Site-directed mutagenesis of histidine-13 and histidine-114 of human angiogenin. Alanine derivatives inhibit angiogenin-induced angiogenesis.
The roles of His-13 and His-114 in the ribonucleolytic and angiogenic activities of human angiogenin have been investigated by site-directed mutagenesis. Replacement of either residue by alanine (H13A and H114A) decreases enzymatic activity toward tRNA by at least 10,000-fold and virtually abolishes 10,000-fold and virtually abolishes angiogenic activity in the chick embryo chorioallantoic membrane assay. Both the H13A and H114A mutant proteins compete effectively with angiogenin in the latter assay; only a 5-fold molar excess of H13A over unmodified protein is required for complete inhibition. The His----Ala substitutions, however, do not have any significant effect on the interaction of angiogenin with human placental ribonuclease inhibitor, an extremely potent inhibitor of angiogenin (Ki approximately 7 x 10(-16 M) previously shown to interact with another active-site residue, Lys-40. The effects of more conservative replacements-glutamine at position 13 and asparagine at position 114--were also examined. While the enzymatic activity of the H114N mutant was at least 3300-fold less than for the unmodified protein, the H13Q derivative had only 300-fold reduced activity toward tRNA and cytidylyl(3'----5') adenosine. Both substitutions substantially decreased angiogenic activity. The parallel effects on ribonucleolytic and biological activities observed with all four mutant proteins provide strong evidence that the latter activity of angiogenin is dependent on a functional enzymatic active site. The capacity of the H13A and H114A derivatives to compete with angiogenin in the chorioallantoic membrane assay suggests several additional features of the biological mode of action of this protein.
Shapiro
R
R
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115.
Vallee
B L
BL
D000409:Alanine / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D043925:Angiogenesis Inducing Agents / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002642:Chick Embryo; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000235:genetics; D015226:Dinucleoside Phosphates / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006133:Growth Substances / Q000235:genetics*; D006639:Histidine / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009154:Mutation; D009389:Neovascularization, Pathologic; D011506:Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D012354:RNA, Transfer, His / Q000378:metabolism; D012259:Ribonuclease, Pancreatic; D012260:Ribonucleases / Q000037:antagonists & inhibitors
D000596:Amino Acids; D043925:Angiogenesis Inducing Agents; D015226:Dinucleoside Phosphates; D006133:Growth Substances; D011506:Proteins; D012354:RNA, Transfer, His; C013907:cytidylyl adenosine; D006639:Histidine; D004247:DNA; D012260:Ribonucleases; C046366:angiogenin; D012259:Ribonuclease, Pancreatic; D000409:Alanine