Replacement of residues 8-22 of angiogenin with 7-21 of RNase A selectively affects protein synthesis inhibition and angiogenesis.
The region of human angiogenin containing residues 8-21 is highly conserved in angiogenins from four mammalian species but differs substantially from the corresponding region of the homologous protein ribonuclease A (RNase A). Regional mutagenesis has been employed to replace this segment of angiogenin with the corresponding RNase A sequence, and the activities of the resulting covalent angiogenin/RNase hybrid, designated ARH-III, have been examined. The ribonucleolytic activity of ARH-III is unchanged toward most substrates, including tRNA, naked 18S and 28S rRNA, CpA, CpG, UpA, and UpG. In contrast, the capacity of ARH-III to inhibit cell-free protein synthesis is decreased 20-30-fold compared to that of angiogenin. The angiogenic activity of ARH-III is also different; it is actually more potent. It induces a maximal response in the chick chorioallantoic membrane assay at 0.1 ng per egg, a 10-fold lower dose than required for angiogenin. In addition, binding of ARH-III to the placental ribonuclease inhibitor is increased by at least 1 order of magnitude (Ki less than or equal to 7 x 10(-17) M) compared to angiogenin. Thus, mutation of a highly conserved region of angiogenin markedly affects those properties likely involved in its biological function(s); it does not, however, alter ribonucleolytic activity toward most substrates.
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009389:Neovascularization, Pathologic; D014176:Protein Biosynthesis; D011487:Protein Conformation; D015202:Protein Engineering; D011506:Proteins / Q000235:genetics*; D012259:Ribonuclease, Pancreatic / Q000494:pharmacology*
D011506:Proteins; C046366:angiogenin; D012259:Ribonuclease, Pancreatic