Yoshiaki Suwa

The Crystal Structure of the Green Tea Polyphenol (―)-Epigallocatechin Gallate―Transthyretin Complex Reveals a Novel Binding Site Distinct from the Thyroxine Binding Site

Amyloid fibril formation is associated with protein misfolding disorders, including neurodegenerative diseases such as Alzheimers, Parkinsons, and Huntingtons diseases. Familial amyloid polyneuropathy (FAP) is a hereditary disease caused by a point mutation of the human plasma protein, transthyretin (TTR), which binds and transports thyroxine (T(4)). TTR variants contribute to the pathogenesis of amyloidosis by forming amyloid fibrils in the extracellular environment. A recent report showed that epigallocatechin 3-gallate (EGCG), the major polyphenol component of green tea, binds to TTR and suppresses TTR amyloid fibril formation. However, structural analysis of EGCG binding to TTR has not yet been conducted. Here we first investigated the crystal structure of the EGCG-V30M TTR complex and found novel binding sites distinct from the thyroxine binding site, suggesting that EGCG has a mode of action different from those of previous chemical compounds that were shown to bind and stabilize the TTR tetramer structure. Furthermore, EGCG induced the oligomerization and monomer suppression in the cellular system of clinically reported TTR variants. Taken together, these findings suggest the possibility that EGCG may be a candidate compound for FAP therapy.

Manadosterols A and B, Sulfonated Sterol Dimers Inhibiting the Ubc13–Uev1A Interaction, Isolated from the Marine Sponge Lissodendryx fibrosa

Two new dimeric sterols, manadosterols A (1) and B (2), were isolated from the marine sponge Lissodendryx fibrosa collected in Indonesia. The two compounds are comprised of two sulfonated sterol cores connected through the respective side chains. Manadosterols A (1) and B (2) inhibited the Ubc13-Uev1A interaction with IC(50) values of 0.09 and 0.13 μM, respectively. They are the second and third natural compounds showing inhibitory activities against the Ubc13-Uev1A interaction and are more potent than leucettamol A (IC(50), 106 μM), the first such inhibitor, isolated from another marine sponge.

Siladenoserinols A-L: new sulfonated serinol derivatives from a tunicate as inhibitors of p53-Hdm2 interaction.

Siladenoserinols A-L were isolated from a tunicate as inhibitors of p53-Hdm2 interaction, a promising target for cancer chemotherapy. Their structures including the absolute configurations were elucidated to be new sulfonated serinol derivatives, each of which contains a 6,8-dioxabicyclo[3.2.1]octane unit and either glycerophosphocholine or glycerophosphoethanolamine moiety. They inhibited p53-Hdm2 interaction with IC(50) values of 2.0-55 μM. Among them, siladenoserinol A and B exhibited the strongest inhibition with an IC(50) value of 2.0 μM.

Role of the glutamic acid 54 residue in transthyretin stability and thyroxine binding

Transthyretin (TTR) is a tetrameric protein associated with amyloidosis caused by tetramer dissociation and monomer misfolding. The structure of two TTR variants (E54G and E54K) with Glu54 point mutation that cause clinically aggressive amyloidosis remains unclear, although amyloidogenicity of artificial triple mutations (residues 53-55) in beta-strand D had been investigated. Here we first analyzed the crystal structures and biochemical and biophysical properties of E54G and E54K TTRs. The direction of the Lys15 side chain in E54K TTR and the surface electrostatic potential in the edge region in both variants were different from those of wild-type TTR. The presence of Lys54 leads to destabilization of tetramer structure due to enhanced electrostatic repulsion between Lys15 of two monomers. Consistent with structural data, the biochemical analyses demonstrated that E54G and E54K TTRs were more unstable than wild-type TTR. Furthermore, the entrance of the thyroxine (T(4)) binding pocket in TTR was markedly narrower in E54K TTR and wider in E54G TTR compared with wild-type TTR. The tetramer stabilization and amyloid fibril formation assays in the presence of T(4) showed lower tetramer stability and more fibril formation in E54K and E54G TTRs than in wild-type TTR, suggesting decreased T(4) binding to the TTR variants. These findings indicate that structural modification by Glu54 point mutation may sufficiently alter tetramer stability and T(4) binding.

