A novel MRGPRX2-targeting antagonistic DNA aptamer inhibits histamine release and prevents mast cell-mediated anaphylaxis.

Anaphylaxis during general anaesthesia is a significant clinical challenge for anaesthesiologists. Approximately 50% of perioperative anaphylaxis cases lack the presence of specific IgE antibodies. Mas-related G-protein coupled receptor X2 (MRGPRX2) in humans and its mouse orthologue Mas-related G-protein coupled receptor B2 (Mrgprb2) are crucial receptors in non-IgE-dependent histamine release. Anaesthetics such as rocuronium and atracurium cause perioperative anaphylaxis by activating histamine release via the Mrgprb2 pathway. We hypothesized that antagonistic DNA aptamers that target MRGPRX2 can prevent perioperative anaphylaxis. Selection of a DNA aptamer that specifically binds MRGPRX2 was achieved by using our modified Systematic Evolution of Ligands by Exponential enrichment (SELEX) approach. Our SELEX process used MRGPRX2-proteoliposomes synthesised by a wheat germ cell-free system as templates. The activity of the selected aptamer to inhibit histamine release from MRGPRX2-activated mast cells and in an anaphylaxis rat model transplanted with this cell line was examined. Our selection process identified aptamer-X35 with the sequence 5'-ATGACCATGACCCTCCACACTGTAGGCACCACGGGTCCCTGGCAGTTAAAAGTACGTTTGTCAGACTGTGGCAGGGAAACA-3'. In silico 2D modelling of aptamer-X35 revealed a structure with a small loop and a long stem. Aptamer-X35 inhibited histamine release from mast cells by 70%. Subcutaneous injection of 30 nmol of aptamer-X35 inhibited the anaphylactic reaction in the rat anaphylaxis model. This study demonstrated that aptamer-X35 selected by the modified SELEX approach reduced histamine release by inhibiting the MRGPRX2 pathway. Overall, our findings establish aptamer-X35 as a potential therapeutic candidate against perioperative anaphylaxis.

Functional G-Protein-Coupled Receptor (GPCR) Synthesis: The Pharmacological Analysis of Human Histamine H1 Receptor (HRH1) Synthesized by a Wheat Germ Cell-Free Protein Synthesis System Combined with Asolectin Glycerosomes.

G-protein-coupled receptors (GPCRs) are membrane proteins distributed on the cell surface, and they may be potential drug targets. However, synthesizing GPCRs in vitro can be challenging. Recently, some cell-free protein synthesis systems have been shown to produce a large amount of membrane protein combined with chemical chaperones that include liposomes and glycerol. Liposomes containing high concentrations of glycerol are known as glycerosomes, which are used in new drug delivery systems. Glycerosomes have greater morphological stability than liposomes. Proteoglycerosomes are defined as glycerosomes that contain membrane proteins. Human histamine H1 receptor (HRH1) is one of the most studied GPCRs. In this study, we synthesized wild-type HRH1 (WT-HRH1) proteoglycerosomes and D107A-HRH1, (in which Asp107 was replaced by Ala) in a wheat germ cell-free protein synthesis system combined with asolectin glycerosomes. The mutant HRH1 has been reported to have low affinity for the H1 antagonist. In this study, the amount of synthesized WT-HRH1 in one synthesis reaction was 434 ± 66.6 µg (7.75 ± 1.19 × 103pmol). The specific binding of [3H]pyrilamine to the WT-HRH1 proteoglycerosomes became saturated as the concentration of the radioligand increased. The dissociation constant (Kd) and maximum density (Bmax) of the synthesized WT-HRH1 were 9.76 ± 1.25 nM and 21.4 ± 0.936 pmol/mg protein, respectively. However, specific binding to D107A-HRH1 was reduced compared with WT-HRH1 and the binding did not become saturated. The findings of this study highlight that HRH1 synthesized using a wheat germ cell-free protein synthesis system combined with glycerosomes has the ability to bind to H1 antagonists.

Efficiency and Safety of CRAC Inhibitors in Human Rheumatoid Arthritis Xenograft Models.

