Dysregulation of murine immune functions on inhalational exposure to ammonia, dimethyl disulfide, 3-methylindole, or propionic acid.

Animal husbandry workers are exposed to various malodorous compounds in the workplace. Although these compounds cause severe nuisance, no systemic investigation of their effects on the immune system has been conducted. To address this issue, we evaluated the effects of inhalational exposure to ammonia, dimethyl disulfide, 3-methylindole (3-MI), and propionic acid (PA), representing four major groups of malodorous compounds, on humoral and cellular immunity in mice. Mice were exposed to the substances (low dose: 10 µL and high dose: 200 µL) for 10 min/day for 4 weeks in a modified standard mouse cage. Neutrophil% and splenic cytotoxic T cell% were significantly lower in the high-dose ammonia group than in the vehicle control. Exposure to ammonia and 3-MI increased immature thymic T lymphocyte% relative to control and concomitantly decreased both mature helper and cytotoxic T-cell populations in the thymus. In the ammonia exposure group, levels of serum immunoglobulin E and immunoglobulin A were elevated, and the IgG2a:IgG1 ratio in the serum was reduced in a dose-dependent manner. Splenic natural killer cell activity was significantly less in the PA exposure group than in the control. Overall, our findings suggest that inhalational exposure to these malodorous substances disturbs immune homeostasis in vivo.

Isolation of antigen-specific, disulphide-rich knob domain peptides from bovine antibodies.

As a novel alternative to established surface display or combinatorial chemistry approaches for the discovery of therapeutic peptides, we present a method for the isolation of small, cysteine-rich domains from bovine antibody ultralong complementarity-determining regions (CDRs). We show for the first time that isolated bovine antibody knob domains can function as autonomous entities by binding antigen outside the confines of the antibody scaffold. This yields antibody fragments so small as to be considered peptides, each stabilised by an intricate, bespoke arrangement of disulphide bonds. For drug discovery, cow immunisations harness the immune system to generate knob domains with affinities in the picomolar to low nanomolar range, orders of magnitude higher than unoptimized peptides from naïve library screening. Using this approach, knob domain peptides that tightly bound Complement component C5 were obtained, at scale, using conventional antibody discovery and peptide purification techniques.

Efficient and selective antibody modification with functionalised divinyltriazines.

A highly efficient disulfide rebridging strategy for the modification of monoclonal antibodies with substituted divinyltriazine linkers is reported. The reaction proceeds efficiently under mild conditions with near stoichiometric quantities of linker. This method of conjugation yields serum stable antibody conjugates with a controlled payload loading of 4.

Role of a noncanonical disulfide bond in the stability, affinity, and flexibility of a VHH specific for the Listeria virulence factor InlB.

A distinguishing feature of camel (Camelus dromedarius) VHH domains are noncanonical disulfide bonds between CDR1 and CDR3. The disulfide bond may provide an evolutionary advantage, as one of the cysteines in the bond is germline encoded. It has been hypothesized that this additional disulfide bond may play a role in binding affinity by reducing the entropic penalty associated with immobilization of a long CDR3 loop upon antigen binding. To examine the role of a noncanonical disulfide bond on antigen binding and the biophysical properties of a VHH domain, we have used the VHH R303, which binds the Listeria virulence factor InlB as a model. Using site directed mutagenesis, we produced a double mutant of R303 (C33A/C102A) to remove the extra disulfide bond of the VHH R303. Antigen binding was not affected by loss of the disulfide bond, however the mutant VHH displayed reduced thermal stability (Tm = 12°C lower than wild-type), and a loss of the ability to fold reversibly due to heat induced aggregation. X-ray structures of the mutant alone and in complex with InlB showed no major changes in the structure. B-factor analysis of the structures suggested that the loss of the disulfide bond elicited no major change on the flexibility of the CDR loops, and revealed no evidence of loop immobilization upon antigen binding. These results suggest that the noncanonical disulfide bond found in camel VHH may have evolved to stabilize the biophysical properties of the domain, rather than playing a significant role in antigen binding.

Empty peptide-receptive MHC class I molecules for efficient detection of antigen-specific T cells.