Albumin domain II mutant with high bilirubin binding affinity has a great potential as serum bilirubin excretion enhancer for hyperbilirubinemia treatment

BACKGROUND 4Z,15Z-bilirubin-IXα (BR), an endogenous toxic compound that is sparingly soluble in water, binds human serum albumin (HSA) with high affinity in a flexible manner. Our previous findings suggest that both Lys195 and Lys199 in subdomain IIA are important for the high-affinity binding of BR, and especially Lys199 in stand-alone domain II plays a prominent role in the renal elimination of BR. Our hypothesis is that HSA-domain II with high BR binding would be a useful therapeutic agent to treat hyperbilirubinemia in patients with impaired liver function. METHODS Unbound BR concentrations were determined using a modified HRP assay. To evaluate the effect of pan3_3-13 domain II mutant in promoting urinary BR excretion, the serum concentration and urinary excretion amount of BR were determined using bile duct ligation mice. RESULTS After three or six rounds of panning, pan3_3-13 and pan6_4 were found to have a significantly higher affinity for BR than wild-type domain II. Administration of pan3_3-13 significantly reduced serum BR level and increased its urinary excretion in the disease model mice as compared to wild-type domain II treatment. CONCLUSIONS These results suggest that pan3_3-13 has great potential as a therapeutic agent that promotes urinary BR excretion in hyperbilirubinemia. GENERAL SIGNIFICANCE This is the first study to be applied to other HSA bound toxic compounds that are responsible for the progression of disease, thereby paving the way for the development of non-invasive and cost effective blood purification treatment methods.

Role of the mobility of antigen binding site in high affinity antibody elucidated by surface plasmon resonance

Antibody is known to exhibit conformational change in the antigen recognition site after forming the initial complex. This structural change, which is widely known as “induced fit”, is believed to be critical for high affinity (Kd of nM range) of antigen-antibody interaction. Elucidation of this ‘induced fit’ process is essential for rational design of high affinity antibody, while it is prevented by limitation of the available biophysical and biochemical data of the initial complex. Here, we performed kinetic and thermodynamic analysis of the interaction between single-chain variable fragment (denoted as scFv) of 64M5 antibody and a (6-4) photoproduct by using surface plasmon resonance (denoted as SPR). It revealed that the 64M5scFv associates the (6-4) photoproduct at initial step by hydrophobic interactions, and enthalpy-driving interactions, hydrogen bonds and van der Waals interactions, were formed by second step structural rearrangement. Furthermore, mutational analysis revealed that the mobility of the antigen-binding site is critical for the second step. It could be assumed that optimization of the mobility of the antigen recognition site is a clue for rational design of high affinity antibody.

Cross-Neutralization Activity of Single-Chain Variable Fragment (scFv) Derived from Anti-V3 Monoclonal Antibodies Mediated by Post-Attachment Binding.

The V3 loop in the envelope (Env) of HIV-1 is one of the major targets of neutralizing antibodies. However, this antigen is hidden inside the Env trimer in most isolates and is fully exposed only during CD4-gp120 interaction. Thus, primary HIV-1 isolates are relatively resistant to anti-V3 antibodies because IgG is too large to access the V3 loop. To overcome this obstacle, we constructed single-chain variable fragments (scFvs) from anti-V3 monoclonal antibodies 0.5γ, 5G2, and 16G6. Enhanced neutralization by 0.5γ and 5G2 scFvs was observed in strains resistant to their IgG counterparts. Neutralization coverage by 0.5γ scFv reached up to 90% of the tested viruses (tier 2 and 3 classes). The temperature-regulated neutralization assay revealed that extensive cross-neutralization of 0.5γ scFv can be explained by post-attachment neutralization. Neutralization assay involving viruses carrying an inter-subunit disulfide bond (SOS virus) showed that the neutralization-susceptible timeframe after attachment was 60 to 120 min. These results indicate that the scFvs efficiently access the V3 loop and subsequently neutralize HIV-1, even after virus attachment to the target cells. Based on its broad and potent neutralizing activity, further development of anti-V3 scFv for therapeutic and preventive strategies is warranted.

Single-chain variable fragment technology in forensic toxicological analysis: production of an antibody to fluvoxamine