Store-operated Ca2+ release-activated Ca2+ (CRAC) channels are involved in the pathogenesis of rheumatoid arthritis (RA) and have been studied as therapeutic targets in the management of RA. We investigated the efficacy and safety of CRAC inhibitors, including a neutralizing Ab (hCRACM1-IgG) and YM-58483, in the treatment of RA. Patient-derived T cell and B cell activity was suppressed by hCRACM1-IgG as well as YM-58483. Systemically constant, s.c. infused CRAC inhibitors showed anti-inflammatory activity in a human-NOD/SCID xenograft RA model as well as protective effects against the destruction of cartilage and bone. hCRACM1-IgG appeared to be safe for systemic application, whereas YM-58483 showed hepatic and renal toxicity in xenograft mice. Treatment with both CRAC inhibitors also caused hyperglycemia in xenograft mice. These results indicate the potential of hCRACM1-IgG and YM-58483 as anti-immunological agents for the treatment of RA. However, some safety issues should be addressed and application methods should be optimized prior to their clinical use.

Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay.

G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-based interaction assay (BiLIA), both of which are developed using wheat cell-free protein synthesis system and liposome technology. Using bilayer-dialysis method, various GPCRs were successfully synthesized with quality and quantity sufficient for immunization. For selection of specific mAb, we designed BiLIA that detects interaction between antibody and membrane protein on liposome. BiLIA prevented denaturation of GPCR, and then preferably selected conformation-sensitive antibodies. Using this approach, we successfully obtained mAbs against DRD1, GHSR, PTGER1 and T1R1. With respect to DRD1 mAb, 36 mouse mAbs and 6 rabbit mAbs were obtained which specifically recognized native DRD1 with high affinity. Among them, half of the mAbs were conformation-sensitive mAb, and two mAbs recognized extracellular loop 2 of DRD1. These results indicated that this approach is useful for GPCR mAb production.

The ligand binding ability of dopamine D1 receptors synthesized using a wheat germ cell-free protein synthesis system with liposomes.

G-protein coupled receptors (GPCRs) share a common seven-transmembrane topology and mediate cellular responses to a variety of extracellular signals. However, structural and functional approaches to GPCRs have often been limited by the difficulty of producing a sufficient amount of receptor protein using conventional expression systems. We synthesized human dopamine D1 receptors using a wheat cell-free protein synthesis system with liposomes and then analyzed their receptor binding ability. We determined the specific binding of [(3)H]SCH23390 to the synthesized receptors generated from a cell-free protein synthesis system or rat striatal membranes. From Scatchard plot analysis, the dissociation constant (Kd) and the maximum density (Bmax) of the synthesized receptors were 6.61±0.06 nM and 1.85±0.05 pmol/mg protein, respectively. The same analysis for rat striatal membrane gave a Kd of 2.67±0.05 nM and Bmax of 0.70±0.10 pmol/mg protein. Using a competition binding assay, Ki values of antagonists, SCH23390, LE300 and SKF83566, for the synthetic receptors were the same as those for rat striatal membranes, but Ki values of agonists, A68930, SKF38393 and dopamine, were 5-17 fold higher than those for rat striatal membranes. These results suggest that the dopamine D1 receptors synthesized in liposomes have a functional binding capacity. The different patterns of binding of antagonists and agonists to the synthetic receptors and rat striatal membranes indicate that G proteins are involved in agonist binding to dopamine D1 receptors. The cell-free protein synthesis method with liposomes will be invaluable for the functional analysis of GPCRs.

Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system.

BACKGROUND: Recently, some groups have reported on cell-free synthesis of functional membrane proteins (MPs) in the presence of exogenous liposomes (liposomes). Previously, we reported synthesis of a functional AtPPT1 plant phosphate transporter that was associated with liposomes during translation. However, it is unclear whether or not lipid/MP complex formation is common to all types of MPs in the wheat cell-free system. RESULTS: AtPPT1 was synthesized using a wheat cell-free system with or without liposomes. AtPPT1 synthesized with liposomes showed high transport activity, but the activity of AtPPT1 synthesized without liposomes was less than 10% activity of that with liposomes. To test whether co-translational association with liposomes is observed in the synthesis of other MPs, we used 40 mammalian MPs having one to 14 transmembrane domains (TMDs) and five soluble proteins as a control. The association rate of all 40 MPs into liposomes was more than 40% (mean value: 59%), while that of the five soluble proteins was less than 20% (mean value: 12%). There were no significant differences in association rate among MPs regardless of the number of TMDs and synthesis yield. These results indicate that the wheat cell-free system is a highly productive method for lipid/MP complex formation and is suitable for large-scale preparation. The liposome association of green fluorescent protein (GFP)-fusion MPs were also tested and recovered as lipid/MP complex after floatation by Accudenz density gradient ultracentrifugation (DGU). Employment of GFP-MPs revealed optimal condition for Accudenz floatation. Using the optimized Accudenz DGU condition, P2RX4/lipid complexes were partially purified and detected as a major band by Coomassie Brilliant Blue (CBB)-staining after SDS-PAGE. CONCLUSION: Formation of lipid/AtPPT1 complex during the cell-free synthesis reaction is critical for synthesis of a functional MP. The lipid/MP complex during the translation was observed in all 40 MPs tested. At least 29 MPs, as judged by their higher productivity compared to GFP, might be suitable for a large-scale preparation. MPs synthesized by this method form lipid/MP complexes, which could be readily partially purified by Accudenz DGU. Wheat cell-free protein synthesis in the presence of liposomes will be a useful method for preparation of variety type of MPs.

