Effect of the light chain C-terminal serine residue on disulfide bond susceptibility of human immunoglobulin G1λ

The light chain cysteine residue that forms an interchain disulfide bond with the cysteine residue in the heavy chain in IgG1κ is the last amino acid. The cysteine residue is followed by a serine residue in IgG1λ. Effect of the serine residue on the susceptibility of disulfide bonds to reduction was investigated in the current study using a method including reduction, differential alkylation using iodoacetic acid with either natural isotopes or enriched with carbon-13, and mass spectrometry analysis. This newly developed method allowed an accurate determination of the susceptibility of disulfide bonds in IgG antibodies. The effect of the serine residue on disulfide bond susceptibility was compared using three antibodies with differences only in the light chain last amino acid, which was either a serine residue, an alanine residue or deleted. The results demonstrated that the presence of the amino acid (serine or alanine) increased the susceptibility of the inter light and heavy chain disulfide bonds to reduction. On the other hand, susceptibility of the two inter heavy chain disulfide bonds and intrachain disulfide bonds was not changed significantly.     

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.     

Suppression of sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample preparation artifacts for analysis of IgG4 half-antibody

Human IgG4 subtype antibodies have often been reported to have a significant portion (5-50%) of a heavy chain-light chain dimer ("half-antibody") on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in which the heavy chain is not covalently linked through the hinge disulfides to another heavy chain. We demonstrate here that there can be artifactual sources of half-antibody. One occurred during SDS-PAGE sample preparation where rapid disulfide scrambling was initiated by preexisting free sulfhydryls in the monoclonal antibody (mAb) and by free sulfhydryl produced by destruction of disulfide bonds during heating. Inclusion of N-ethylmaleimide in the sample buffer prevented the disulfide scrambling. Presumably, cyclization of the flexible IgG4 hinge during this disulfide scrambling leads to the preferential separation of heavy chains. A second condition producing half-antibody was reoxidation after exposure to reductant, where 46% of the antibody was trapped in the intrachain disulfide form. The amount of half-antibody was reduced to 4% by reoxidation in the presence of a mixture of oxidized and reduced glutathione. When the improved sample preparation conditions were used, IgG4 mAb freshly isolated from cells contained 4.5-15% half-antibody, indicating that equilibration of the interchain and intrachain hinge disulfide pairing was not always attained in cells.     

Proof of a disulfide bridge accessible to disulfide exchange between the heavy chains of IgG

The paper deals with the direct experimental proof that human immunoglobulin G1 (IgG1) contains a reactive disulfide bond that can be opened by 3,3'-dithiobis(6-nitrobenzoate) (DTNB) within 24 h by a SH-catalysed disulfide exchange reaction. These results were obtained with the purified IgG1 myeloma protein and confirm earlier indirect evidence based on correlation analysis of DTNB reactivity and quantitative IgG1 determination. The reactive disulfide bond is most likely the one between Cys235 of the heavy chains in the "hinge"-region, activated for the disulfide exchange by the protonated amino groups of Lys231 as turned out by analysis of IgG1. As with the whole molecule, one mol of reactive disulfide was found per mol of the Fc-fragment. 0.8 mol of labile S-S bonds was detected per mol of F(ab)2. After separation of the excess of reagent, the sedimentation pattern still corresponded with the dimer. The unaltered antigenic properties as well as the crystallizability speak against any severe conformational changes. Therefrom it was concluded that in approximately 80% of the F(ab)2 molecules one of the two inter heavy chain-bridges was opened. With the isolated F(ab)-fragment a reaction with DTNB was ascertained to an extent of 20%, which is probably due to an altered stability of the heavy-light chain-SS-bridge. However, no influence on the sedimentation pattern was observed. The intrachainar disulfide bonds of neither the heavy nor the light chain reacted with DTNB to a measurable extent.     

