Structural basis for differential recognition of phosphohistidine-containing peptides by 1-pHis and 3-pHis monoclonal antibodies.

In 2015, monoclonal antibodies (mAbs) that selectively recognize the 1-pHis or 3-pHis isoforms of phosphohistidine were developed by immunizing rabbits with degenerate Ala/Gly peptides containing the nonhydrolyzable phosphohistidine (pHis) analog- phosphotriazolylalanine (pTza). Here, we report structures of five rabbit mAbs bound to cognate pTza peptides: SC1-1 and SC50-3 that recognize 1-pHis, and their 3-pHis-specific counterparts, SC39-4, SC44-8, and SC56-2. These cocrystal structures provide insights into the binding modes of the pTza phosphate group that are distinct for the 1- and 3-pHis mAbs with the selectivity arising from specific contacts with the phosphate group and triazolyl ring. The mode of phosphate recognition in the 3-pHis mAbs recapitulates the Walker A motif, as present in kinases. The complementarity-determining regions (CDRs) of four of the Fabs interact with the peptide backbone rather than peptide side chains, thus conferring sequence independence, whereas SC44-8 shows a proclivity for binding a GpHAGA motif mediated by a sterically complementary CDRL3 loop. Specific hydrogen bonding with the triazolyl ring precludes recognition of pTyr and other phosphoamino acids by these mAbs. Kinetic binding experiments reveal that the affinity of pHis mAbs for pHis and pTza peptides is submicromolar. Bound pHis mAbs also shield the pHis peptides from rapid dephosphorylation. The epitope-paratope interactions illustrate how these anti-pHis antibodies are useful for a wide range of research techniques and this structural information can be utilized to improve the specificity and affinity of these antibodies toward a variety of pHis substrates to understand the role of histidine phosphorylation in healthy and diseased states.

The many ways that nature has exploited the unusual structural and chemical properties of phosphohistidine for use in proteins.

Histidine phosphorylation is an important and ubiquitous post-translational modification. Histidine undergoes phosphorylation on either of the nitrogens in its imidazole side chain, giving rise to 1- and 3- phosphohistidine (pHis) isomers, each having a phosphoramidate linkage that is labile at high temperatures and low pH, in contrast with stable phosphomonoester protein modifications. While all organisms routinely use pHis as an enzyme intermediate, prokaryotes, lower eukaryotes and plants also use it for signal transduction. However, research to uncover additional roles for pHis in higher eukaryotes is still at a nascent stage. Since the discovery of pHis in 1962, progress in this field has been relatively slow, in part due to a lack of the tools and techniques necessary to study this labile modification. However, in the past ten years the development of phosphoproteomic techniques to detect phosphohistidine (pHis), and methods to synthesize stable pHis analogues, which enabled the development of anti-phosphohistidine (pHis) antibodies, have accelerated our understanding. Recent studies that employed anti-pHis antibodies and other advanced techniques have contributed to a rapid expansion in our knowledge of histidine phosphorylation. In this review, we examine the varied roles of pHis-containing proteins from a chemical and structural perspective, and present an overview of recent developments in pHis proteomics and antibody development.

Polydisperse molecular architecture of connexin 26/30 heteromeric hemichannels revealed by atomic force microscopy imaging.

Connexin (Cx) protein forms hemichannels and gap junctional channels, which play diverse and profound roles in human physiology and diseases. Gap junctions are arrays of intercellular channels formed by the docking of two hemichannels from adjacent cells. Each hexameric hemichannel contains the same or different Cx isoform. Although homomeric Cxs forms have been largely described functionally and structurally, the stoichiometry and arrangement of heteromeric Cx channels remain unknown. The latter, however, are widely expressed in human tissues and variation might have important implications on channel function. Investigating properties of heteromeric Cx channels is challenging considering the high number of potential subunit arrangements and stoichiometries, even when only combining two Cx isoforms. To tackle this problem, we engineered an HA tag onto Cx26 or Cx30 subunits and imaged hemichannels that were liganded by Fab-epitope antibody fragments via atomic force microscopy. For Cx26-HA/Cx30 or Cx30-HA/Cx26 heteromeric channels, the Fab-HA binding distribution was binomial with a maximum of three Fab-HA bound. Furthermore, imaged Cx26/Cx30-HA triple liganded by Fab-HA showed multiple arrangements that can be derived from the law of total probabilities. Atomic force microscopy imaging of ringlike structures of Cx26/Cx30-HA hemichannels confirmed these findings and also detected a polydisperse distribution of stoichiometries. Our results indicate a dominant subunit stoichiometry of 3Cx26:3Cx30 with the most abundant subunit arrangement of Cx26-Cx26-Cx30-Cx26-Cx30-Cx30. To our knowledge, this is the first time that the molecular architecture of heteromeric Cx channels has been revealed, thus providing the basis to explore the functional effect of these channels in biology.

