Erythropoietin (Ep) is the glycoprotein hormone which is responsible for maintaining erythropoiesis in animals and man. Of many putative growth factors affecting hematopoiesis, Ep is the only one with a defined physiological role. However, understanding of the mechanisms of Ep's action on responsive cells has been limited due to 1) insufficient quantities of pure Ep, 2) lack of appropriate laboratory probes and 3) difficulties in obtaining sufficiently enriched populations of target cells for study. We have purified Ep in microgram amounts and we propose to utilize recent developments in molecular and physiological interactions to study the biochemistry of Ep and its molecular and cellular-biology with responsive cells. We will use monoclonal and polyvalent antibodies to Ep and to defined polypeptide sequences to further purify Ep by immunoaffinity chromatography. As larger quantities of Ep become available, we will determine its biochemical properties and amino acid sequence. We will develop site-specific antibodies to Ep, both polyvalent and monoclonal, to immunologically characterize the molecule. Functional properties will be correlated with antibody-defined structural domains. We will clone the Ep coding sequences from anemic baboon kidney. The cDNA libraries will be screened by immunological methods using anti-Ep antibodies and oligonucleotide probes developed from amino acid sequence data. As the protein sequence is determined, it will be compared to the nucleotide sequences for Ep coding regions. Concurrently, we will attempt to develop labeled, biologically active Ep probes using different iodination schemes, tritium labeling of carbohydrate and amino groups, photoaffinity labeling, and chemical derivatization. Alternatively, we will use labeled or flouresceinated antibody or Fab fragments to identify Ep. With the acquisition of additional purified Ep as well as biologically active probes we will purify populations of Ep-responsive cells and begin studies to identify the Ep receptor and its properties. From the sequence of experiments outlined in this proposal we can expect greater understanding of Ep, its protein structure, its genomic coding sequences, identification of target cells bearing presumed receptors and characterization of Ep interactions with the receptor. These studies may serve as a model for understanding the function of other hematopoietic growth factors and, ultimately, may lead to therapeutic benefit in disorders accompanied by Ep deficiency.
| Broudy, V C; Tait, J F; Powell, J S (1988) Recombinant human erythropoietin: purification and analysis of carbohydrate linkage. Arch Biochem Biophys 265:329-36 |