Human IgG2 antibodies display disulfide-mediated structural isoforms

In this work, we present studies of the covalent structure of human IgG2 molecules. Detailed analysis showed that recombinant human IgG2 monoclonal antibody could be partially resolved into structurally distinct forms caused by multiple disulfide bond structures. In addition to the presently accepted structure for the human IgG2 subclass, we also found major structures that differ from those documented in the current literature. These novel structural isoforms are defined by the light chain constant domain (C(L)) and the heavy chain C(H)1 domain covalently linked via disulfide bonds to the hinge region of the molecule. Our results demonstrate the presence of three main types of structures within the human IgG2 subclass, and we have named these structures IgG2-A, -B, and -A/B. IgG2-A is the known classic structure for the IgG2 subclass defined by structurally independent Fab domains and hinge region. IgG2-B is a structure defined by a symmetrical arrangement of a (C(H)1-C(L)-hinge)(2) complex with both Fab regions covalently linked to the hinge. IgG2-A/B represents an intermediate form, defined by an asymmetrical arrangement involving one Fab arm covalently linked to the hinge through disulfide bonds. The newly discovered structural isoforms are present in native human IgG2 antibodies isolated from myeloma plasma and from normal serum. Furthermore, the isoforms are present in native human IgG2 with either kappa or lambda light chains, although the ratios differ between the light chain classes. These findings indicate that disulfide structural heterogeneity is a naturally occurring feature of antibodies belonging to the human IgG2 subclass.     

Biologic significance of disulfide bonds in human IgE molecules.

E myeloma protein, PS, was reduced in different concentrations of dithiothreitol (DTT) for 1 hr followed by alkylation with 14C-iodoacetamide. The affinity of the reduced-alkylated molecules for target cells was evaluated by their ability 1) to sensitize primate skin in a reversed P-K reaction, 2) to sensitize human basophils in a reversed-type histamine release and 3) to block passive sensitization with reaginic antibody. Antibody-epsilon0 antibody was employed for reversed type reactions to avoid participation of cell-bound normal IgE in the reactions. The sensitizing activity of IgE did not change following reduction in 1 mM DTT, which split inter-heavy-light chain disulfide bond. The activity of IgE significantly diminished after reduction in 2 mM DTT followed by alkylation. This treatment resulted in the cleavage of two intra-epsilon-chain disulfide bonds, which are present between the hinge and the Fd portion of the molecules. The reduced-alkylated protein was capable of sensitizing primate skin and human basophils, however, a much higher concentration of the reduced-alkylated protein than the native protein was required for passive sensitization. The optimal sensitization period for the reversed P-K reaction was 3 hr with the reduced-alkylated protein. The protein had the ability to block passive sensitization with reaginic antibody. The reduced-alkylated protein and the native protein were labeled with 125I, and binding of these proteins with human basophils was examined by autoradiography. The results showed that affinity of the reduced-alkylated protein for basophils was less than that of native protein. Since the disulfide bonds split by 2 mM DTT were not included in the Fc portion of the molecules, the Fc fragment was obtained from the reduced-alkylated protein and was tested for affinity for basophils. It was found that the Fc fragment had higher affinity than the reduced-alkylated protein. Recovery of the affinity by papain digestion strongly suggested that cleavage of disulfide bonds in the Fab portion of the molecules induced conformational changes in the Fc portion which is involved in binding to the target cells. Reduction of IgE with 10 mM DTT followed by alkylation resulted in cleavage of 5 disulfide bonds, which is accompanied by a loss of both sensitizing and blocking activities. The fifth disulfide bond which was cleaved by 10 mM DTT, but not by 2 mM DTT, appears to be an inter-heavy chain disulfide bond in the Fc portion of the epsilon-chains. Neither epsilon1 nor epsilon2 determinants in the Fc portion of epsilon-chains were degraded by this treatment.     

Engineering disulfide bonds within an antibody

Antibodies have evolved to function in oxidative, extracellular environments. A pair of cysteines in close proximity will oxidatively react to form a disulfide bond that fixes and stabilizes the tertiary structure of a protein. Immunoglobulin G (IgG) includes several disulfide bonds, and the patterns of inter-chain disulfide bonds characterize different IgG sub-classes. Moreover, the Ig-fold domains are characterized by a buried intra-domain disulfide bond, which is important for its structural stability. However, the intra-domain disulfide bond can be replaced without crucial effects on the structure and function, if the domain structure is intrinsically stable or has been stabilized by protein engineering. In previous studies, disulfide bonds were removed by amino-acid substitution indicating that Val and/or Ala (i.e. Ala–Ala, Ala–Val, Val–Ala, and Val–Ala) pairs were preferred for cysteine replacement in the Ig-fold domain. As such, these mutations may be useful for the intracellular use of antibodies. Recently, additional intra-domain disulfide bonds have been shown to stabilize Ig-fold domains and whole IgGs. In heavy chain variable or light chain variable domains, the introduction of additional disulfide bonds into the framework region did not reduce antigen-binding affinity, suggesting that generating disulfide bonds may be a method for stabilizing IgG and antibody fragments, such as the antigen-binding fragment, and single-chain and single-domain antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Engineering an improved IgG4 molecule with reduced disulfide bond heterogeneity and increased Fab domain thermal stability