N-Terminal Modification of Gly-His-Tagged Proteins with Azidogluconolactone.

Site-specific protein modifications are vital for biopharmaceutical drug development. Gluconoylation is a non-enzymatic, post-translational modification of N-terminal HisTags. We report high-yield, site-selective in vitro α-aminoacylation of peptides, glycoproteins, antibodies, and virus-like particles (VLPs) with azidogluconolactone at pH 7.5 in 1 h. Conjugates slowly hydrolyse, but diol-masking with borate esters inhibits reversibility. In an example, we multimerise azidogluconoylated SARS-CoV-2 receptor-binding domain (RBD) onto VLPs via click-chemistry, to give a COVID-19 vaccine. Compared to yeast antigen, HEK-derived RBD was immunologically superior, likely due to observed differences in glycosylation. We show the benefits of ordered over randomly oriented multimeric antigen display, by demonstrating single-shot seroconversion and best virus-neutralizing antibodies. Azidogluconoylation is simple, fast and robust chemistry, and should accelerate research and development.

Efficient application of a baculovirus-silkworm larvae expression system for obtaining porcine circovirus type 2 virus-like particles for a vaccine.

Porcine circovirus type 2 (PCV2) is a major pathogen associated with swine diseases. It is the smallest single-stranded DNA virus, and its genome contains four major open reading frames (ORFs). ORF2 encodes the major structural protein Cap, which can self-assemble into virus-like particles (VLPs) in vitro and contains the primary antigenic determinants. In this study, we developed a high-efficiency method for obtaining VLPs and optimized the purification conditions. In this method, we expressed the protein Cap with a 6× His tag using baculovirus-infected silkworm larvae as well as the E. coli BL21(DE3) prokaryotic expression system. The PCV2 Cap proteins produced by the silkworm larvae and E. coli BL21(DE3) were purified. Cap proteins purified from silkworm larvae self-assembled into VLPs in vitro, while the Cap proteins purified from bacteria were unable to self-assemble. Transmission electron microscopy confirmed the self-assembly of VLPs. The immunogenicity of the VLPs produced using the baculovirus system was demonstrated using an enzyme-linked immunosorbent assay (ELISA). Furthermore, the purification process was optimized. The results demonstrated that the expression system using baculovirus-infected silkworm larvae is a good choice for obtaining VLPs of PCV2 and has potential for the development of a low-cost and efficient vaccine.

Diagnosing the RGS11 Lung Cancer Biomarker: The Integration of Competitive Immunoassay and Isothermal Nucleic Acid Exponential Amplification Reaction.

Lung cancer is the primary cause of cancer-associated mortality worldwide, which makes the identification of reliable lung cancer biomarkers a pressing need for early diagnosis and prognosis. RGS11, which is a regulator of G-protein signaling and also a lung cancer biomarker, plays an important role in cancer-related metastasis. However, trace levels of RGS11 (in the range of pg/mL) in serum samples make it difficult to quantify using currently available enzyme-linked immunosorbent assay (ELISA) kits and, therefore, this hinders progress in the discovery of new approaches for treating lung cancer. The aim of this study is to develop a rapid, sensitive, and reliable platform for the detection of RGS11 lung cancer biomarker based on a suspension immunoassay coupled with an isothermal exponential amplification strategy. Our study was initiated by the functionalization of magnetic beads with anti-RGS11 antibodies (Ab-MB) by EDC (1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide)/NHS ( N-hydroxysulfosuccinimide) activation. Ab-MB served as a sensing probe for the competitive immunorecognitions between known concentrations of His-tag RGS11 and unknown concentrations of target RGS11 in serum. The reporter anti-His antibodies, which were modified with primers that induced an isothermal exponential amplification reaction, were subsequently introduced to the reaction mixture that resulted in the formation of immunosandwich complexes. The exponentially amplified DNA duplex that was intercalated with SYBR Green was designated as a signal reporter for the assessment of RGS11 in an inversely proportional relationship. The sensing platform was excellent for the determination of RGS11 with an exceptional detection limit of 148 fg/mL and a linear dynamic range of 0.1-10 pg/mL using a minimal sample volume (20 µL) and with a reaction time of 1.5 h. In addition, we challenged the sensing platform with RGS11-spiked samples (in 2× diluted serum), and an acceptable recovery rate (>90%) was observed. Finally, 24 clinical samples acquired from patients with advanced lung cancer (C), inflammation (I), and heart failure (H) were analyzed by this newly developed sensing platform and a commercial ELISA kit for validation. This sensing platform has potential in biomedical applications for clinically diagnosing liquid biopsy samples for patients with lung cancer. Moreover, the universal design of our proposed system is easily adapted to detect any other protein if a His-tag recombinant protein is available.