Immunoassay techniques are widely used for drug screening in the fields of forensic toxicology and emergency medicine, because of their simple procedures and rapid outcome of results [1, 2]. To create an immunoassay method for a compound, the most laborious step is the production of an antibody that is specific to the compound. Here, we present a new recombinant antibody technology for producing a single-chain variable fragment (scFv). scFv is a small antibody molecule that retains high antigen specificity and binding activity of the original whole immunoglobulin [3]. One advantage of scFv is its simple production using bacterial cell cultures, which also provide variable functionalities by introducing mutations. Furthermore, creating scFv phage libraries, which display various scFvs, might be useful in searching for drug-reactive elements. This seems to be a powerful tool for rapid creation of new antidrug antibodies without immunizing animals. Recently, the frequency of psychotropic drug poisoning has increased in Japan [4]. Among such drugs, fluvoxamine (FLV), one of the selective serotonin reuptake inhibitor (SSRI) antidepressant drugs, is widely used for treatment of depression and obsessive compulsive disorders [5]. Although SSRIs are relatively safe as compared with conventional antidepressant drugs, such as tricyclic antidepressants and monoamine oxidase inhibitors [6], the number of FLV poisoning cases has increased according to the increase in its prescription [7, 8]. In this study, we created an anti-FLV scFv using novel recombinant antibody technology. FLV malate was obtained from Sigma-Aldrich (St. Louis, MO, USA). Mercaptosuccinyl bovine serum albumin (MS-BSA) was a generous gift from Prof. K. Fujiwara, Sojo University (Kumamoto, Japan). All other solvents and chemicals were of analytical grade, and purchased through local suppliers. BALB/c mice (female, 4 weeks old; Kyudo, Kumamoto, Japan) were maintained in the Center for Animal Resources and Development, Kumamoto University, Kumamoto, Japan, and were kept in an environmentally controlled room (22 ± 2 C, 50–70 % humidity, illuminated from 0700 to 1900 hours). All procedures were approved by the Kumamoto University Ethics Review Committee for Animal Experimentation. The immunogen (BSA–FLV) for induction of anti-FLV antibodies was prepared as described previously with a slight modification [9]. Briefly, FLV maleate (4.3 mg; approximately 10 lmol) in 1.0 ml of 50 mM phosphate buffer (pH 7.0) was mixed with N-[c-maleimidobutyryloxy] succinimide (GMBS) (0.25 mg; approximately 0.89 lmol), and incubated at room temperature with stirring. MS-BSA, estimated to contain 18 thiol groups per BSA molecule, was diluted with 3.0 ml of 0.1 M phosphate buffer (pH 7.0), and added immediately to the This article is for the special issue TIAFT2012 edited by Osamu Suzuki.

Preparation, crystallization and preliminary X-ray diffraction analysis of the DNA-binding domain of the Ets transcription factor in complex with target DNA.

The Ets2 transcription factor is a member of the Ets transcription-factor family. Ets2 plays a role in the malignancy of cancer and in Downs syndrome by regulating the transcription of various genes. The DNA-binding domain of Ets2 (Ets domain; ETSD), which contains residues that are highly conserved among Ets transcription-factor family members, was expressed as a GST-fusion protein. The aggregation of ETSD produced after thrombin cleavage could be prevented by treatment with NDSB-195 (nondetergent sulfobetaine 195). ETSD was crystallized in complex with DNA containing the Ets2 target sequence (GGAA) by the hanging-drop vapour-diffusion method. The best crystals were grown using 25% PEG 3350, 80 mM magnesium acetate, 50 mM sodium cacodylate pH 5.0/5.5 as the reservoir at 293 K. The crystals belonged to space group C2, with unit-cell parameters a = 85.89, b = 95.52, c = 71.89 A, beta = 101.7 degrees and a V(M) value of 3.56 A(3) Da(-1). Diffraction data were collected to a resolution of 3.0 A.

Production of Single-Chain Fv Antibodies Specific for GA-Pyridine, an Advanced Glycation End-Product (AGE), with Reduced Inter-Domain Motion

Due to their lower production cost compared with monoclonal antibodies, single-chain variable fragments (scFvs) have potential for use in several applications, such as for diagnosis and treatment of a range of diseases, and as sensor elements. However, the usefulness of scFvs is limited by inhomogeneity through the formation of dimers, trimers, and larger oligomers. The scFv protein is assumed to be in equilibrium between the closed and open states formed by assembly or disassembly of VH and VL domains. Therefore, the production of an scFv with equilibrium biased to the closed state would be critical to overcome the problem in inhomogeneity of scFv for industrial or therapeutic applications. In this study, we obtained scFv clones stable against GA-pyridine, an advanced glycation end-product (AGE), by using a combination of a phage display system and random mutagenesis. Executing the bio-panning at 37 °C markedly improved the stability of scFvs. We further evaluated the radius of gyration by small-angle X-ray scattering (SAXS), obtained compact clones, and also visualized open–close dynamics of these scFvs by high-speed atomic force microscopy (HS-AFM), revealing that one of the compact clones was biased to the closed state. Finally, nuclear magnetic resonance (NMR) analysis revealed that peak intensity and line width became homogeneous, supporting that dynamic features and/or formation of oligomers was improved in the thus-obtained clone. These findings should contribute to the future industrial and therapeutic use of scFvs.