The integrity of antibody structure, stability, and biophysical characterization are becoming increasingly important as antibodies receive increasing scrutiny from regulatory authorities. We altered the disulfide bond arrangement of an IgG4 molecule by mutation of the Cys at the N terminus of the heavy chain constant domain 1 (C(H)1) (Kabat position 127) to a Ser and introduction of a Cys at a variety of positions (positions 227-230) at the C terminus of C(H)1. An inter-LC-C(H)1 disulfide bond is thus formed, which mimics the disulfide bond arrangement found in an IgG1 molecule. The antibody species present in the supernatant following transient expression in Chinese hamster ovary cells were analyzed by immunoblot to investigate product homogeneity, and purified product was analyzed by a thermofluor assay to determine thermal stability. We show that the light chain can form an inter-LC-C(H)1 disulfide bond with a Cys when present at several positions on the upper hinge (positions 227-230) and that such engineered disulfide bonds can consequently increase the Fab domain thermal stability between 3 and 6.8 °C. The IgG4 disulfide mutants displaying the greatest increase in Fab thermal stability were also the most homogeneous in terms of disulfide bond arrangement and antibody species present. Importantly, mutations did not affect the affinity for antigen of the resultant molecules. In combination with the previously described S241P mutation, we present an IgG4 molecule with increased Fab thermal stability and reduced product heterogeneity that potentially offers advantages for the production of IgG4 molecules.     

Non–antigen-contacting region of an asymmetric bispecific antibody to factors IXa/X significantly affects factor VIII-mimetic activity

While antibody engineering improves the properties of therapeutic antibodies, optimization of regions that do not contact antigens has been mainly focused on modifying the effector functions and pharmacokinetics of antibodies. We recently reported an asymmetric anti-FIXa/FX bispecific IgG₄ antibody, ACE910, which mimics the cofactor function of FVIII by placing the two factors into spatial proximity for the treatment of hemophilia A. During the optimization process, we found that the activity was significantly affected by IgG subclass and by modifications to the inter-chain disulfide bonds, upper hinge region, elbow hinge region, and Fc glycan, even though these regions were unlikely to come into direct contact with the antigens. Of these non–antigen-contacting regions, the tertiary structure determined by the inter-chain disulfide bonds was found to strongly affect the FVIII-mimetic activity. Interestingly, IgG₄-like disulfide bonds between Cys131 in the heavy chain and Cys114 in the light chain, and disulfide bonds between the two heavy chains at the hinge region were indispensable for the high FVIII-mimetic activity. Moreover, proline mutations in the upper hinge region and removal of the Fc glycan enhanced the FVIII-mimetic activity, suggesting that flexibility of the upper hinge region and the Fc portion structure are important for the FVIII-mimetic activity. This study suggests that these non–antigen-contacting regions can be engineered to improve the biological activity of IgG antibodies with functions similar to ACE910, such as placing two antigens into spatial proximity, retargeting effector cells to target cells, or co-ligating two identical or different antigens on the same cell.     

Non–antigen-contacting region of an asymmetric bispecific antibody to factors IXa/X significantly affects factor VIII-mimetic activity

While antibody engineering improves the properties of therapeutic antibodies, optimization of regions that do not contact antigens has been mainly focused on modifying the effector functions and pharmacokinetics of antibodies. We recently reported an asymmetric anti-FIXa/FX bispecific IgG₄ antibody, ACE910, which mimics the cofactor function of FVIII by placing the two factors into spatial proximity for the treatment of hemophilia A. During the optimization process, we found that the activity was significantly affected by IgG subclass and by modifications to the inter-chain disulfide bonds, upper hinge region, elbow hinge region, and Fc glycan, even though these regions were unlikely to come into direct contact with the antigens. Of these non–antigen-contacting regions, the tertiary structure determined by the inter-chain disulfide bonds was found to strongly affect the FVIII-mimetic activity. Interestingly, IgG₄-like disulfide bonds between Cys131 in the heavy chain and Cys114 in the light chain, and disulfide bonds between the two heavy chains at the hinge region were indispensable for the high FVIII-mimetic activity. Moreover, proline mutations in the upper hinge region and removal of the Fc glycan enhanced the FVIII-mimetic activity, suggesting that flexibility of the upper hinge region and the Fc portion structure are important for the FVIII-mimetic activity. This study suggests that these non–antigen-contacting regions can be engineered to improve the biological activity of IgG antibodies with functions similar to ACE910, such as placing two antigens into spatial proximity, retargeting effector cells to target cells, or co-ligating two identical or different antigens on the same cell.     

