Mutagenesis of residues flanking Lys-40 enhances the enzymatic activity and reduces the angiogenic potency of angiogenin.
The primary structure of the blood vessel inducing protein angiogenin is 35% identical with that of pancreatic ribonuclease (RNase) and contains counterparts for the critical RNase active-site residues His-12, Lys-41, and His-119. Although angiogenin is a ribonucleolytic enzyme, its activity toward conventional substrates is lower than that of pancreatic RNase by several orders of magnitude. Comparison of the amino acid sequences of RNase and angiogenin reveals several striking differences in the region flanking the active-site lysine, including a deletion and a transposition of aspartic acid and proline residues. In order to examine how these sequence changes alter the functional properties of angiogenin, an angiogenin/RNase hybrid protein (ARH-II), in which residues 38-41 of angiogenin (Pro-Cys-Lys-Asp) have been replaced by the corresponding segment of bovine pancreatic RNase (Asp-Arg-Cys-Lys-Pro), was prepared by regional mutagenesis. Compared to angiogenin, ARH-II has markedly diminished angiogenic activity on the chick embryo chorioallantoic membrane but 5-75-fold greater enzymatic activity toward a variety of polynucleotide and dinucleotide substrates. In addition, the specificity of ARH-II toward dinucleotide substrates differs from that of angiogenin and is qualitatively similar to that of pancreatic RNase. Thus, non-active-site residues near Lys-40 in angiogenin appear to play a significant role in determining enzymatic specificity and reactivity as well as angiogenic potency. An additional angiogenin/RNase hybrid protein (ARH-IV), in which residues 59-71 of ARH-II have been replaced by the corresponding segment of pancreatic RNase, was also prepared.(ABSTRACT TRUNCATED AT 250 WORDS)
Harper
J W
JW
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115.
Fox
E A
EA
Shapiro
R
R
Vallee
B L
BL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002642:Chick Embryo; D005813:Genes, Synthetic; D006801:Humans; D008969:Molecular Sequence Data; D009389:Neovascularization, Pathologic; D015202:Protein Engineering; D011506:Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D011506:Proteins; D011993:Recombinant Fusion Proteins; C046366:angiogenin; D012259:Ribonuclease, Pancreatic