Erythropoietin Structure-Function Relationships: High Degree of Sequence Homology Among Mammals

• Published in: Blood 1993-09, Vol.82 (5), p.1507-1516
• Main Authors: Wen, Danyi, Boissel, Jean-Paul R., Tracy, Timothy E., Gruninger, Robert H., Mulcahy, Linda S., Czelusniak, John, Goodman, Morris, Bunn, H. Franklin
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Biological and medical sciences; Cats; Cell differentiation, maturation, development, hematopoiesis; Cell physiology; Cricetinae; DNA - analysis; Dogs; Dolphins; Erythropoietin - chemistry; Erythropoietin - genetics; Exons; Fundamental and applied biological sciences. Psychology; Gene Amplification; Horses; Humans; Lions; Macaca mulatta; Molecular and cellular biology; Molecular Sequence Data; Polymerase Chain Reaction; Rats; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Sheep; Structure-Activity Relationship; Swine
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V82.5.1507.1507

To investigate structure-function relationships of erythropoietin (Epo), we have obtained cDNA sequences that encode the mature Epo protein of a variety of mammals. A first set of primers, corresponding to conserved nucleotide sequences between mouse and human DNAs, allowed us to amplify by polymerase chain reaction (PCR) intron 1/ exon 2 fragments from genomic DNA of the hamster, cat, lion, dog, horse, sheep, dolphin, and pig. Sequencing of these fragments permitted the design of a second generation of species-specific primers. RIMA was prepared from anemic kidneys and reverse-transcribed. Using our battery of species-specific 5’ primers, we were able to successfully PCR-amplify Epo cDNA from Rhesus monkey, rat, sheep, dog, cat, and pig. Deduced amino acid sequences of mature Epo proteins from these animals, in combination with known sequences for human, Cynomolgus monkey, and mouse, showed a high degree of homology, which explains the biologic and immunological cross-reactivity that has been observed in a number of species. Human Epo is 91 % identical to monkey Epo, 85% to cat and dog Epo, and 80% to 82% to pig, sheep, mouse, and rat Epos. There was full conservation of (1) the disulfide bridge linking the NH2 and COOH termini; (2) N-glycosylation sites; and (3) predicted amphipathic α-helices. In contrast, the short disulfide bridge (C29/C33 in humans) is not invariant. Cys33 was replaced by a Pro in rodents. Most of the amino acid replacements were conservative. The C-terminal part of the loop between the C and D helices showed the most variation, with several amino acid substitutions, deletions, and/or insertions. Calculations of maximum parsimony for intron 1/exon 2 sequences as well as coding sequences enabled the construction of cladograms that are in good agreement with known phylogenetic relationships.