The role of disulfide bonds in the conformational stability and catalytic activity of phytase.

Previous studies have predicted five disulfide bonds in Aspergillus niger phytase (phy A). To investigate the role of disulfide bonds, intrinsic fluorescence spectra, far-ultraviolet circular dichroism (CD) spectra, and an enzyme activity assay were used to compare the differences of catalytic activity and conformational stability of phytase during denaturation in urea in the presence and absence of dithiothreitol (DTT). In the presence of 2 mM DTT, the inactivation and unfolding were greatly enhanced at the same concentration of denaturant. The fluorescence emission maximum red shift and decreases of ellipticity at 222 nm were in accord with the changes of catalytic activity. The kinetics of the unfolding courses were a biphasic process consisting of two first-order reactions in the absence of DTT and a monophasic process of a first-order reaction in the presence of DTT. The results suggested that the loss of enzymatic activity was most likely because of a conformational change, and that disulfide bonds played an important role in three-dimensional structure and catalytic activity.     

Free sulfhydryl in recombinant monoclonal antibodies

Monoclonal antibody (mAb) therapy applications have been growing rapidly in recent years. Like other recombinant protein drugs, therapeutic mAb's need to be well characterized to ensure their structural and functional integrity. IgG mAb's are composed of two heavy and two light chains covalently linked by interchain disulfide bonds. Each domain of the heavy or light chain contains one additional disulfide bond. Native IgG mAb's, with completely formed disulfide bonds, should not bear any free sulfhydryl. This report describes detection and quantification of free sulfhydryl in recombinant mAb's produced in Chinese hamster ovary (CHO) cells using a fluorescent technique. The method utilizes the fluorescent probe N-(1-pyrenyl)maleimide (NPM). The purified mAb's appear to be homogeneous under native conditions with approximately 0.02 mol of free sulfhydryl per mole of protein. Upon denaturation, minor species related to the mAb's are observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the free sulfhydryl level is determined to be approximately 0.1 mol/mol of protein. These results suggest that a small portion of these recombinant mAb's lack in intermolecular disulfide bonds but remain noncovalently associated under native conditions. The formation of the free sulfhydryl containing mAb species is likely to occur during the culture process and/or protein folding process in the endoplasmic reticulum (ER).     

Formation of intermolecular disulfide bonds on nascent immunoglobulin polypeptides.

The initial step of intermolecular covalent assembly of immunoglobulins molecules involves formation of heavy chain-light chain or heavy chain-heavy chain disulfide bonds. Using QAE-Sephadex chromatography to isolate microsomal nascent polypeptides, we have shown that this initial step of intermolecular covalent assembly occurs, to a substantial extent, on nascent heavy chains, as well as on completed heavy chains as previously demonstrated by others. In MPC 11 mouse myeloma cells, completed light chains are assembled covalently to nascent heavy chains, whereas in MOPC 21 mouse myeloma cells, completed heavy chains are assembled covalently to nascent heavy chains. These results are consisted with the heavy-light half-molecule being the major initial intermediate in the assembly of MPC 11 IgG2b and heavy-heavy dimer being the major initial intermediate formed in assembly of MOPC 21 IgG1. The nascent MPC 11 heavy chain must be at least 38,000 daltons in size before assembly with the light chain occurs, even though the heavy chain cysteine involved in this disulfide bond is 131 residues (approximately 15,000 daltons) from the NH2 terminus. In addition, pulse-chase labeling studies of MPC 11 cells have shown that the assembly of completed light chains with the nascent heavy chain must occur within a few minutes of the synthesis of the light chain even though a large excess of unassembled MPC 11 light chains remain inside the cell for an average time of 2 h before being secreted.     

