Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: a novel strategy exploiting disulfide assisted protein folding

The aim of this study has been to develop a strategy for purifying correctly oxidized denatured major histocompability complex class I (MHC-I) heavy-chain molecules, which on dilution, fold efficiently and become functional. Expression of heavy-chain molecules in bacteria results in the formation of insoluble cellular inclusion bodies, which must be solubilized under denaturing conditions. Their subsequent purification and refolding is complicated by the fact that (1). correct folding can only take place in combined presence of beta(2)-microglobulin and a binding peptide; and (2). optimal in vitro conditions for disulfide bond formation ( approximately pH 8) and peptide binding ( approximately pH 6.6) are far from complementary. Here we present a two-step strategy, which relies on uncoupling the events of disulfide bond formation and peptide binding. In the first phase, heavy-chain molecules with correct disulfide bonding are formed under non-reducing denaturing conditions and separated from scrambled disulfide bond forms by hydrophobic interaction chromatography. In the second step, rapid refolding of the oxidized heavy chains is afforded by disulfide bond-assisted folding in the presence of beta(2)-microglobulin and a specific peptide. Under conditions optimized for peptide binding, refolding and simultaneous peptide binding of the correctly oxidized heavy chain was much more efficient than that of the fully reduced molecule.     

Stability and structure-forming properties of the two disulfide bonds of alpha-conotoxin GI

alpha-Conotoxin GI is a 13 residue snail toxin peptide cross-linked by Cys2-Cys7 and Cys3-Cys13 disulfide bridges. The formation of the two disulfide bonds by thiol/disulfide exchange with oxidized glutathione (GSSG) has been characterized. To characterize formation of the first disulfide bond in each of the two pathways by which the two disulfide bonds can form, two model peptides were synthesized in which Cys3 and Cys13 (Cono-1) or Cys2 and Cys7 (Cono-2) were replaced by alanines. Equilibrium constants were determined for formation of the single disulfide bonds of Cono-1 and Cono-2, and an overall equilibrium constant was measured for formation of the two disulfide bonds of alpha-conotoxin GI in pH 7.00 buffer and in pH 7. 00 buffer plus 8 M urea using concentrations obtained by HPLC analysis of equilibrium thiol/disulfide exchange reaction mixtures. The results indicate a modest amount of cooperativity in the formation of the second disulfide bond in both of the two-step pathways by which alpha-conotoxin GI folds into its native structure at pH 7.00. However, when considered in terms of the reactive thiolate species, the results indicate substantial cooperativity in formation of the second disulfide bond. The solution conformational and structural properties of Cono-1, Cono-2, and alpha-conotoxin GI were studied by 1H NMR to identify structural features which might facilitate formation of the disulfide bonds or are induced by formation of the disulfide bonds. The NMR data indicate that both Cono-1 and Cono-2 have some secondary structure in solution, including some of the same secondary structure as alpha-conotoxin GI, which facilitates formation of the second disulfide bond by thiol/disulfide exchange. However, both Cono-1 and Cono-2 are considerably less structured than alpha-conotoxin GI, which indicates that formation of the second disulfide bond to give the Cys2-Cys7, Cys3-Cys13 pairing induces considerable structure into the backbone of the peptide.     

A convenient method for the synthesis of oligonucleotide-cationic peptide conjugates

A method was developed for the synthesis of oligonucleotide-cationic peptide conjugates in solution phase by disulfide bond formation. Precipitation was avoided by the easily removable triethylammonium trifluoroacetate (TEATFAc) salt which served at the same time as a buffer of the reaction mixture. The fast and high yielding disulfide bond formation was due to the Npys thio protecting and activating group of Cys. A solution of the free 5-thiol modified oligonucleotide obtained from Poly-Pak purification was used for conjugation.     

Independent and synergistic effects of disulfide bond formation, beta 2-microglobulin, and peptides on class I MHC folding and assembly in an in vitro translation system.

