Erythropoietin (EPO) produced by the kidney and the liver (in fetuses) stimulates erythropoiesis. In the central nervous system, neurons express EPO receptor (EPOR) and astrocytes produce EPO. EPO has been shown to protect primary cultured neurons from N-methyl-D-aspartate (NMDA) receptor-mediated glutamate toxicity. Here we report in vivo evidence that EPO protects neurons against ischemia-induced cell death. Infusion of EPO into the lateral ventricles of gerbils prevented ischemia-induced learning disability and rescued hippocampal CA1 neurons from lethal ischemic damage. The neuroprotective action of exogenous EPO was also confirmed by counting synapses in the hippocampal CA1 region. Infusion of soluble EPOR (an extracellular domain capable of binding with the ligand) into animals given a mild ischemic treatment that did not produce neuronal damage, caused neuronal degeneration and impaired learning ability, whereas infusion of the heatdenatured soluble EPOR was not detrimental, demonstrating that the endogenous brain EPO is crucial for neuronal survival. The presence of EPO in neuron cultures did not repress a NMDA receptor-mediated increase in intracellular Ca 2؉ , but rescued the neurons from NO-induced death. Taken together EPO may exert its neuroprotective effect by reducing the NO-mediated formation of free radicals or antagonizing their toxicity.Mammals respond to oxygen deficiency in many different ways (1). One strategy for survival of the individual cells under hypoxic conditions is the induction of glycolytic enzymes, facilitating ATP production by glycolysis rather than mitochondrial oxidative phosphorylation. In response to the systemic oxygen deficiency due to anemia or decreasedenvironmental oxygen concentration, erythropoietin (EPO) production is stimulated. EPO is a glycoprotein that stimulates differentiation and proliferation of erythroid precursor cells, and hypoxic induction of EPO production increases red blood cells, leading to better oxygen supply to tissues (2, 3). The action of EPO is mediated by binding to the specific receptor that belongs to a new family of cytokine receptors that have no tyrosine kinase domain (4). EPO regulating erythropoiesis is mainly produced by the kidney in adults and by the liver at fetal stages (2, 3).Stimulation of red blood cell formation was thought to be the sole physiological function of EPO, but a different function in the central nervous system has been proposed (5-7). Neuronal cell lines such as PC12 and SN6 express EPO receptor (EPOR), and binding of EPO to PC12 cells increases the intracellular concentration of monoamines (8). Immunochemical staining with anti-EPOR antibody showed that EPOR is expressed in murine hippocampal and cerebral cortical areas, and also in primary cultured hippocampal and cortical neurons (6, 9). With the use of radioiodinated EPO, specific EPO binding sites were found in some defined areas of the murine brain including the hippocampus and cerebral cortex (10). Because the blood-brain barrier prevents neurons fr...

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Vitelline membrane outer layer protein I (VMO-I) tightly bound to ovomucin fibrils of hen's egg yolk membrane was characterized in terms of its amino acid sequence and structural stability. The deduced sequence of VMO-I using the conventional sequencing method is: RTREYTSVITVPNGGHWGKWGIRQFCHSGYANGFALKVEPSQFGRDDTALNGIRLRCLD- GSVIESLVGKWGTWTSFLVCPTGYLVSFSLRSEKSQGGGDDTAANNIQFRCSDEAVLVGD- DLSWGRFGPWSKRCKICGLQTKVESPQGLRDDTALNNVRFFCCK. Thus, VMO-I is composed of 163 amino acid residues with a calculated molecular weight of 17,979. The sequence confirms the cDNA sequence of VMO-I we recently determined and does not show any significant similarity to proteins compiled in the NBRF database. Two of the four disulfide bonds found in VMO-I were estimated to lie between Cys26 and Cys57 and between Cys79 and Cys110. The sequence analyses show that VMO-I contains three 53-residue internal repeats that contain distinctive regions of turns flanked by beta-sheets consistent with the recent finding that the molecule contains a new beta-fold motif, the beta-prism. The molecular characteristics of VMO-I in solution were examined by CD spectroscopy in the far and near ultraviolet regions, NMR spectroscopy, and high sensitive differential scanning calorimetry (DSC). CD spectra in the far UV region at room temperature were similar to that assigned to a random coil, while in the near UV region, small positive peaks were observed. The ellipticity in both regions decreased on raising the temperature. Proton NMR experiments showed the native structure unfolds to unordered conformations at 70 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

