Satoshi Abe

Production and ligand-binding characteristics of the soluble form of murine erythropoietin receptor.

A recombinant soluble form (sEPO-R) of erythropoietin (EPO) receptor (EPO-R) was produced by Chinese hamster ovary cells and isolated in high yield with the EPO-fixed gel. Ligand binding assays were done using three methods; precipitation of sEPO-R radiolabeled EPO complex and competition of sEPO-R for the binding of radiolabeled EPO with the cellular EPO-R. The results showed a Kd of 17 nM which was much lower than those for cellular EPO-R. One N-glycosylation site exists in sEPO-R but the glycosylation did not affect the binding affinity to EPO. A complex with a molecular size that corresponded to a 1:1 complex of EPO and sEPO-R was detected.

Porous protein crystals as reaction vessels for controlling magnetic properties of nanoparticles

Magnetic bimetallic CoPt nanoparticles are synthesized in the solvent channels of hen egg white lysozyme crystals by the reduction of Co(2+) and Pt(2+) ions pre-organized on the interior surface of the solvent channels. By using different lysozyme crystal systems, the magnetic properties of CoPt nanoparticles can be controlled.

Elucidation of Metal‐Ion Accumulation Induced by Hydrogen Bonds on Protein Surfaces by Using Porous Lysozyme Crystals Containing RhIII Ions as the Model Surfaces

Metal-ion accumulation on protein surfaces is a crucial step in the initiation of small-metal clusters and the formation of inorganic materials in nature. This event is expected to control the nucleation, growth, and position of the materials. There remain many unknowns, as to how proteins affect the initial process at the atomic level, although multistep assembly processes of the materials formation by both native and model systems have been clarified at the macroscopic level. Herein the cooperative effects of amino acids and hydrogen bonds promoting metal accumulation reactions are clarified by using porous hen egg white lysozyme (HEWL) crystals containing Rh(III) ions, as model protein surfaces for the reactions. The experimental results reveal noteworthy implications for initiation of metal accumulation, which involve highly cooperative dynamics of amino acids and hydrogen bonds: i) Disruption of hydrogen bonds can induce conformational changes of amino-acid residues to capture Rh(III) ions. ii) Water molecules pre-organized by hydrogen bonds can stabilize Rh(III) coordination as aqua ligands. iii) Water molecules participating in hydrogen bonds with amino-acid residues can be replaced by Rh(III) ions to form polynuclear structures with the residues. iv) Rh(III) aqua complexes are retained on amino-acid residues through stabilizing hydrogen bonds even at low pH (approximately 2). These metal-protein interactions including hydrogen bonds may promote native metal accumulation reactions and also may be useful in the preparation of new inorganic materials that incorporate proteins.

Artificial Metalloproteins Exploiting Vacant Space: Preparation, Structures, and Functions

Molecular design of artificial metalloproteins is one of the most attractive subjects in bioinorganic chemistry. Protein vacant space has been utilized to prepare artificial metalloproteins because it provides a unique chemical environment for application to catalysts and to biomaterials bearing electronic, magnetic, and medical properties. Recently, X-ray crystal structural analysis has increased in this research area because it is a powerful tool for understanding the interactions of metal complexes and protein scaffolds, and for providing rational design of these composites. This chapter reviews the recent studies on the preparation methods and X-ray crystal structural analyses of metal/protein composites, and their functions as catalysts, metal-drugs, etc.

An extra high dose of erythropoietin fails to support the proliferation of erythropoietin dependent cell lines

Erythropoietin is responsible for the red blood cell formation by stimulating the proliferation and the differentiation of erythroid precursor cells. Erythropoietin triggers the conformational change in its receptor thereby induces the phosphorylation of JAK2. In this study, we show that an extra high dose of erythropoietin, however, fails to activate the erythropoietin receptor, to stimulate the phosphorylation of JAK2 and to support the cell proliferation of Ep-FDC-P2 cell. Moreover, high dose of EPO also inhibited the proliferation of various erythropoietin-dependent cell lines, suggesting that excess amount of EPO could not trigger the conformational change of the receptor. In the presence of an extra high dose of erythropoietin as well as in the absence of erythropoietin, the cells caused the DNA fragmentation, a typical symptom of apoptosis. The impairment of cell growth and the DNA fragmentation at the extremely high concentration of EPO was rescued by the addition of erythropoietin antibody or soluble form of erythropoietin receptor by titrating the excess erythropoietin. These results suggest that two erythropoietin binding sites on erythropoietin receptor dimer should be occupied by a single erythropoietin molecule for the proper conformational change of the receptor and the signal transduction of erythropoietin, instead, when two erythropoietin binding sites on the receptor are shared by two erythropoietin molecules, it fails to evoke the conformational change of erythropoietin receptor adequate for signal transduction.

Production and Characterization of Recombinant Soluble form Erythropoietin Receptor

A recombinant soluble form (sEPO-R) of erythropoietin (EPO) receptor (EPO-R) was produced by Chinese hamster ovary cells. One subclone, N14.2 could produce sEPO-R more than 40µg/106cells/day maximally. sEPO-R was isolated easily with EPO fixed gel in a high yield. Affinity of sEPO-R to EPO was determined by precipitating sEPO-R·radiolabeled EPO complex using anti EPO-R antibody and polyethylene glycol. The results showed a Kd of 13 nM which was much lower than those for cellular EPO-R. One N-glycosylation site exists in sEPO-R but the glycosylation did not affect the binding affinity to EPO. A complex with a molecular size that corresponded to a 1:1 complex of EPO and sEPO-R was detected by gel filtration analysis.