Importance of asparagine-61 and asparagine-109 to the angiogenic activity of human angiogenin

• Published in: Biochemistry (Easton) Vol. 31; no. 34; pp. 8022 - 8029
• Main Authors: Hallahan, Terrence W, Shapiro, Robert, Strydom, Daniel J, Vallee, Bert L
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01-09-1992
• Subjects: activity; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; angiogenin; Animals; asparagine; Asparagine - chemistry; Base Sequence; Binding Sites; Biological and medical sciences; Chick Embryo; Chromatography, High Pressure Liquid; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Hydrolases; man; Molecular Sequence Data; Molecular Structure; Mutagenesis, Site-Directed; Neovascularization, Pathologic; Peptide Fragments - chemistry; Peptide Mapping; Proteins - chemistry; Proteins - genetics; Proteins - pharmacology; Ribonuclease, Pancreatic; role; Structure-Activity Relationship; Human; Angiogenesis; Enzymatic activity; Structure activity relation; Enzyme; Site directed mutagenesis; Ribonuclease; In vitro
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00149a036

Two distinct regions of angiogenin are critical for angiogenic activity: a catalytic site capable of cleaving RNA and a noncatalytic site, encompassing residues 60-68, which may bind to an endothelial cell-surface receptor [Hallahan, T. W., Shapiro, R., & Vallee, B. L. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2222-2226]. We have now shown that Asn-61 is an essential residue within the cell-binding site and that in addition a segment containing Asn-109 is part of this site. Both asparagines undergo nonenzymatic deamidation during long-term storage or treatment at alkaline pH. While the isolated desamido-61 and desamido-109 derivatives retain nearly full enzymatic activity, their angiogenic activity on the chicken embryo chorioallantoic membrane is markedly attenuated and they do not inhibit angiogenin-induced neovascularization. Tryptic peptide mapping and Edman degradation demonstrate that the isolated deamidated derivatives primarily contain isoaspartic rather than aspartic acid at the positions in question (83% for desamido-61, greater than 99% for desamido-109). Aspartic acid replacement of Asn-61 and Asn-109 by site-directed mutagenesis results in the same ribonucleolytic and angiogenic activities as those of the spontaneous deamidation products. However, the aspartyl derivatives differ strikingly from their isoaspartyl counterparts in that they do inhibit angiogenin-induced angiogenesis. These results indicate that the combination of ribonucleolytic activity and receptor-binding capacity is not sufficient for angiogenic activity and that Asn-61 and Asn-109 within the noncatalytic site are required for some additional function, as yet undefined.