Oxidation of M252 but not M428 in hu-IgG1 is responsible for decreased binding to and activation of hu-FcγRIIa (His131).

Oxidation of monoclonal therapeutic antibodies (mAbs) can affect binding to Fc-receptors and potentially influence pharmacokinetics or effector functions like e.g. antibody dependent cellular phagocytosis (ADCP). Recently, it has been demonstrated that binding to FcγRIIa (H131) is affected by methionine oxidation of the Fc-portion but it is currently unknown which methionine is responsible for decreased binding. We separated an oxidized IgG1 monoclonal antibody based on the oxidation state of methionine 252 and analyzed fractionated material in receptor binding experiments as well as in functional (cell-based) assays. Although the unfractionated mixture demonstrated weaker interaction/activation of the receptor, differently oxidized isolated subspecies can lead both to stronger as well as weaker binding and activation of the histidine variant of FcγRIIa.

Simultaneous detection of assembly and disassembly of multivalent HA tag and anti-HA antibody in single in-capillary assay.

Herein, we have developed an in-capillary assay for simultaneous detection of the assembly and disassembly of the multivalent HA tag peptide and antibody. HA tag with hexahistidine at C terminus (YPYDVPDYAG4 H6 , termed YPYDH6 ) was conjugated with quantum dots (QDs) by metal-affinity force to form a multivalent HA tag (QD-YPYDH6 ). QD-YPYDH6 and monoclonal anti-HA antibody (anti-HA) were sequentially injected into the capillary. They were mixed and assembled inside the capillary. The reaction products were online discriminated and detected by fluorescence coupled capillary electrophoresis (CE-FL). For the in-capillary assay, the binding efficiency of the multivalent HA tag and antibody on was influenced by the molar ratio and injection time. Such novel assay could even give out the self-assembly kinetic constant of QDs and YPYDH6 as KD of 34.1 µM with n (binding cooperativeness) of 2.2 by Hill equation. More importantly, the simultaneous detection of the assembly and imidazole (Im) induced disassembly of the QD-YPYDH6 -anti-HA complex was achieved in a single in-capillary assay. Our study demonstrated a new method for the online detection of antigen-antibody interactions.

RETRACTED: Identification of an antitumor immune response of polyhistidine through a toll-like receptor 4-dependent manner.

Polyhistidine is widely used for the delivery of nucleic acids and antibodies into the cell cytoplasm. However, little attention has been concerned on the effect of polyhistidine on the immune system. In this work, we identify a novel function of polyhistidine as an activator of the immune system. Single-molecule fluorescence imaging and single-molecule force measurements show that polyhistidine binds specifically to the toll-like receptor 4 (TLR4), inducing receptor dimerization and activation. Moreover, in a B16 melanoma model we demonstrate that polyhistidine treatment inhibits tumor growth in TLR4(+/+) but not TLR4(-/-) mice. These results suggest the potential use of polyhistidine for cancer immunotherapy.

Reversible major histocompatibility complex I-peptide multimers containing Ni(2+)-nitrilotriacetic acid peptides and histidine tags improve analysis and sorting of CD8(+) T cells.

MHC-peptide multimers containing biotinylated MHC-peptide complexes bound to phycoerythrin (PE) streptavidin (SA) are widely used for analyzing and sorting antigen-specific T cells. Here we describe alternative T cell-staining reagents that are superior to conventional reagents. They are built on reversible chelate complexes of Ni(2+)-nitrilotriacetic acid (NTA) with oligohistidines. We synthesized biotinylated linear mono-, di-, and tetra-NTA compounds using conventional solid phase peptide chemistry and studied their interaction with HLA-A*0201-peptide complexes containing a His(6), His(12), or 2×His(6) tag by surface plasmon resonance on SA-coated sensor chips and equilibrium dialysis. The binding avidity increased in the order His(6) < His(12) < 2×His(6) and NTA(1) < NTA(2) < NTA(4), respectively, depending on the configuration of the NTA moieties and increased to picomolar K(D) for the combination of a 2×His(6) tag and a 2×Ni(2+)-NTA(2). We demonstrate that HLA-A2-2×His(6)-peptide multimers containing either Ni(2+)-NTA(4)-biotin and PE-SA- or PE-NTA(4)-stained influenza and Melan A-specific CD8+ T cells equal or better than conventional multimers. Although these complexes were highly stable, they very rapidly dissociated in the presence of imidazole, which allowed sorting of bona fide antigen-specific CD8+ T cells without inducing T cell death as well as assessment of HLA-A2-peptide monomer dissociation kinetics on CD8+ T cells.

