Primary structure of human plasma fibronectin--characterization of the 6,000 dalton C-terminal fragment containing the interchain disulfide bridges

The carboxy terminal fragment of human plasma fibronectin has been isolated after tryptic digestion and separation by DEAE-cellulose chromatography and gel filtration on Sephadex G-50. It has a molecular weight of 6,000 which changes to 3,000 after reduction indicating that the fragment is a dimer. We have determined the amino acid sequence of the 6kDa fragment and showed that it contains 26 residues including two half-cystines which form two interchain disulfide bridges. The 6kDa fragment is not phosphorylated as in bovine fibronectin although its amino acid sequence is identical to that reported for bovine plasma fibronectin. When compared to the sequence deduced from a rat cDNA, one amino acid substitution can be found. It appears that the carboxyl end of fibronectin is highly conserved among species.     

Elongation of the interchain disulfide bridges of insulin. A synthetic analog.

The sythesis and isolation in purified form of an analog of insulin with the interchain disulfide bridges elongated by a methylene group is described. This analog differs from the parent molecule in that the cystein residues occupying positions A-7 and A-20 and involved in the formation of the two interchain disulfide bridges of insulin have been replaced by homocysteine residues. For the synthesis of this compound the Hcy-7, 20-A chain of sheep insulin was chemically synthesized and isolated in the S-sulfonated form. Conversion of the latter product to the sulfhydryl derivative and combination with the S-sulfonated form of the B chain of sheep insulin yielded the [Hcy-7, 20-A] insulin. Isolation of the analog from the combination mixture was effected by chromatography on a carboxymethylcellulose column with acetate buffer (pH 3.3) and an exponential sodium chloride gradient. This analog, by the mouse convulsion assay methods and in doses at least 40-fold higher than those normally used for insulin assay, was inactive. By the radioimmunoassay method this synthetic analog was found to possess a potency of 2 i.u./mg. It is concluded that the biological activity of insulin depends critically on a particular geometry conferred on the molecule by the proper placement of the A and B chains.     

Effect of reduction of the interchain disulfide bridge of human thrombin on its enzymatic activity and structure

It was shown that selective hydrolysis of the disulfide bridge between the A- and B-chains of human thrombin in the absence of denaturating agents decrease its proteolytic (e.g., fibrinogen-binding), esterase and amidase activities. Both chains remain bound by non-covalent interactions. A preparation of partially reduced thrombin was obtained and its kinetic parameters were determined. The experimental results suggest that the S-S bond connecting the A- and B-chains of thrombin is involved in the stabilization of the enzyme active center.     

Importance of the disulfide bridges in the antibacterial activity of human hepcidin

Hepcidin was first identified as an antimicrobial peptide present in human serum and urine. It was later demonstrated that hepcidin is the long sought hormone that regulates iron homeostasis in mammals. The native peptide of 25 amino acids (Hepc25) contains four disulfide bridges that maintain a β-hairpin motif. The aim of the present study was to assess whether the intramolecular disulfide bridges are necessary for Hepc25 antimicrobial activity. We show that a synthetic peptide corresponding to human Hepc25, and which contains the four disulfide bridges, has an antibacterial activity against several strains of Gram-positive and Gram-negative bacteria. On the contrary, a synthetic peptide where all cysteines were replaced by alanines (Hepc25-Ala) had no detectable activity against the same strains of bacteria. In a further step, the mode of action of Hepc25 on Escherichia coli was studied. SYTOX Green uptake was used to assess bacterial membrane integrity. No permeabilization of the membrane was observed with Hepc25, indicating that this peptide does not kill bacteria by destroying their membranes. Gel retardation assay showed that the Hepc25 binds to DNA with high efficiency, and that this binding ability is dependent on the presence of the intramolecular disulfide bridges. Reduction of Hepc25 or replacement of the eight cysteines by alanine residues led to peptides that were no longer able to bind DNA in the in vitro assay. Altogether, these results demonstrate that Hepc25 should adopt a three-dimensional structure stabilized by the intramolecular disulfide bridges in order to have antibacterial activity.     

Characterization and bioactivity of hepcidin-2 in zebrafish: Dependence of antibacterial activity upon disulfide bridges

Hepcidin is an antimicrobial peptide and iron-regulatory molecule with highly conserved disulfide bridges among vertebrates, but structural insights into the function in fish remains largely missing. We demonstrate here that recombinant hepcidin-2 from zebrafish is capable of inhibiting the growth of the Gram-negative bacteria Escherichia coli and Vibrio anguillarum, and the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis with minimum inhibitory concentrations (MICs) of 18, 15, 13 and 9 μM, respectively. We also show by TEM examination that recombinant hepcidin-2 is directly cidal to the cells of E. coli and S. aureus. Moreover, we find that hepcidin-2 displays affinity to LPS, LTA and PGN. All these data indicate that hepcidin-2 is both a pattern recognition molecule, capable of identifying LPS, LTA and PGN, and an antibacterial effector, capable of inhibiting the growth of bacteria. The data also show that the antibacterial activity of hepcidin-2 depends upon the disulfide bridges.     

Optical activity of disulfide bonds in proteins. 1. Studies on plasmin modified human somatotropin.

The reduction and alkylation of the two disulfide bonds in a preparation of human pituitary growth hormone which had been previously modified by limited proteolysis with the enzyme plasmin have been studied. Quantitative and selective reduction of the carboxyl-terminal disulfide, as well as total reduction of both disulfides, has been achieved in the absence of denaturants. Circular dichroism spectra of the various reduced and reduced-alkylated derivatives have provided sufficient information to allow an estimation of the individual contributions of each disulfide bond to the total optical activity of the protein. These contributions were found to represent a significant portion of the total optical activity between 290 and 250 nm. The carboxyl-termimal bond exhibits negative dichroism with an apparent center near 258 nm ([theta]M,258nm = 2100 deg cm2 dmol-1). By comparison, the contribution of the remaining disulfide is red-shifted to 273 nm, is also negative in sign, and somewhat more intense ([theta]M,273nm = 3200 deg cm2 dmol-1). Circular dichroism measurements have also been used to approximate the rate of reduction of the protein.     