The peptide-dependent stability of MHC class I molecules poses a substantial challenge for their use in peptide-MHC multimer-based approaches to comprehensively analyze T cell immunity. To overcome this challenge, we demonstrate the use of functionally empty MHC class I molecules stabilized by a disulfide bond to link the α1 and α2 helices close to the F pocket. Peptide-loaded disulfide-stabilized HLA-A*02:01 shows complete structural overlap with wild-type HLA-A*02:01. Peptide-MHC multimers prepared using disulfide-stabilized HLA-A*02:01, HLA-A*24:02, and H-2Kb can be used to identify antigen-specific T cells, and they provide a better staining index for antigen-specific T cell detection compared with multimers prepared with wild-type MHC class I molecules. Disulfide-stabilized MHC class I molecules can be loaded with peptide in the multimerized form without affecting their capacity to stain T cells. We demonstrate the value of empty-loadable tetramers that are converted to antigen-specific tetramers by a single-step peptide addition through their use to identify T cells specific for mutation-derived neoantigens and other cancer-associated antigens in human melanoma.

Effects of the SOS (A501C/T605C) and DS (I201C/A433C) Disulfide Bonds on HIV-1 Membrane Envelope Glycoprotein Conformation and Function.

Most broadly neutralizing antibodies and many entry inhibitors target the pretriggered (state 1) conformation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env). Here we examine two previously reported Env mutants designed to be stabilized in this conformation by the introduction of artificial disulfide bonds: A501C/T605C (called SOS) and I201C/A433C (called DS). SOS Env supported virus entry and cell-cell fusion only after exposure to a reducing agent, dithiothreitol (DTT). Deletion of the Env cytoplasmic tail improved the efficiency with which the SOS Env supported virus infection in a reducing environment. The antigenicity of the SOS Env was similar to that of the unmodified Env, except for greater sensitivity to some state 1-preferring ligands. In contrast, viruses with the DS Env were not infectious, even after DTT treatment. The proteolytic maturation of the DS Env on both cell surfaces and virions was severely compromised compared with that of the unmodified Env. The DS Env exhibited detectable cell-fusing activity when DTT was present. However, the profiles of cell-surface Env recognition and cell-cell fusion inhibition by antibodies differed for the DS Env and the unmodified Env. Thus, the DS Env appears to be stabilized in an off-pathway conformation that is nonfunctional on the virus. The SOS change exerted more subtle, context-dependent effects on Env conformation and function.IMPORTANCE The human immunodeficiency virus type 1 (HIV-1) envelope proteins (Envs) bind receptors on the host cell and change shape to allow the virus to enter the cell. Most virus-inhibiting antibodies and drugs recognize a particular shape of Env called state 1. Disulfide bonds formed by cysteine residues have been introduced into soluble forms of the flexible envelope proteins in an attempt to lock them into state 1 for use in vaccines and as research tools. We evaluated the effect of these cysteine substitutions on the ability of the membrane Env to support virus entry and on susceptibility to inhibition by antibodies and small molecules. We found that the conformation of the envelope proteins with the cysteine substitutions differed from that of the unmodified membrane envelope proteins. Awareness of these effects can assist efforts to create stable HIV-1 Env complexes that more closely resemble the state 1 conformation.

Acid pH promotes bispecific antibody formation by the redox procedure.

Bispecific antibodies (BsAbs), are potential theranostics. Chemical procedures of preparation of BsAbs, in which two monospecific antibodies are split into half molecules and heterodimerized, continue to attract attention in view of their simplicity. Poor dissociation of antibodies with reduced inter-heavy chain disulfides into half molecules under neutral conditions however restricts the BsAbs formation. In this study, we report that the heterodimerization of antibodies can be improved leading to over 6-fold increase in the yield of BsAbs, by carrying out the redox procedure at pH 4.0. In view of improvement in heterodimerization, BsAbs could be conveniently prepared starting from partially purified ion-exchange fraction of the antiserum and purified by twin affinity chromatography on antigen supports. The UV, CD, intrinsic and extrinsic fluorescence spectral analysis of BsAbs prepared by the modified redox procedure were comparable with the native IgG, which suggest the absence of significant acid-pH-induced damage. ThT binding studies and native size exclusion chromatography ruled out amyloid fibril formation.