We have examined the post-translational processing, intrachain disulfide bond formation, folding, and assembly of MHC class I H chains with beta 2-microglobulin after coupled in vitro translation of homogeneous mRNA and transport of nascent chains into canine microsomal vesicles. The formation of native alpha 3 domain conformation was dependent on conditions that optimized intrachain disulfide bond formation, and efficient folding of the alpha 1 alpha 2 domain required exposure to antigenic peptide. beta 2-microglobulin and peptide acted synergistically in forming native alpha 1 alpha 2 domain structure, and a small proportion of molecules with native alpha 1 alpha 2, but non-native alpha 3 structure were detected, indicating that alpha 3 domain folding is not an absolute prerequisite for the formation of native alpha 1 alpha 2 domain structure.     

A CONVENIENT METHOD FOR THE SYNTHESIS OF OLIGONUCLEOTIDE-CATIONIC PEPTIDE CONJUGATES

A method was developed for the synthesis of oligonucleotide-cationic peptide conjugates in solution phase by disulfide bond formation. Precipitation was avoided by the easily removable triethylammonium trifluoroacetate (TEATFAc) salt which served at the same time as a buffer of the reaction mixture. The fast and high yielding disulfide bond formation was due to the Npys thio protecting and activating group of Cys. A solution of the free 5′-thiol modified oligonucleotide obtained from Poly-Pak? purification was used for conjugation.     

Cyclic peptides. XXVI. Synthesis of AM-toxin II analogs by cyclization through ester bond formation

In order to explore the route for the preparation of cyclodepsipeptide by cyclization through an ester bond formation, two analogs of AM-toxin II, cyclotetradepsipeptide, were synthesized. As a preliminary experiment, synthesis of [L-Phe3, L-Ser(Bzl)4]-AM-toxin II, containing L-Phe and L-Ser(Bzl) in place of L-App (2-amino-5-phenyl-pentanoic acid) and delta Ala (alpha, beta-dehydroalanine), respectively, was attempted. Cyclization of H-L-Hmb-L-Phe-L-Ser(Bzl)-L-Ala-OH in CH2Cl2 at 10 mM concentration using water-soluble carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP) successfully gave a cyclic monomer in 16% yield. Cyclization of H-L-Hmb-L-App-L-Ser(Bzl)-L-Ala-OH under the same conditions also afforded a cyclic monomer, [L-Ser(Bzl)4]AM-toxin II, in 19% yield. Analytical parameters of these cyclic monomers obtained were identical to those of the authentic samples obtained by cyclization through a peptide bond formation.     

Protein disulfide bond formation in the cytoplasm during oxidative stress

The majority of disulfide-linked cytosolic proteins are thought to be enzymes that transiently form disulfide bonds while catalyzing oxidation-reduction (redox) processes. Recent evidence indicates that reactive oxygen species can act as signaling molecules by promoting the formation of disulfide bonds within or between select redox-sensitive proteins. However, few studies have attempted to examine global changes in disulfide bond formation following reactive oxygen species exposure. Here we isolate and identify disulfide-bonded proteins (DSBP) in a mammalian neuronal cell line (HT22) exposed to various oxidative insults by sequential nonreducing/reducing two-dimensional SDS-PAGE combined with mass spectrometry. By using this strategy, several known cytosolic DSBP, such as peroxiredoxins, thioredoxin reductase, nucleoside-diphosphate kinase, and ribonucleotide-diphosphate reductase, were identified. Unexpectedly, a large number of previously unknown DSBP were also found, including those involved in molecular chaperoning, translation, glycolysis, cytoskeletal structure, cell growth, and signal transduction. Treatment of cells with a wide range of hydrogen peroxide concentrations either promoted or inhibited disulfide bonding of select DSBP in a concentration-dependent manner. Decreasing the ratio of reduced to oxidized glutathione also promoted select disulfide bond formation within proteins from cytoplasmic extracts. In addition, an epitope-tagged version of the molecular chaperone HSP70 forms mixed disulfides with both beta4-spectrin and adenomatous polyposis coli protein in the cytosol. Our findings indicate that disulfide bond formation within families of cytoplasmic proteins is dependent on the nature of the oxidative insult and may provide a common mechanism used to control multiple physiological processes.     

Synthesis and characterization of the human CC chemokine HCC-2.