A hybridoma cell line producing the monoclonal antibody against erythropoietin receptor (EpoR) was established using the soluble ectodomain of mouse erythropoietin receptor (sEpoR) as an antigen. The monoclonal antibody termed 1G3 bound to the denatured sEpoR. Epitope mapping with peptide library revealed that 1G3 recognized the amino terminal region including the hexapeptide (positions 6 to 11; LeuProAspProLysPhe). The amino acid sequence in this hexapeptide was identical in mice, rats, and humans, and therefore 1G3 bound to EpoR from all of these sources. Using 1G3, we evaluated sEpoR by a sandwich enzyme-linked immunoassay, and EpoR in the solubilized membrane preparation was detected by Western blotting. The cells expressing EpoR were identified with immunochemical staining. We confirmed the presence of EpoR in a neuronal cell line and PC12 cells, and EpoR was expressed in primary cultured hippocampal neurons.

. apacity by the epidermal growth factor receptor is glycosylaion-dependent [6,7]. N-Glycosylation at a specific site of the nsulin receptor fl subunit appears to be important for the eceptor activation and transmembrane signaling [4,5]. Degly-:osylation of the basic fibroblast growth factor receptor results n the loss of the ligand-binding [8] and sialylation of the Ninked sugars in the somatostatin receptor is required for mainenance of the high-affinity binding state [9].EPO transduces the proliferation and differentiation signal hrough its receptor on erythroid precursor cells and some )ther lineage cells [16,17]. By molecular cloning of murine 18,19] and human [20,21] EPOR, the mature 53-kDa EPOR ms been predicted to consist of an extracellular 225-226 amino lcid domain, 22 amino acid transmembrane region, and an ntracellular 236 amino acid domain. A single N-glycosylation ;ite is present in the extracellular domain in EPOR. Studies on "Corresponding author. Fax: (81) (75) 753-6274.4bbreviations: EPO, erythropoietin; EPOR, erythropoietin receptor; BHK, baby hamster kidney; IL-3, interleukin 3; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. the IL-3-dependent Ba/F3 cells that were stably transfected with the wild-type EPOR and became EPO-responsive indicated that the EPOR with 66 kDa was the mature glycosylated form exposed on the cell surface [22] and that the 72-kDa EPOR could be an EPO-induced phosphorylated form of 66-kDa EPOR [23,24]. BHK cells stably transfected with the wildtype EPOR cDNA and the N-glycosylation-defective mutant expressed 68-kDa and 66-kDa EPOR, respectively and the two forms of EPOR were not different in the ligand-binding assay [14]. More recently, Sawyer and Hankins [25] reported that an EPO-dependent murine erythroleukemia cell line, which was highly sensitive to EPO, expressed the 78-kDa high molecular mass form of EPOR and that the 78-kDa EPOR was a functional form of EPOR because it was correlated well with cell surface expression, endocytosis and EPO-induced phosphorylation. They also indicated that the 78-kDa EPOR resulted from high N-glycosylation rather than phosphorylation, suggesting the importance of N-glycosylation for expression of the functional EPOR or the ligand-sensitive EPOR. These findings of EPOR prompted us to examine the role of the receptor glycosylation using the N-glycosylation-defective EPOR. Herein, we prepared EPO-dependent cell lines expressing wild-type or mutant EPOR, and compared the ligand-saturation curves with respect to EPO binding and EPO-dependent cell proliferation. Materials and methods I. EPO-dependent cells expressing mouse wild-o,pe EPOR and Nglveosylation-defective EPORThe plasmid for expression of N-glycosylation-defective EPOR (Asn~Gln) was prepared [14] from the plasmid for expression of wild-type EPOR (pXM 190) [18]. IL-3-dependent BaF-BO3 cells, a subclone of the mouse pro-B cell line (Ba/F3), were maintained in the medium consisting of RPMI1640, 10% fetal calf serum and 10% WEHI3B conditioned medium as a sourc...