Purification of eukaryotic translation factors from wheat germ for reconstitution of protein synthesis.

The wheat germ cell-free protein synthesis is a powerful and versatile method for preparation of proteins based on the accumulated DNA sequence information. As the cell extract used for it contains many factors that are unknown or do not directly involve in protein synthesis, details of the translation reaction is yet to be understood. Therefore, we have decided to try reconstitution of protein synthesis, which would be useful for better understanding of the mechanisms supporting eukaryotic protein synthesis and translational regulation and probably applicable to synthetic biology. In the present study, we fractionated an extract from crude wheat germ according to published protocols to obtain the fractions containing the eukaryotic elongation factors (eEFs) 1A, 1B, and 2. The eEF1A and eEF2 fractions supported polyphenylalanine synthesis.

Detection of structural changes in a cofactor binding protein by using a wheat germ cell-free protein synthesis system coupled with unnatural amino acid probing.

A cell-free protein synthesis system is a powerful tool with which unnatural amino acids can be introduced into polypeptide chains. Here, the authors describe unnatural amino acid probing in a wheat germ cell-free translation system as a method for detecting the structural changes that occur in a cofactor binding protein on a conversion of the protein from an apo-form to a holo-form. The authors selected the FMN-binding protein from Desulfovibrio vulgaris as a model protein. The apo-form of the protein was synthesized efficiently in the absence of FMN. The purified apo-form could be correctly converted to the holo-form. Thus, the system could synthesize the active apo-form. Gel filtration chromatography, analytical ultracentrifugation, and circular dichroism-spectra studies suggested that the FMN-binding site of the apo-form is open as compared with the holo-form. To confirm this idea, the unnatural amino acid probing was performed by incorporating 3-azido-L-tyrosine at the Tyr35 residue in the FMN-binding site. The authors optimized three steps in their system. The introduced 3-azido-L-tyrosine residue was subjected to specific chemical modification by a fluorescein-triarylphosphine derivative. The initial velocity of the apo-form reaction was 20 fold faster than that of the holo-form, demonstrating that the Tyr35 residue in the apo-form is open to solvent.

A novel way of amino acid-specific assignment in (1)H-(15)N HSQC spectra with a wheat germ cell-free protein synthesis system.

For high-throughput protein structural analyses, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones utilizing E. coli cells, have been developed, a lot of proteins functioning in solution still were synthesized as insoluble forms. Recently, a novel wheat germ cell-free protein synthesis system was developed, and many of such proteins were synthesized as soluble forms. This means that the applicability of this protein synthesis method to determination of the functional structures of soluble proteins. In our previous work, we synthesized (15)N-labeled proteins with this wheat germ cell-free system, and confirmed this applicability on the basis of the strong similarity between the (1)H-(15)N HSQC spectra for native proteins and the corresponding ones for synthesized ones. In this study, we developed a convenient and reliable method for amino acid selective assignment in (1)H-(15)N HSQC spectra of proteins, using several inhibitors for transaminases and glutamine synthase in the process of protein synthesis. Amino acid selective assignment in (1)H-(15)N HSQC spectra is a powerful means to monitor the features of proteins, such as folding, intermolecular interactions and so on. This is also the first direct experimental evidence of the presence of active transaminases and glutamine synthase in wheat germ extracts.

Formation of circular polyribosomes in wheat germ cell-free protein synthesis system.