Human CC chemokine 2 (HCC-2) is a novel member of the chemokine peptide family that induces chemotaxis of monocytes, T lymphocytes and eosinophils via activation of the CCR-1 and CCR-3 receptors. Fmoc chemistry was optimized and used to synthesize the biologically active 66-residue peptide HCC-2-(48-113). Introduction of the three disulfide bonds was achieved by oxidative folding in the presence of the redox system cysteine/cystine. Alternatively, a semiselective approach utilizing a mixed Acm/Trt protection scheme for disulfide formation was applied. It was found that, without participation of the two HCC-2-specific cysteine residues in positions 64 and 104, the two typical chemokine disulfides are formed predominantly during oxidative folding. In addition, the mutant [Ala64,104]HCC-2-(48-113) lacking the third disulfide bond that discriminates HCC-2 from most other chemokines was synthesized. For disulfide bond formation, oxidative folding was compared with the use of Acm/Trt protection. HCC-2-(48-113) and the mutant [Ala64,104]HCC-2-(48-113) were further analyzed by CD and one-dimensional 1H NMR-spectroscopy. Both peptides adopt a similar stable secondary and tertiary structure in solution.     

Chemical synthesis of La1 isolated from the venom of the scorpion Liocheles australasiae and determination of its disulfide bonding pattern

La1 is a 73‐residue cysteine‐rich peptide isolated from the scorpion Liocheles australasiae venom. Although La1 is the most abundant peptide in the venom, its biological function remains unknown. Here, we describe a method for efficient chemical synthesis of La1 using the native chemical ligation (NCL) strategy, in which three peptide components of less than 40 residues were sequentially ligated. The peptide thioester necessary for NCL was synthesized using an aromatic N‐acylurea approach with Fmoc‐SPPS. After completion of sequential NCL, disulfide bond formation was carried out using a dialysis method, in which the linear peptide dissolved in an acidic solution was dialyzed against a slightly alkaline buffer to obtain correctly folded La1. Next, we determined the disulfide bonding pattern of La1. Enzymatic and chemical digests of La1 without reduction of disulfide bonds were analyzed by liquid chromatography/mass spectrometry (LC/MS), which revealed two of four disulfide bond linkages. The remaining two linkages were assigned based on MS/MS analysis of a peptide fragment containing two disulfide bonds. Consequently, the disulfide bonding pattern of La1 was found to be similar to that of a von Willebrand factor type C (VWC) domain. To our knowledge, this is the first report of the experimental determination of the disulfide bonding pattern of peptides having a single VWC domain as well as their chemical synthesis. La1 synthesized in this study will be useful for investigation of its biological role in the venom. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational analysis of a toxic peptide from Trimeresurus wagleri which blocks the nicotinic acetylcholine receptor.

The 22-residue toxic peptide (WTX1) from the venom of the Southeast Asian snake Trimeresurus wagleri has multiple sites of action, but its lethal effect has been attributed to blocking the postsynaptic acetylcholine receptor at the neuromuscular junction. The 3-dimensional structure of WTX1 was studied using 2-dimensional nuclear magnetic resonance spectroscopy, circular dichroism, and computer simulations. In aqueous solution, WTX1 was shown to have extended and flexible "tails" defined by a short, rigid disulfide-bonded loop. The flexible regions can undergo structural rearrangement when moved from an aqueous to a less polar environment and may contribute to its effectiveness at different receptor sites. By substituting Gly or Phe for His at position 10, significant effects on the disulfide bond formation and, thereby, the activity of the peptide were observed. These results suggest that even subtle differences in single residues can have profound effects on the dynamics of folding, disulfide bond formation, and activity of this toxic peptide.     

Formation of a major histocompatibility complex class I tapasin disulfide indicates a change in spatial organization of the peptide-loading complex during assembly

