Role of lysines in human angiogenin: chemical modification and site-directed mutagenesis.
The role of lysines in the ribonucleolytic and angiogenic activities of human angiogenin has been examined by chemical modification and site-directed mutagenesis. It was demonstrated previously [Shapiro, R., Weremowicz, S., Riordan, J.F., & Vallee, B.L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8783-8787] that extensive treatment with lysine reagents markedly decreases the ribonucleolytic activity of angiogenin. In the present study, limited chemical modification with 1-fluoro-2,4-dinitrobenzene followed by C18 high-performance liquid chromatography yielded several (dinitrophenyl)angiogenin derivaties. The major derivative formed had slightly increased enzymatic activity compared with the unmodified protein. Tryptic peptide mapping demonstrated the site of modification to be Lys-50. A second derivative, modified at Lys-60, was 34% active. Analysis of a third derivative indicated that modification of Lys-82 did not decrease activity. Thus, Lys-50 and Lys-82 are unessential for enzymatic activity while Lys-60 may play a minor role. No pure derivative modified at Lys-40, corresponding to the active-site residue Lys-41 of the homologous protein ribonuclease A, could be obtained by chemical procedures. Therefore, we employed oligonucleotide-directed mutagenesis to replace this lysine with glutamine or arginine. The Gln-40 derivative had less than 0.05% enzymatic activity compared with the unmodified protein and substantially reduced angiogenic activity when examined with the chick embryo chorioallantoic membrane assay. These results suggest that the angiogenic activity of the protein is dependent on an intact enzymatic active site. The Arg-40 derivative had 2.2% ribonucleolytic activity compared with unmodified angiogenin. The effects of reductive methylation of this derivative indicate that no lysines other than Lys-40 are critical.(ABSTRACT TRUNCATED AT 250 WORDS)
Shapiro
R
R
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115.
Fox
E A
EA
Riordan
J F
JF
D000596:Amino Acids / Q000032:analysis; D043925:Angiogenesis Inducing Agents / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004139:Dinitrofluorobenzene / Q000494:pharmacology; D006133:Growth Substances / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012259:Ribonuclease, Pancreatic; D014357:Trypsin
D000596:Amino Acids; D043925:Angiogenesis Inducing Agents; D006133:Growth Substances; D010446:Peptide Fragments; D011506:Proteins; D011994:Recombinant Proteins; D004139:Dinitrofluorobenzene; C046366:angiogenin; D012259:Ribonuclease, Pancreatic; D014357:Trypsin; D008239:Lysine