We report a morphological study of functioning ribosomes in a efficient and robust cell-free protein synthesis system prepared from wheat embryos. Sucrose density gradient analysis of translated mixtures programmed with luciferase mRNAs having different 5' and 3' untranslated regions showed formation of large polysomes. Electron microscopic examination of translation mixtures programmed with those of capped and polyadenylated mRNA revealed that ribosomes assemble into a circular-type polysome in vitro. Furthermore, a series of experiments using mRNAs lacking either cap, poly(A) tail or both also resulted in the formation of circular polysomes, which are indistinguishable from those with the original mRNA. The wheat germ cell-free system may provide a good experimental system for understanding functional ribosomes at the molecular level.

Application of cell-free translation systems to studies of cofactor binding proteins.

To develop applications of in vitro cell-free translation systems for production and characterization of cofactor binding proteins, we investigate the production of apo- or holo-forms of Flavin Mono Nucleotide (FMN)-binding protein from Desulfovibrio vulgaris (Miyazaki F) and purified them. The redox potential analysis and measurements of UV-, visible, and fluorescent spectra of reconstructed holo-protein showed that the FMN correctly bound to the FMN binding site. On the other hand, contrary to our expectation, we found that the apo-protein formed a dimer structure and the incorporation of the FMN led the conformational alterations of the protein. These studies demonstrate the utility of cell-free translation systems to analyses of cofactor-binding proteins.

Aquifex aeolicus tRNA (Gm18) methyltransferase has unique substrate specificity. TRNA recognition mechanism of the enzyme.

Transfer RNA (guanosine-2')-methyltransferase (Gm-methylase) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine to 2'-OH of G18 in the D-loop of tRNA. Based on their mode of tRNA recognition, Gm-methylases can be divided into the following two types: type I having broad specificity toward the substrate tRNA, and type II that methylates only limited tRNA species. Protein synthesized by in vitro cell-free translation revealed that Gm-methylase encoded in the Aquifex aeolicus genome is a novel type II enzyme. Experiments with chimeric tRNAs and mini- and micro-helix RNAs showed that the recognition region of this enzyme is included within the D-arm structure of tRNALeu and that a bulge is essentially required. Variants of tRNALeu, tRNASer, and tRNAPhe revealed that a combination of certain base pairs in the D-stem is strongly recognized by the enzyme, that 4 bp in the D-stem enhance methyl acceptance activity, and that the Py16Py17G18G19 sequence is important for efficient methyl transfer. The methyl acceptance activities of all the A. aeolicus tRNA genes, which can be classified into 14 categories on the basis of their D-arm structure, were tested. The results clearly showed that the substrate recognition mechanism elucidated by the variant experiments was applicable to their native substrates.

A cell-free protein synthesis system for high-throughput proteomics.

We report a cell-free system for the high-throughput synthesis and screening of gene products. The system, based on the eukaryotic translation apparatus of wheat seeds, has significant advantages over other commonly used cell-free expression systems. To maximize the yield and throughput of the system, we optimized the mRNA UTRs, designed an expression vector for large-scale protein production, and developed a new strategy to construct PCR-generated DNAs for high-throughput production of many proteins in parallel. The resulting system achieves high-yield expression and can maintain productive translation for 14 days. Additionally, in the integration of a PCR-directed system for template creation, at least 50 genes can be translated in parallel, yielding between 0.1 and 2.3 mg of protein by one person within 2 days. Assessment of correct protein folding by the products of this high-throughput protein-expression system were performed by enzymatic assays of kinases and by NMR spectroscopic analysis. The cell-free system, reported here, bypasses many of the time-consuming cloning steps of conventional expression systems and lends itself to a robotic automation for the high-throughput expression of proteins.

A bilayer cell-free protein synthesis system for high-throughput screening of gene products.

A high-throughput cell-free protein synthesis method has been described. The methodology is based on a bilayer diffusion system that enables the continuous supply of substrates, together with the continuous removal of small byproducts, through a phase between the translation mixture and substrate mixture. With the use of a multititer plate the system was functional for a prolonged time, and as a consequence yielded more than 10 times that of the similar batch-mode reaction. Combining this method with a wheat germ cell-free translation system developed by us, the system could produce a large amount of protein sufficient for carrying out functional analyses. This novel bilayer-based cell-free protein synthesis system with its simplicity, minimum time and low cost may be useful practical methodology in the post-genome era.