Interleukin 4 is inactivated via selective disulfide-bond reduction by extracellular thioredoxin.

Thioredoxin 1 (TRX), an essential intracellular redox regulator, is also secreted by mammalian cells. Recently, we showed that TRX activates extracellular transglutaminase 2 via reduction of an allosteric disulfide bond. In an effort to identify other extracellular substrates of TRX, macrophages derived from THP-1 cells were treated with NP161, a small-molecule inhibitor of secreted TRX. NP161 enhanced cytokine outputs of alternatively activated macrophages, suggesting that extracellular TRX regulated the activity of interleukin 4 (IL-4) and/or interleukin 13 (IL-13). To test this hypothesis, the C35S mutant of human TRX was shown to form a mixed disulfide bond with recombinant IL-4 but not IL-13. Kinetic analysis revealed a kcat/KM value of 8.1 µM-1⋅min-1 for TRX-mediated recognition of IL-4, which established this cytokine as the most selective partner of extracellular TRX to date. Mass spectrometry identified the C46-C99 bond of IL-4 as the target of TRX, consistent with the essential role of this disulfide bond in IL-4 activity. To demonstrate the physiological relevance of our biochemical findings, recombinant TRX was shown to attenuate IL-4-dependent proliferation of cultured TF-1 erythroleukemia cells and also to inhibit the progression of chronic pancreatitis in an IL-4-driven mouse model of this disease. By establishing that IL-4 is posttranslationally regulated by TRX-promoted reduction of a disulfide bond, our findings highlight a novel regulatory mechanism of the type 2 immune response that is specific to IL-4 over IL-13.

Disulfide bond mapping of Pfs25, a recombinant malaria transmission blocking vaccine candidate.

A liquid chromatography tandem-mass spectrometry method was developed to map the eleven disulfide bonds in Pfs25, a malaria transmission-blocking vaccine candidate. The compact and complex nature of Pfs25 has led to difficulties in prior peptide mapping efforts. Here, we report confirmation of proper disulfide pairing of a recombinant Pfs25, by optimizing denaturation and digestion with trypsin/Lys-C. The digested peptides were separated by reversed phase HPLC to obtain the peptide map and elucidate the disulfide linkages. MSE fragmentation confirmed the digested peptides and disulfide bonds. The eleven disulfide bonds and locations matched the predicted Pvs25 crystal structure, a Pfs25 homologue.

Human Fc receptor-like 5 distinguishes IgG2 disulfide isoforms and deamidated charge variants.

Human Fc receptor-like 5 (FCRL5) is a novel IgG receptor. We reported that IgG2 samples display a thousand-fold range affinity for FCRL5, indicating that attributes beyond the isotype affect binding. We hypothesized that the complex interaction could be exploited to identify distinct changes in the IgG2 molecule. We investigated using surface plasmon resonance two factors that might affect the interaction between IgG2 and FCRL5; heterogeneity related to disulfide isoforms and charge variants. We found that panitumumab and denosumab samples enriched for the more flexible A disulfide isoform bound FCRL5 with two-fold and 82-fold higher apparent affinity, respectively, than the B isoform. We next assessed whether FCRL5 binding can distinguish panitumumab charge variants which increase during storage, using two approaches. First, samples were stored at 40°C to promote acidic variants. Heat stressed panitumumab had up to four-fold higher apparent affinity for FCRL5. Next, we used conditions that promoted deamidation, a common cause of acidic variants. We found that deamidated panitumumab had up to 14-fold higher apparent affinity for FCRL5, indicating that deamidation promotes the interaction. Statistical analyses of kinetic parameters and similarity scores obtained from sensogram comparisons indicated that IgG2 disulfide isoforms, heat stressed and deamidated samples each bind FCRL5 differently. We conclude that based on FCRL5 binding, we can discern distinct changes in the IgG2 molecule, including the disulfide isoform structure and charge variants related to deamidation. Since both IgG2 deamidation and conversion of disulfide isoforms occur in vivo, these findings elucidate the biological FCRL5 ligand.

Impact of antibody subclass and disulfide isoform differences on the biological activity of CD200R and ßklotho agonist antibodies.

