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Takasaki, Japan

Kuwada T.,Nihon University | Hasegawa T.,Rigaku Corporation | Takagi T.,Peptide Door Co. | Sakae T.,Nihon University | And 2 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2011

Monomeric haemoglobin component V (Hb V) from the larva of the midge Propsilocerus akamusi shows high Cl - affinity under high salt concentrations at acidic pH. In order to understand the structural changes that depend on Cl - binding, crystal structures of Hb V were determined under acidic high-salt conditions and the structural changes arising from different haem-bound ligands were simulated. Crystal structures of Hb V under acidic high-salt conditions indicated that the side chain of ArgE10 on the distal face of the haem contributes to stabilizing haem-bound Cl -. The conformation of the Arg side chain in the Cl --bound form was almost identical to that in ligated Hb V at neutral pH but not to that in met Hb V under acidic salt-free conditions. Furthermore, preliminary molecular-dynamics simulations also indicated that the swinging of the Arg side chain into the haem pocket depends on Cl - ligation. This result suggests that, like pH change, Cl - binding affects the location of the distal Arg residue. Owing to the increased positive electrostatic potential observed in the haem pocket at acidic pH, it was concluded that electrostatic changes caused by pH change and anionic ligand binding may affect the behaviour of the polar Arg residue. © 2011 International Union of Crystallography. Source


Suzuki M.M.,Peptide Door Co. | Matsumoto M.,Peptide Door Co. | Yamamoto A.,Japan National Institute of Infectious Diseases | Ochiai M.,Japan National Institute of Infectious Diseases | And 5 more authors.
Journal of Microbiological Methods | Year: 2010

Lipopolysaccharide (LPS), a major constituent of the outer membrane of Gram-negative bacteria, is highly toxic and can cause sepsis or septic shock. Therefore, detection of LPS and the ability to neutralize its toxicity is important. We previously obtained a strong LPS-binding peptide, Li5-001, using the phage display method (Matsumoto et al., 2010. J. Microbiol. Methods. 82, 54-58). We modified the sequence the amino acid sequence of this peptide (KNYSSSISSIHAC), by replacing and deleting amino acids to obtain higher LPS-binding affinity and greater resistance to protease digestion. Consequently we obtained a dodecapeptide, Li5-025 (K'YSSSISSIRAC', K' and C' are D-forms of K and C, respectively) which showed a high affinity for LPS, approximately 1000 folds higher affinity than Li5-001 and Kd value of 0.01 nM. By replacing both N- and C-terminal amino acids from L-type to D-type, the peptide was rendered resistant to protease digestion without altering its overall binding capacity. © 2010 Elsevier B.V. Source


Matsumoto M.,Peptide Door Co. | Horiuchi Y.,National Institute of Infection Disease | Yamamoto A.,National Institute of Infection Disease | Ochiai M.,National Institute of Infection Disease | And 4 more authors.
Journal of Microbiological Methods | Year: 2010

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. It has strong toxicity and might cause sepsis or septic shock. Thus early detection of LPS and neutralization of LPS toxicity are required. We obtained several new LPS-binding peptides using a phage display method. We synthesized 3 of these peptides and analyzed their binding affinity and capacity to LPS. One of these peptides, named Li5-001, showed high binding affinity to LPS and lipid A; the Kd values were 10 and 1nM, respectively. Li5-001 showed a high binding capacity to LPS, and was estimated to bind 130ng LPS/mg, which is higher than that of polymyxin B (80ng LPS/mg); however, its LPS-neutralizing activity was low. Li5-001 coupled with beads will be useful for eliminating endotoxin contamination from pharmaceuticals. Its low LPS-neutralizing activity allows to be used in the Limulus amebocyte lysate test without eluting LPS from the Li5-001 coupled beads. © 2010 Elsevier B.V. Source


Kuwada T.,Nihon University | Hasegawa T.,Rigaku Corporation | Takagi T.,Tohoku University | Takagi T.,Peptide Door Co. | And 2 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2010

Haemoglobin component V (Hb V) from the midge larva Propsilocerus akamusi exhibits oxygen affinity despite the replacement of HisE7 and a pH-dependence of its functional properties. In order to understand the contribution of the distal residue to the ligand-binding properties and the pH-dependent structural changes in this insect Hb, the crystal structure of Hb V was determined under five different pH conditions. Structural comparisons of these Hb structures indicated that at neutral pH ArgE10 contributes to the stabilization of the haem-bound ligand molecule as a functional substitute for the nonpolar E7 residue. However, ArgE10 does not contribute to stabilization at acidic and alkaline pH because of the swinging movement of the Arg side chain under these conditions. This pH-dependent behaviour of Arg results in significant differences in the hydrogen-bond network on the distal side of the haem in the Hb V structures at different pH values. Furthermore, the change in pH results in a partial movement of the F helix, considering that coupled movements of ArgE10 and the F helix determine the haem location at each pH. These results suggested that Hb V retains its functional properties by adapting to the structural changes caused by amino-acid replacements. © 2010 International Union of Crystallography. Source


Suzuki M.M.,Peptide Door Co. | Matsumoto M.,Peptide Door Co. | Omi H.,Peptide Door Co. | Kobayashi T.,Peptide Door Co. | And 4 more authors.
Journal of Microbiological Methods | Year: 2014

We previously reported the generation of lipopolysaccharide (LPS)-binding peptides by phage display and chemical modification. Among them, a dodecapeptide designated Li5-025 (K'YSSSISSIRAC'; K' and C' denote d-lysine and d-cysteine, respectively) showed a high binding affinity for LPS and was resistant to protease digestion (Suzuki et al., 2010). In the current study, Li5-025-bound silica beads, hereafter referred to as P-beads, were generated and found to be devoid of LPS-neutralizing activity. Thus, LPS bound to the P-beads could be directly used in the Limulus amebocyte lysate (LAL) assay. P-beads bound LPS dissolved in solutions of ethanol, pH. 4, pH. 10, and 0.5. M NaCl and LPS bound to the P-beads was quantitatively assayed. The sensitivity of this assay was observed to be approximately 0.1. pg/mL LPS. P-beads bound LPS dissolved in antithrombin III (AT III) solution which is a strong inhibitor of activated factors C and B as well as the clotting enzyme in the LAL assay; the inhibitory effect of AT III was completely reversed upon washing the P-beads with 25% acetonitrile. This was employed as the first step for the detection of free LPS in plasma using the LAL assay. LPS added to human plasma at 0. °C followed by application to the P-beads and subsequent washing with 25% acetonitrile resulted in low LPS activity as detected by the LAL assay. However, further washing of the P-beads with 0.1% Triton X100 in 25% acetonitrile resulted in high LPS activity. This is the first instance of quantitative detection of free LPS in plasma using the LAL assay, and the sensitivity of this method was observed to be 1. pg/mL of LPS. The proteins eluted in the 0.1% Triton X-100 wash were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Two protein bands of 28. kDa and 18. kDa were predominantly observed. Mass spectrometry analysis revealed that the 28. kDa and 18. kDa bands corresponded to apolipoprotein A-I (apoA-I) and apolipoprotein A-II (apoA-II), respectively. ApoA-I and apoA-II are components of high density lipoprotein (HDL). Thus, it is likely that the P-beads-bound LPS was sequestered by HDL, resulting in neutralization of its toxicity. This study showed that by using P-beads, free LPS in plasma can be quantitatively measured by the LAL assay at a concentration of 1. pg/mL. © 2014 Elsevier B.V. Source

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