大肠杆菌L-酒石酸脱氢酶b亚基体外分子交联

通过PCR技术扩增大肠杆菌L-酒石酸脱氢酶b亚基(L-tartrate dehydratase beta subunit, TtdB)野生型与Cys/Ser突变型目的基因, 构建带6×His标签的诱导型表达载体pTrcHisC-TtdB。重组蛋白以包含体形式存在, 应用TALON固定化金属亲和树脂(Immobilized metal affinity chromatography, IMAC)以变性的方法纯化, 通过分步透析逐步去除变性剂的方法复性, 复性率可达70%。将复性后的两种蛋白通过热诱导去折叠和氧化重折叠方法进行体外蛋白质分子交联实验。SDS-PAGE分析表明: 野生型TtdB在其变性的临界温度反应时, 出现交联二聚体和多聚体; 在氧化重折叠后SDS-PAGE前加入100 mmol/L DTT时, 交联强度明显减弱。这种DTT打不开的交联即为异肽键交联; 若在其氧化重折叠反应液中加入DTT则没有任何交联。突变型TtdB在与野生型TtdB相同的热诱导去折叠条件下, 完全没有二聚体和多聚体的形成。     

Characterization of lower molecular weight artifact bands of recombinant monoclonal IgG1 antibodies on non-reducing SDS-PAGE

SDS-PAGE under non-reducing conditions is one of the most commonly used techniques for recombinant monoclonal antibody purity and stability indicating assay. On non-reducing SDS-PAGE, bands with a lower molecular weight than the intact antibody are routinely observed and is a common feature of IgG molecules. These fragments were analyzed by in-gel digestion followed by matrix-assisted-laser-desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry, Western blot and by comparing the banding pattern of sample prepared in the presence of a reducing reagent. The fragments bands were identified as antibody lacking one light chain, two heavy chains, one light chain and one heavy chain, free heavy chain and free light chain. Sensitivity of fragmentation to sample buffer pH, incubation time, reducing reagent and alkylation reagents indicated that fragments were formed during sample preparation, but not present in the samples analyzed. Disulfide bond scrambling and β-elimination are the two major mechanisms of the formation antibody fragments. Mass spectrometry analysis suggested that disulfide bond scrambling can be prevented by specifically modifying free sulhydryl using alkylation and thus reduced the amount of artifacts on non-reducing SDS-PAGE. Breakage of disulfide bonds by β-elimination was evidenced by the detection of dehydroalanine using mass spectrometry.     

Dynamics of Inter-heavy Chain Interactions in Human Immunoglobulin G (IgG) Subclasses Studied by Kinetic Fab Arm Exchange

Interdomain interactions between the CH3 domains of antibody heavy chains are the first step in antibody assembly and are of prime importance for maintaining the native structure of IgG. For human IgG4 it was shown that CH3-CH3 interactions are weak, resulting in the potential for half-molecule exchange (“Fab arm exchange”). Here we systematically investigated non-covalent interchain interactions for CH3 domains in the other human subclasses, including polymorphisms (allotypes), using real-time monitoring of Fab arm exchange with a FRET-based kinetic assay. We identified structural variation between human IgG subclasses and allotypes at three amino acid positions (Lys/Asn-392, Val/Met-397, Lys/Arg-409) to alter the strength of inter-domain interactions by >6 orders of magnitude. Each substitution affected the interactions independent from the other substitutions in terms of affinity, but the enthalpic and entropic contributions were non-additive, suggesting a complex interplay. Allotypic variation in IgG3 resulted in widely different CH3 interaction strengths that were even weaker for IgG3 than for IgG4 in the case of allotype G3m(c3c5*/6,24*), whereas G3m(s*/15*) was equally stable to IgG1. These interactions are sufficiently strong to maintain the structural integrity of IgG1 during its normal life span; for IgG2 and IgG3 the inter-heavy chain disulfide bonds are essential to prevent half-molecule dissociation, whereas the labile hinge disulfide bonds favor half-molecule exchange in vivo for IgG4.     

Disulfide cross-linked polymer capsules: en route to biodeconstructible systems

Hydrogen-bonded multilayer thin films were constructed using poly(vinylpyrrolidone) and poly(methacrylic acid) functionalized with cysteamine. The resulting films included thiol moieties that were cross-linked to render the films stable at physiological pH. Film buildup was followed using quartz crystal microgravimetry, which was also used to demonstrate the improved stability imparted by reacting the thiol moieties to form disulfide bonds. Films without disulfide bonds were readily deconstructed at physiological pH, while those with disulfide bonds were swollen upon exposure to this pH (7) but remained intact. Addition of a common thiol-disulfide exchange reagent, dithiothreitol (DTT) at pH 7 led to disassembly of the multilayer films. The films were also prepared on colloidal substrates (as demonstrated using confocal microscopy) and were used to retain a model drug (fluorescently labeled transferrin) and release this molecule when triggered by the addition of DTT. This approach has potential for the in vivo applications of hollow capsules, as thiol-disulfide exchange leading to deconstruction of the capsules can occur with the assistance of intracellular proteins.     