The assembly and peptide loading of major histocompatibility complex Class I molecules within the endoplasmic reticulum are essential for antigen presentation at the cell surface and are facilitated by the peptide-loading complex. The formation of a mixed disulfide between the heavy chain of Class I and components of the loading complex (ERp57, protein disulfide isomerase, and tapasin) suggests that these molecules are involved in the redox regulation of components during assembly and peptide loading. We demonstrate here that a disulfide formed between heavy chain and tapasin can occur between cysteine residues located in the cytosolic regions of these proteins following translation of heavy chain in an in vitro translation system. The formation of this disulfide occurs after assembly into the loading complex and is coincident with the stabilization of the alpha2 disulfide bond within the peptide binding grove. A ternary complex between heavy chain, ERp57, and tapasin was observed and shown to be stabilized by a disulfide between both tapasinheavy chain and tapasin-ERp57. No disulfides were observed between ERp57 and heavy chain within the loading complex. The results provide a detailed evaluation of the various transient disulfides formed within the peptide-loading complex during biosynthesis. In addition, the absence of the disulfide between tapasin and heavy chain in TAP-deficient cells indicates that a change in the spatial organization of tapasin and heavy chain occurs following assembly into the loading complex.     

The nature of the bond between peptide and carrier molecule determines the immunogenicity of the construct.

The influence of the nature of the bond between a peptide and a (lipidic) carrier molecule on the immunogenicity of that construct was investigated. As types of bonds a thioester-, a disulfide-, an amide- and a thioether bond were investigated. As carrier molecules a peptide, an N-palmitoylated peptide or a C(16)-hydrocarbon chain were used. The biostability of the bond between peptide and carrier molecule is thioether > amide > disulfide > thioester. However, the immunogenic potency of the constructs used was found to be thioester > disulfide > amide > thioether. In conclusion, a construct with a bond between peptide and (lipidic) carrier molecule that is more susceptible to biological degradation is more immunogenic when used in a peptide-based vaccine than a bond that is less susceptible to biological degradation.     

Functions of ERp57 in the folding and assembly of major histocompatibility complex class I molecules

ERp57 is a thiol oxidoreductase of the endoplasmic reticulum that appears to be recruited to substrates indirectly through its association with the molecular chaperones calnexin and calreticulin. However, its functions in living cells have been difficult to demonstrate. During the biogenesis of class I histocompatibility molecules, ERp57 has been detected in association with free class I heavy chains and, at a later stage, with a large complex termed the peptide loading complex. This implicates ERp57 in heavy chain disulfide formation, isomerization, or reduction as well as in the loading of peptides onto class I molecules. In this study, we show that ERp57 does indeed participate in oxidative folding of the heavy chain. Depletion of ERp57 by RNA interference delayed heavy chain disulfide bond formation, slowed folding of the heavy chain alpha(3) domain, and caused slight delays in the transport of class I molecules from the endoplasmic reticulum to the Golgi apparatus. In contrast, heavy chain-beta(2)-microglobulin association kinetics were normal, suggesting that the interaction between heavy chain and beta(2) -microglobulin does not depend on an oxidized alpha(3) domain. Likewise, the peptide loading complex assembled properly, and peptide loading appeared normal upon depletion of ERp57. These studies demonstrate that ERp57 is involved in disulfide formation in vivo but do not support a role for ERp57 in peptide loading of class I molecules. Interestingly, depletion of another thiol oxidoreductase, ERp72, had no detectable effect on class I biogenesis, consistent with a specialized role for ERp57 in this process.     

Effects of proteolysis and reduction on phosphatase and ROS-generating activity of human tartrate-resistant acid phosphatase

Osteoclasts and macrophages express high amounts of tartrate-resistant acid phosphatase (TRACP), an enzyme with unknown biological function. TRACP contains a disulfide bond, a protease-sensitive loop peptide, and a redox-active iron that can catalyze formation of reactive oxygen species (ROS). We studied the effects of proteolytic cleavage by trypsin, reduction of the disulfide bond by beta-mercaptoethanol, and reduction of the redox-active iron by ascorbate on the phosphatase and ROS-generating activity of baculovirus-generated recombinant human TRACP. Ascorbate alone and trypsin in combination with beta-mercaptoethanol increased k(cat)/K(m) of the phosphatase activity seven- to ninefold. The pH-optimum was changed from 5.4-5.6 to 6.2-6.4 by ascorbate and trypsin cleavage. Trypsin cleavage increased k(cat)/K(m) of the ROS-generating activity 2.5-fold without affecting the pH-optimum (7.0). These results suggest that the protease-sensitive loop peptide, redox-active iron, and disulfide bond are important regulatory sites in TRACP, and that the phosphatase and ROS-generating activity are performed with different reaction mechanisms.     