Agonism of cell surface receptors by monoclonal antibodies is dependent not only on its ability to bind the target, but also to deliver a biological signal through receptors to the cell. Immunoglobulin G2 antibodies (IgG2s) are made up of a mixture of distinct isoforms (IgG2-A, -B and A/B), which differ by the disulfide connectivity at the hinge region. When evaluating panels of agonistic antibodies against CD200 receptor (CD200R) or ßklotho receptor (ßklotho), we noticed striking activity differences of IgG1 or IgG2 antibodies with the same variable domains. For the CD200R antibody, the IgG2 antibody demonstrated higher activity than the IgG1 or IgG4 antibody. More significantly, for ßklotho, agonist antibodies with higher biological activity as either IgG2 or IgG1 were identified. In both cases, ion exchange chromatography was able to isolate the bioactivity to the IgG2-B isoform from the IgG2 parental mixture. The subclass-related increase in agonist activity was not correlated with antibody aggregation or binding affinity, but was driven by enhanced avidity for the CD200R antibody. These results add to the growing body of evidence that show that conformational differences in the antibody hinge region can have a dramatic impact on the antibody activity and must be considered when screening and engineering therapeutic antibody candidates. The results also demonstrate that the IgG1 (IgG2-A like) or the IgG2-B form may provide the most active form of agonist antibodies for different antibodies and targets.

Disulfide-modified antigen for detection of celiac disease-associated anti-tissue transglutaminase autoantibodies.

A simple and rapid immunosensor for the determination of the celiac disease-related antibody, anti-tissue transglutaminase, was investigated. The antigenic protein tissue transglutaminase was chemically modified, introducing disulfide groups through different moieties of the molecule (amine, carboxylic, and hydroxyl groups), self-assembled on gold surfaces, and used for the detection of IgA and IgG autoantibodies. The modified proteins were evaluated using enzyme-linked immunosorbent assay and surface plasmon resonance, which showed that only introduction of the disulfide groups through amine moieties in the tissue transglutaminase preserved its antigenic properties. The disulfide-modified antigen was co-immobilized via chemisorption with a poly(ethylene glycol) alkanethiol on gold electrodes. The modified electrodes were then exposed to IgA anti-tissue transglutaminase antibodies and subsequently to horseradish peroxidase-labeled anti-idiotypic antibodies, achieving a detection limit of 260 ng ml-1. Immunosensor performance in the presence of complex matrixes, including clinically relevant serum reference solutions and real patient samples, was evaluated. The introduction of disulfides in the antigenic protein enabled a simple and convenient one-step surface immobilization procedure involving only spontaneous gold-thiol covalent binding. Complete amperometric assay time was 30 min.

Modulating Therapeutic Activity and Toxicity of Pyrrolobenzodiazepine Antibody-Drug Conjugates with Self-Immolative Disulfide Linkers.

A novel disulfide linker was designed to enable a direct connection between cytotoxic pyrrolobenzodiazepine (PBD) drugs and the cysteine on a targeting antibody for use in antibody-drug conjugates (ADCs). ADCs composed of a cysteine-engineered antibody were armed with a PBD using a self-immolative disulfide linker. Both the chemical linker and the antibody site were optimized for this new bioconjugation strategy to provide a highly stable and efficacious ADC. This novel disulfide ADC was compared with a conjugate containing the same PBD drug, but attached to the antibody via a peptide linker. Both ADCs had similar efficacy in mice bearing human tumor xenografts. Safety studies in rats revealed that the disulfide-linked ADC had a higher MTD than the peptide-linked ADC. Overall, these data suggest that the novel self-immolative disulfide linker represents a valuable way to construct ADCs with equivalent efficacy and improved safety. Mol Cancer Ther; 16(5); 871-8. ©2017 AACR.

Identification and Characterization of Complex Glycosylated Peptides Presented by the MHC Class II Processing Pathway in Melanoma.