Identification and function of disulfide bridges in the extracellular domains of the angiotensin II type 2 receptor.

The angiotensin II (AngII) receptor family is comprised of two subtypes, type 1 (AT(1)) and type 2 (AT(2)). Although sharing low homology (only 34%), mutagenesis has identified some key residues that are conserved between both subtypes, including four extracellular cysteines. Previous AT(1) mutagenesis demonstrated that the cysteines form two disulfide bonds, one linking the first and second extracellular loops and another connecting the amino terminus to the third extracellular loop. The importance of these AT(1) disulfides in ligand binding is supported by the effect of dithiothreitol (DTT). DTT breaks disulfide bonds, thereby strongly inhibiting ligand binding in AT(1) receptors. Despite retaining the same cysteines, AT(2) receptor ligand binding is paradoxically enhanced by DTT. Thus, we constructed a series of AT(2) cysteine mutations, either individually or paired, to establish the role of the cysteines and the source of DTT's effects. The AT(2) cysteine mutants surprisingly confirmed that the cysteines form disulfide bonds in the same manner as in the AT(1) subtype. However, breaking the AT(2) disulfide bridges yielded two responses. As in AT(1) receptors, mutations disrupting the disulfide bond between the first and second extracellular loops reduced AT(2) binding by 4-fold. In contrast, mutations breaking the disulfide bridge between the amino terminus and the third extracellular loop increased AT(2) binding, mimicking DTT's effect on this subtype. Further analysis of AT(1)/AT(2) chimeric exchange mutants of these domains suggested that the AT(2) amino terminus and third extracellular loop may possess latent binding epitopes that are only uncovered after DTT exposure.     

Evidence for the presence of disulfide bridges in opioid receptors essential for ligand binding. Possible role in receptor activation

The mobility of purified mu opioid binding protein in SDS-polyacrylamide gek electrophoresis is sensitive to the presence of reducing agents. In the presence of increasing concentrations of DTT the apparent molecular weight increases in a stepwise fashion from 53 kDa to 65 kDa. This reduction in mobility is attributed to the successive breakage of disulfide bridges, resulting in an increasingly asymmetric molecule. Treatment of cell membranes from various brain areas with reducing agents, such as DTT, produced a concentration-dependent inhibition of opioid binding. Sensitivity to DTT inhibition varied between receptor types, mu greater than delta much greater than kappa. For mu receptors, agonist binding was considerably more sensitive to DTT than antagonist binding. Inhibition by DTT is readily reversible and is unaffected by Na+ and/or Mg2+ ions. Reversibility may be partially prevented by the inclusion of a low concentration of a reducing reagent such as glutathione which does not inhibit binding but blocks reformation of disulfide bonds. Scatchard analysis of saturation data shows that DTT causes a pronounced decrease in binding affinity with little effect on receptor number. It is suggested that disulfide bonds are essential for ligand binding and that cleavage of one or more of these bonds may play a role in opioid receptor activation by agonists.     

Intracellular manipulation of disulfide bond formation in rotavirus proteins during assembly.

Rotavirus undergoes a unique mode of assembly in the rough endoplasmic reticulum (RER) of infected cells. Luminal RER proteins undergo significant cotranslational and posttranslational modifications, including disulfide bond formation. Addition of a reducing agent (dithiothreitol [DTT]) to rotavirus-infected cells did not significantly inhibit translation or disrupt established disulfide bonds in rotavirus proteins but prevented the formation of new disulfide bonds and infectious viral progeny. In DTT-treated, rotavirus-infected cells, all vp4, vp6, and ns28 epitopes but no vp7 epitopes were detected by immunohistochemical staining with a panel of monoclonal antibodies. When oxidizing conditions were reestablished in DTT-treated cells, intramolecular disulfide bonds in vp7 were rapidly and correctly established with the restoration of antigenicity, although prolonged DTT treatment led to the accumulation of permanently misfolded vp7. Electron microscopy revealed that cytosolic assembly of single-shelled particles and budding into the ER was not affected by DTT treatment but that outer capsid assembly was blocked, leading to the accumulation of single-shelled and enveloped intermediate subviral particles in the RER lumen.