Amyloidogenic synthetic peptides of beta2-microglobulin--a role of the disulfide bond

To search for the essential regions responsible for the beta2-microglobulin (beta2-m) amyloid fibril formation, we synthesized six peptides corresponding to six of the seven beta-sheets in the native structure of beta2-m, and examined their amyloidogenicity. Among the peptides examined, peptide (21-31) (strand B) and the mixture of peptide (21-31) and (78-86) (strand F) showed fibril formation at both pH 2.5 and 7.5. Peptide (21-31) is the N-terminal half of the previously reported proteolytic fragment of beta2-m, Ser21-Lys41 (K3), suggesting that this region may be the essential core. Interestingly, the dimer formation of peptide (21-31) by the disulfide bond substantially facilitated the fibril formation, indicating that the disulfide bond is important for the structural stability of the fibrils.     

Synthetic approaches to multivalent lipopeptide dendrimers containing cyclic disulfide epitopes of foot-and-mouth disease virus

The synthesis of a multiantigenic peptide dendrimer incorporating four copies of a cyclic disulfide epitope has been undertaken. Since standard chemoselective ligation procedures involving thioether formation are inadvisable in the presence of a preformed disulfide, conjugation through a peptide bond between the lipidated branched lysine scaffold and a suitably protected version of the cyclic disulfide has been used instead. Several synthetic approaches to the partially protected cyclic disulfide peptide have been explored. The most effective involves building a minimally protected version of the peptide by Boc solid phase synthesis, using fluorenyl-based anchorings and cysteine protecting groups. Peptide-resin cleavage and cysteine deprotection/oxidation are performed simultaneously by base-promoted elimination. The cyclic disulfide epitope is readily obtained in sufficient amounts by this procedure and subsequently incorporated to the lipidated lysine core by peptide bond formation in solution. A final acid deprotection step in anhydrous HF yields a peptide construction containing a maximum of three copies of the cyclic disulfide epitope, the lower substitution being attributable to steric constraints. This immunogen has been successfully used in an experimental vaccination trial against foot-and-mouth disease virus.     

Intrachain disulfide bond in the core hinge region of human IgG4.

IgG is a tetrameric protein composed of two copies each of the light and heavy chains. The four-chain structure is maintained by strong noncovalent interactions between the amino-terminal half of pairs of heavy-light chains and between the carboxyl-terminal regions of the two heavy chains. In addition, interchain disulfide bonds link each heavy-light chain and also link the paired heavy chains. An engineered human IgG4 specific for human tumor necrosis factor-alpha (CDP571) is similar to human myeloma IgG4 in that it is secreted as both disulfide bonded tetramers (approximately 75% of the total amount of IgG) and as tetramers composed of nondisulfide bonded half-IgG4 (heavy chain disulfide bonded to light chain) molecules. However, when CDP571 was genetically engineered with a proline at residue 229 of the core hinge region rather than serine, CDP571 (S229P), or with an IgG1 rather than IgG4 hinge region, CDP571(gamma 1), only trace amounts of nondisulfide bonded half-IgG tetramers were observed. Trypsin digest reversephase HPLC peptide mapping studies of CDP571 and CDP571(gamma 1) with on-line electrospray ionization mass spectroscopy supplemented with Edman sequencing identified the chemical factor preventing inter-heavy chain disulfide bond formation between half-IgG molecules: the two cysteines in the IgG4 and IgG1 core hinge region (CPSCP and CPPCP, respectively) are capable of forming an intrachain disulfide bond. Conformational modeling studies on cyclic disulfide bonded CPSCP and CPPCP peptides yielded energy ranges for the low-energy conformations of 31-33 kcal/mol and 40-42 kcal/mol, respectively. In addition, higher torsion and angle bending energies were observed for the CPPCP peptide due to backbone constraints caused by the extra proline. These modeling results suggest a reason why a larger fraction of intrachain bonds are observed in IgG4 rather than IgG1 molecules: the serine in the core hinge region of IgG4 allows more hinge region flexibility than the proline of IgG1 and thus may permit formation of a stable intrachain disulfide bond more readily.     