The MHC class II (MHCII) processing pathway presents peptides derived from exogenous or membrane-bound proteins to CD4+ T cells. Several studies have shown that glycopeptides are necessary to modulate CD4+ T cell recognition, though glycopeptide structures in these cases are generally unknown. Here, we present a total of 93 glycopeptides from three melanoma cell lines and one matched EBV-transformed line with most found only in the melanoma cell lines. The glycosylation we detected was diverse and comprised 17 different glycoforms. We then used molecular modeling to demonstrate that complex glycopeptides are capable of binding the MHC and may interact with complementarity determining regions. Finally, we present the first evidence of disulfide-bonded peptides presented by MHCII. This is the first large scale study to sequence glyco- and disulfide bonded MHCII peptides from the surface of cancer cells and could represent a novel avenue of tumor activation and/or immunoevasion.

Targeting synthetic Human Papillomavirus (HPV) L2 disulfide-induced N-terminus conformational epitopes for pan-HPV vaccine development.

BACKGROUND: Current vaccines against Human Papillomavirus (HPV) are highly effective and based on recombinant virus-like particles (VLPs) of the major capsid protein L1. Since these vaccines are HPV type-specific and expensive for global implementation, an alternative, broader-spectrum immunogen would be the N-terminus of the minor capsid protein L2 that induces low titered broadly cross-neutralizing antibodies. Here we analyzed the reactivity of different synthetic L2 peptides containing N-terminus amino acids 17-36 in order to test their antigenicity. METHODS: Different synthetic peptides were designed to target the 17-36 amino acid sequences, present in highly antigenic amino-terminus of L2 protein. Six different peptides including Cys22-Cys28 disulfide bonded cyclized L2 peptide were examined for their antigenicity against mouse monoclonal antibody RG-1 and rabbit polyclonal antisera to HPV L2 by enzyme-linked immunosorbent assay (ELISA). RESULTS: Here we report that the cyclized form of synthetic L2 peptide, which is formed through Cys22-Cys28 disulfide bridges, has the highest reactivity to antibodies than other synthetic L2 peptides. CONCLUSION: A cyclized L2 peptide has potential to be an excellent candidate to formulate a low-cost, broadly protective pan-oncogenic HPV vaccine.

Diverse cellular and organismal functions of the lysosomal thiol reductase GILT.

Gamma-interferon-inducible lysosomal thiol reductase (GILT) is the only enzyme known to catalyze disulfide bond reduction in the endocytic pathway. GILT facilitates the presentation of a subset of epitopes from disulfide bond-containing antigens. Enhanced presentation of MHC class II-restricted epitopes alters central tolerance and modulates CD4+ T cell-mediated autoimmunity. Improved cross-presentation of viral epitopes results in improved cross-priming of viral-specific CD8+ T cells. GILT regulates the cellular redox state. In GILT-/- cells, there is a shift from the reduced to the oxidized form of glutathione, resulting in mitochondrial autophagy, decreased superoxide dismutase 2, and elevated superoxide levels. GILT expression diminishes cellular activation, including decreased phosphorylated ERK1/2, and decreases cellular proliferation. GILT enhances the activity of bacterial hemolysins, such as listeriolysin O, and increases bacterial replication and infection. GILT expression in cancer cells is associated with improved patient survival. These diverse roles of GILT are discussed.

The C-terminal disulfide bonds of Helicobacter pylori GroES are critical for IL-8 secretion via the TLR4-dependent pathway in gastric epithelial cells.

Helicobacter pylori GroES (HpGroES), a potent immunogen, is a secreted virulence factor that stimulates production of proinflammatory cytokines and may contribute to gastric carcinogenesis. HpGroES is larger than other bacterial orthologs because of an additional C-terminal region, known as domain B. We found that the HpGroES-induced IL-8 release by human gastric epithelial cells was dependent on activation of the MAPK and NF-κB pathways. HpGroES lacking domain B was unable to induce IL-8 release. Additionally, a TLR4 inhibitor significantly inhibited IL-8 secretion and reduced HpGroES-induced activation of MAPKs. Furthermore, HpGroES-induced IL-8 release by primary gastric epithelial cells from TLR4(-/-) mice was significantly lower than from wild-type mice. We also found that HpGroES bound to TLR4 in cell lysates and colocalized with TLR4 on the cell membrane only when domain B was present. We then constructed two deletion mutants lacking C-terminal regions and mutants with point mutations of two of the four cysteine residues, C111 and C112, in domain B and found that the deletion mutants and a double mutant lacking the C94-C111 and C95-C112 disulfide bonds were unable to interact with TLR4 or induce IL-8 release. We conclude that HpGroES, in which a unique conformational structure, domain B, is generated by these two disulfide bonds, induces IL-8 secretion via a TLR4-dependent mechanism.

