Structural role of amino acids 99-110 in recombinant human erythropoietin.
Erythropoietin is the prime regulator of red blood cell production. Previous studies demonstrated that antipeptide antibodies to amino acids 99-119 and 111-129 bind to two non-overlapping domains and inhibit the hormone's action (Sytkowski, A.J. & Donahue, K. A. (1987) J. Biol. Chem. 262, 1161-1165). Oligonucleotide-directed mutagenesis now shows that amino acids 99-110 (domain 1) but not 119-129 (domain 2) are important to erythropoietin's structure and function. Mutagenesis of wild-type human erythropoietin cDNA was used to produce a series of mutant proteins with sequential deletion of three adjacent amino acids and insertion of the sequence Glu-Phe across the two domains. Transient expression in COS-7 cells revealed 2.0-kb transcripts encoded by all of the cDNAs. Domain 2 mutants exhibited specific biological activities similar to that of the wild type. In contrast, domain 1 mutants were not secreted. In vitro transcription and translation of the domain 1, domain 2 and wild-type cDNAs resulted in the isolation of 23.5-kDa and 32-kDa proteins in the absence or presence of pancreatic microsomes, respectively, consistent with efficient translation of all of the mutants and equivalent post-translational processing of each protein. The data suggest that mutation within domain 1 results in the intracellular biosynthesis of erythropoietins with altered structure, rendering them subject to rapid degradation. The bioassay of erythropoietins synthesized entirely in vitro demonstrated that domain 1 mutants were inactive, whereas both wild type and domain 2 mutant hormones exhibited biologic activity. The results are consistent with a critical role for amino acids 99-110 in the structure of human erythropoietin.
Chern
Y J
YJ
Laboratory for Cell and Molecular Biology, New England Deaconess Hospital, Boston, MA 02215.
Chung
T W
TW
Sytkowski
A J
AJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004921:Erythropoietin / Q000737:chemistry* / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D000596:Amino Acids; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004921:Erythropoietin; D004247:DNA