Cell-free synthesis and assembly of prolyl 4-hydroxylase: the role of the beta-subunit (PDI) in preventing misfolding and aggregation of the alpha-subunit.

Prolyl 4-hydroxylase (P4-H) catalyses a vital post-translational modification in the biosynthesis of collagen. The enzyme consists of two distinct polypeptides forming an alpha 2 beta 2 tetramer (alpha = 64 kDa, beta = 60 kDa), the beta-subunit being identical to the multifunctional enzyme protein disulfide isomerase (PDI). By studying the cell-free synthesis of the rat alpha-subunit of P4-H we have shown that the alpha-subunit can be translocated, glycosylated and the signal peptide cleaved by dog pancreatic microsomal membranes to yield both singly and doubly glycosylated forms. When translations were carried out under conditions which prevent disulfide bond formation, the product synthesized formed aggregates which were associated with the immunoglobulin heavy chain binding protein (BiP). Translations carried out under conditions that promote disulfide bond formation yielded a product that was not associated with BiP but formed a complex with the endogenous beta-subunit (PDI). Complex formation was detected by co-precipitation of the newly synthesized alpha-subunit with antibodies raised against PDI, by sucrose gradient centrifugation and by chemical cross-linking. When microsomal vesicles were depleted of PDI, BiP and other soluble endoplasmic reticulum proteins, no complex formation was observed and the alpha-subunit aggregated even under conditions that promote disulfide bond formation. We have therefore demonstrated that the enzyme P4-H can be assembled at synthesis in a cell-free system and that the solubility of the alpha-subunit is dependent upon its association with PDI.     

Transpeptidation during the analytical proteolysis of proteins

Since peptide mapping with proteolytic enzymes such as trypsin and Staphylococcus aureus V8 protease is a powerful tool for the characterization of proteins, investigators should be cognizant of possible artifacts due to the technique itself. This article describes the identification of minor peaks found in the maps of recombinant human relaxin and insulin-like growth factor I as transpeptidation products. Both proteins have some homology to insulin with relaxin being composed of two chains designated A and B, while insulin-like growth factor I is composed of a single polypeptide chain. Digestion of relaxin with trypsin at pH 7.2 yields two peptides, T2,3(A10-18) and T7(B10-13), linked together by a disulfide bond. An unexpected component at a 10% level was identified to be the T2-T7 peptide pair where T3(ArgA18) has formed a peptide bond with the amino-terminal LeuB10 of the T7 peptide. It was also observed that the digestion of insulin-like growth factor I with V8 protease normally yields two peptides V4(13-20) and V9(59-70) linked by a disulfide bridge. A minor peak at a 1 to 2% level was identified to be a single polypeptide resulting from the formation of a peptide bond between the amino-terminal Met59 of V9 and the carboxyl-terminal Asp20 of V4, with the disulfide bond intact. These transpeptidation products were isolated by reversed-phase HPLC and identified using amino-terminal sequence and mass spectrometric analyses.     

Chemical synthesis and folding pathways of large cyclic polypeptides: studies of the cystine knot polypeptide kalata B1.

Kalata B1 is a member of a new family of polypeptides, isolated from plants, which have a cystine knot structure embedded within an amide-cyclized backbone. This family of molecules are the largest known cyclic peptides, and thus, the mechanism of synthesis and folding is of great interest. To provide information about both these phenomena, we have synthesized kalata B1 using two distinct strategies. In the first, oxidation of the cysteine residues of a linear precursor peptide to form the correct disulfide bonds results in folding of the three-dimensional structure and preorganization of the termini in close proximity for subsequent cyclization. The second approach involved cyclization prior to oxidation. In the first method, the correctly folded peptide was produced only in the presence of partially hydrophobic solvent conditions. These conditions are presumably required to stabilize the surface-exposed hydrophobic residues. However, in the synthesis involving cyclization prior to oxidation, the cyclic reduced peptide folded to a significant degree in the absence of hydrophobic solvents and even more efficiently in the presence of hydrophobic solvents. Cyclization clearly has a major effect on the folding pathway and facilitates formation of the correctly disulfide-bonded form in aqueous solution. In addition to facilitating folding to a compact stable structure, cyclization has an important effect on biological activity as assessed by hemolytic activity.