Hapten-directed spontaneous disulfide shuffling: a universal technology for site-directed covalent coupling of payloads to antibodies.

Humanized hapten-binding IgGs were designed with an accessible cysteine close to their binding pockets, for specific covalent payload attachment. Individual analyses of known structures of digoxigenin (Dig)- and fluorescein (Fluo) binding antibodies and a new structure of a biotin (Biot)-binder, revealed a "universal" coupling position (52(+2)) in proximity to binding pockets but without contributing to hapten interactions. Payloads that carry a free thiol are positioned on the antibody and covalently linked to it via disulfides. Covalent coupling is achieved and driven toward complete (95-100%) payload occupancy by spontaneous redox shuffling between antibody and payload. Attachment at the universal position works with different haptens, antibodies, and payloads. Examples are the haptens Fluo, Dig, and Biot combined with various fluorescent or peptidic payloads. Disulfide-bonded covalent antibody-payload complexes do not dissociate in vitro and in vivo. Coupling requires the designed cysteine and matching payload thiol because payload or antibody without the Cys/thiol are not linked (<5% nonspecific coupling). Hapten-mediated positioning is necessary as hapten-thiol-payload is only coupled to antibodies that bind matching haptens. Covalent complexes are more stable in vivo than noncovalent counterparts because digoxigeninylated or biotinylated fluorescent payloads without disulfide-linkage are cleared more rapidly in mice (approximately 50% reduced 48 hour serum levels) compared with their covalently linked counterparts. The coupling technology is applicable to many haptens and hapten binding antibodies (confirmed by automated analyses of the structures of 140 additional hapten binding antibodies) and can be applied to modulate the pharmacokinetics of small compounds or peptides. It is also suitable to link payloads in a reduction-releasable manner to tumor- or tissue-targeting delivery vehicles.

Two-dimensional layered MoS2 biosensors enable highly sensitive detection of biomolecules.

We present a MoS2 biosensor to electrically detect prostate specific antigen (PSA) in a highly sensitive and label-free manner. Unlike previous MoS2-FET-based biosensors, the device configuration of our biosensors does not require a dielectric layer such as HfO2 due to the hydrophobicity of MoS2. Such an oxide-free operation improves sensitivity and simplifies sensor design. For a quantitative and selective detection of PSA antigen, anti-PSA antibody was immobilized on the sensor surface. Then, introduction of PSA antigen, into the anti-PSA immobilized sensor surface resulted in a lable-free immunoassary format. Measured off-state current of the device showed a significant decrease as the applied PSA concentration was increased. The minimum detectable concentration of PSA is 1 pg/mL, which is several orders of magnitude below the clinical cut-off level of ~4 ng/mL. In addition, we also provide a systematic theoretical analysis of the sensor platform - including the charge state of protein at the specific pH level, and self-consistent channel transport. Taken together, the experimental demonstration and the theoretical framework provide a comprehensive description of the performance potential of dielectric-free MoS2-based biosensor technology.

Barley γ3-hordein: glycosylation at an atypical site, disulfide bridge analysis, and reactivity with IgE from patients allergic to wheat.

Post translational modifications of a seed storage protein, barley γ3-hordein, were determined using immunochemical and mass spectrometry methods. IgE reactivity towards this protein was measured using sera from patients diagnosed with allergies to wheat. N-glycosylation was found at an atypical Asn-Leu-Cys site. The observed glycan contains xylose. This indicates that at least some γ3-hordein molecules trafficked through the Golgi apparatus. Disulfide bridges in native γ3-hordein were almost the same as those found in wheat γ46-gliadin, except the bridge involving the cysteine included in the glycosylation site. IgE reacted more strongly towards the recombinant than the natural γ3-hordein protein. IgE binding to γ3-hordein increased when the protein sample was reduced. Glycosylation and disulfide bridges therefore decrease epitope accessibility. Thus the IgE from patients sensitized to wheat cross-react with γ3-hordein due to sequence homology with wheat allergens rather than through shared carbohydrate determinants.