Substrates Modulate Charge-Reorganization Allosteric Effects in Protein-Protein Association.

Protein function may be modulated by an event occurring far away from the functional site, a phenomenon termed allostery. While classically allostery involves conformational changes, we recently observed that charge redistribution within an antibody can also lead to an allosteric effect, modulating the kinetics of binding to target antigen. In the present work, we study the association of a polyhistidine tagged enzyme (phosphoglycerate kinase, PGK) to surface-immobilized anti-His antibodies, finding a significant Charge-Reorganization Allostery (CRA) effect. We further observe that PGK's negatively charged nucleotide substrates modulate CRA substantially, even though they bind far away from the His-tag-antibody interaction interface. In particular, binding of ATP reduces CRA by more than 50%. The results indicate that CRA is affected by the binding of charged molecules to a protein and provide further insight into the significant role that charge redistribution can play in protein function.

Toward the limits of sandwich immunoassay of very low molecular weight molecules.

A model study aiming at exploring the limits of sandwich immunoassays of very small molecules is described. Combinatorial association of antibody couples to detect small molecules constituted by two small epitopes connected via different linear spacers was used to investigate the minimum size of compounds susceptible to be simultaneously bound by two distinct antibodies. The results clearly indicated that despite the fact that below 10 carbon atoms unfavorable interactions between antibodies took place, molecules bearing two epitopes separated by only 5 carbon atoms might be directly detected by sandwich immunoassays.

Use of a semisynthetic epitope to probe histidine kinase activity and regulation.

Histidine-aspartic acid phosphotransfer pathways are central components of prokaryotic signal transduction pathways and are also found in many eukaryotes. Tools to study histidine kinases, however, are currently quite limited. In this article, we present a new tool to study histidine-aspartic acid phosphotransfer pathways. We show that many histidine kinases will accept ATPgammaS as a substrate to form a stable thiophosphohistidine even when they do not form stable phosphohistidines using the natural substrate ATP. An antibody that has previously been used to detect thiophosphorylated serine, threonine, and tyrosine residues is shown to recognize thiophosphohistidine and thiophosphoaspartic acid residues. Histidine kinase autothiophosphorylation is regulated by other protein sensor domains in the same way as autophosphorylation, and thiophosphate is transferred to downstream aspartic acid containing response regulators.

A high-throughput differential filtration assay to screen and select detergents for membrane proteins.

Structural studies on integral membrane proteins are routinely performed on protein-detergent complexes (PDCs) consisting of purified protein solubilized in a particular detergent. Of all the membrane protein crystal structures solved to date, a subset of only four detergents has been used in more than half of these structures. Unfortunately, many membrane proteins are not well behaved in these four detergents and/or fail to yield well-diffracting crystals. Identification of detergents that maintain the solubility and stability of a membrane protein is a critical step and can be a lengthy and "protein-expensive" process. We have developed an assay that characterizes the stability and size of membrane proteins exchanged into a panel of 94 commercially available and chemically diverse detergents. This differential filtration assay (DFA), using a set of filtered microplates, requires sub-milligram quantities of purified protein and small quantities of detergents and other reagents and is performed in its entirety in several hours.

Immunosensor incorporating anti-His (C-term) IgG F(ab') fragments attached to gold nanorods for detection of His-tagged proteins in culture medium.

Immunosensors based on gold electrodes (electrochemical) or gold discs (optical) modified with 1,6-hexanedithiol, gold nanorods and Anti-His (C-term) monoclonal antibody F(ab') fragment are described. The antigen detected by the sensing platform is a recombinant histidine-tagged silk proteinase inhibitor (rSPI2-His(6)). Electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR) techniques were used as methods for detection of the antigen. This approach allows to detect the antigen protein in concentration of 10 pg per mL (0.13 pM) of culture medium. The immunosensor shows good reproducibility due to covalent immobilization of F(ab') fragments to gold nanorods layer.

Evaluation of protective immunity responses against pneumococcal PhtD and its C-terminal in combination with outer-membrane vesicles as adjuvants.

