C83/91, because a native disulfide bond Cys77-Cys95 was found not necessary for correct folding in vivo (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). The resultant mutant (AC83/91) was secreted as two proteins (AC83/91-a and AC83/91-b) with different specific activities. Amino acid and peptide mapping analyses showed that two glutathiones appeared to be attached to the thiol groups of the cysteine residues introduced into AC83/91-a and that four disulfide bonds including an artificial disulfide bond existed in the AC83/91-b molecule. The presence of cysteine residues modified with glutathione may indicate that the non-native disulfide bond Cys83-Cys91 is not so easily formed as a native disulfide bond. These results suggest that the introduction of Cys83 and Cys91 may act to suppress the process of native disulfide bond formation through disulfide bond interchange in the folding of human lysozyme.