Enhanced stability of human prion proteins with two disulfide bridges

We compare the folding equilibrium of the globular domain of the human prion protein with two variants of this domain, for which an additional disulfide bond was introduced into the location where it is found in the naturally occurring doppel protein. We find that the unfolding transition midpoint of the variants is shifted toward higher denaturant concentration, indicating that the engineered disulfide bond significantly stabilizes the global protein structure. Our results further reveal that the two-disulfide variant proteins, while possessing the same global fold as the wild-type, display marked differences in their folding pathway-in particular, the absence of a characteristic alpha-helix to beta-sheet transition, which is a fundamental feature associated with misfolding of proteins into amyloid fibrils, especially in the context of prion diseases. These surprising characteristics of disulfide mutant prion proteins have important implications for the understanding of the generic aberrant processes leading to amyloid fibril formation and protein aggregation, as well as providing insight into possible therapeutic strategies.     

The role of interchain disulfide bond in a recombinant human interleukin-17A variant

Interleukin-17A (IL-17A) is the prototype of IL-17 family and has been implicated in the pathogenesis of a variety of autoimmune diseases. Therefore its structural and functional properties are of great medical interest. During our research on a recombinant human IL-17A (rhIL-17A) variant, four isoforms were obtained when it was refolded. While isoforms 1 and 2 represented non-covalent dimers, isoforms 3 and 4 were determined to be covalent dimers. All four isoforms were structurally similar by Circular Dichroism and fluorescence spectroscopy studies, but differential scanning calorimetry demonstrated thermal stability in the order of isoform 1 = isoform 2 < isoform 4 < isoform 3. In addition, compared to covalent dimers (isoform 3 and 4), the non-covalent dimers (isoforms 1 and 2) are slightly less active in a receptor-binding assay but at least 5-fold less active in a cell-based assay.     

Beta-endorphin. Synthesis and biological activity of analogs with disulfide bridges

Two analogs of human beta-endorphin (beta-EP) which contain cystine bridges, [Cys15-Cys26,Phe27,Gly31]-beta-EP (I) and [Cys16-Cys26,Phe27,Gly31]-beta-EP (II), were synthesized by the solid-phase method. Peptides I and II were shown to contain 2-2.5 times the opiate receptor binding activity of beta-endorphin. We also synthesized two analogs with reduced alkylated cysteine residues and these peptides, [Arg9,19,24,28,29 Cys(Cam)11,26,Phe27,Gly31] and [Arg9,19,24,28,29,Cys-(Cam)12,26,Phe27,Gly31], were shown to have approximately the same opiate receptor activity as beta-endorphin.     

Induced interchain disulfide bonding in cAMP-dependent protein kinase II

Cupric phenanthroline was used to catalyze the formation of disulfide bonds in cAMP-dependent protein kinase II. Incubation of holoenzyme alone with cupric phenanthroline resulted in no disulfide bond formation. In contrast, when holoenzyme was preincubated with cAMP prior to treatment with cupric phenanthroline, specific interchain disulfide bonding was found between the regulatory (R) and catalytic (C) subunits. Formation of the R-C dimer was independent of the phosphorylation state of R. Experiments with R that had been freed of bound cyclic nucleotide suggest that a ternary complex of R, C, and cAMP is necessary for the formation of this cross-linked species. When the dimeric R-subunit alone was incubated with cupric phenanthroline, the two protomers of the R-dimer were frequently cross-linked. Phosphorylation of R did not affect the formation of R-R dimers. In contrast to the R-subunit of the type I protein kinase, R-R dimers of the type II protein kinase were not normally observed in the absence of an added catalyst. Factors which favor disulfide bond formation in the R-dimer have not been determined.     

Subunits of human alpha 2-macroglobulin produced by specific reduction of interchain disulfide bonds with thioredoxin

Disulfide bonds in alpha 2-macroglobulin (alpha 2M) were reduced with the thioredoxin system from Escherichia coli. Under the conditions selected, 3.5-4.1 disulfide bonds were cleaved in each alpha 2M molecule, as determined by the consumption of NADPH during the reaction and by the incorporation of iodo[3H]acetate into the reaction product. This extent of disulfide bond reduction, approximately corresponding to that expected from specific cleavage of all four interchain disulfide bonds of the protein, coincided with the nearly complete dissociation of the intact alpha 2M molecule to a species migrating as an alpha 2M subunit in gel electrophoresis, under both denaturing and nondenaturing conditions. The dissociation was accompanied by only small changes of the spectroscopic properties of the subunits, which thus retain a near-native conformation. Reaction of isolated subunits with methylamine or trypsin led to the appearance of approximately 0.55 mol of thiol group/mol of subunits, indicating that the thio ester bonds are largely intact. Moreover, the rate of cleavage of these bonds by methylamine was similar to that in the whole alpha 2M molecule. Although the bait region was specifically cleaved by nonstoichiometric amounts of trypsin, the isolated subunits had minimal proteinase binding ability. Reaction of subunits with methylamine or trypsin produced changes of farultraviolet circular dichroism and near-ultraviolet absorption similar to those induced in the whole alpha 2M molecule, although in contrast with whole alpha 2M no fluorescence change was observed. The methylamine- or trypsin-treated subunits reassociated to a tetrameric species, migrating as the "fast" form of whole alpha 2M in gradient gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)