Introduction. Streptococcus pneumoniae is a significant bacterial pathogen in humans. Currently, there are two types of pneumococcal vaccines, but there are concerns regarding their application.Aim. Since many pneumococcal proteins are serotype-independent, polyhistidine triad protein D (PhtD) has been selected as a vaccine candidate.Methodology. We prepared recombinant PhtD and its C-terminal fragment (PhtD-C) using alum and outer-membrane vesicles (OMVs) as adjuvants. The combinations were injected intraperitoneally into mice, and then total immunoglobulin G (IgG) and specific IgG, IgG1 and IgG2a were measured. A serum bactericidal assay and opsonophagocytosis were also performed as complementary tests. Meningococcal OMVs were used as an adjuvant.Results. The levels of specific IgG and IgG1 against combinations of PhtD and its C-terminal with OMVs and alum as adjuvants increased at the time of the third mouse immunization on day 35. Forty per cent and 60% of S. pneumoniae ATCC 6303 (serotype 3) as a virulent pneumococcal strain, respectively, were killed in the opsonophagocytosis test and these results could also be observed in the serum bactericidal assay. Mice mmunized iwith PhtD and its C-terminal with OMVs and alum as adjuvants survived after 10 days of pneumococcal challenge.Conclusion. The combination of PhtD and PhtD-C with alum produced optimal results, but the combination of PhtD and PhtD-C with OMVs produced minimal results by comparison. The survival rates were also measured, and these corresponded with the results of the immunological assessments. Our findings showed that mice receiving PhtD and PhtD-C plus OMV and alum had higher survival rates than the mice in the other groups.

An iBody-based lateral flow assay for semi-quantitative determination of His-tagged protein concentration.

The polyhistidine tag (His-tag) is one of the most commonly used epitope tags in protein engineering. While His-tagged proteins can be detected reliably using immunological methods such as ELISA and Western blot, these methods are costly and time-intensive, necessitating more facile solutions for preliminary qualitative determination and concentration estimation. To this end, we present a rapid test strip assay based on iBody antibody mimetics that target the His-tag. We compare this strategy to commercial antibody-based assays and discuss the advantages and caveats of lateral flow assay design. Our test strip detected a panel of His-tagged proteins with different tag attachment strategies with a visual detection limit of 1 µM and densitometric detection limit of 0.5 µM. Due to its chemical nature, the presented assay exhibits wide reagent compatibility in comparison to antibody-based assays.

Comparison of carboxylated and Penta-His microspheres for semi-quantitative measurement of antibody responses to His-tagged proteins.

The Luminex system is a flow cytometry based tool that permits the simultaneous measurement of many analytes from just a single serum sample. The technology uses microspheres, which are available in different colors and can be coated with different kinds of biomolecules. For the immobilisation of His-tagged proteins, two types of beads can be used: chemically activated carboxylated beads or Penta-His beads, which have antibodies against His-tags on their surface. In this study, we compared carboxylated and Penta-His beads. For carboxylated as compared to Penta-His beads, the non-specific background is lower (Median Fluorescence Intensity; MFI>250, 0% versus 15%), the specific signal intensity is higher (mean MFI 2860 versus 722) and not dependent on the configuration of the protein. Above all, the protein coupled carboxylated beads are useful over longer periods of time. Therefore, we conclude that for developing a multiplex assay for semi-quantitative measurement of antibody responses against His-tagged proteins the best microspheres to use are the carboxylated ones.

Improved method to raise polyclonal antibody using enhanced green fluorescent protein transgenic mice.

Recombinant fusion protein is widely used as an antigen to raise antibodies against the epitope of a target protein. However, the concomitant anticarrier antibody in resulting antiserum reduces the production of the desired antibody and brings about unwanted non-specific immune reactions. It is proposed that the carrier protein transgenic animal could be used to solve this problem. To validate this hypothesis, enhanced green fluorescent protein (EGFP) transgenic mice were produced. By immunizing the mice with fusion protein His6HAtag-EGFP, we showed that the antiserum from the transgenic mice had higher titer antibody against His6HA tag and lower titer antibody against EGFP compared with that from wild-type mice. Therefore, this report describes an improved method to raise high titer antipeptide polyclonal antibody using EGFP transgenic mice that could have application potential in antibody preparation.

One-step separation of IgG subclasses of DNA hydrolyzing autoantibodies: a study on sera of patients with systemic lupus erythematosus and primary antiphospholipid syndrome by histidine ligand affinity chromatography.

A method based on histidine ligand affinity chromatography has been utilized for the separation of DNA hydrolyzing autoantibodies from sera of patients suffering from systemic lupus erythematosus and primary antiphospholipid syndrome using the gel, histidyl-aminohexyl-sepharose. The separation of autoantibodies was carried out under mild chromatographic conditions. Human IgG subclass distribution in the different fractions separated on the column was studied by enzyme-linked immunosorbent assay. The purified DNA hydrolyzing autoantibodies were shown to hydrolyze plasmid DNA.