Replacing a surface loop endows ribonuclease A with angiogenic activity.
Angiogenin (ANG) promotes the formation of blood vessels in animals. This hormone is a small, monomeric protein that is homologous to bovine pancreatic ribonuclease A (RNase). ANG is a poor ribonuclease but its ribonucleolytic activity is essential for its angiogenic activity. RNase is not angiogenic. A hybrid protein was produced in which 13 residues of a divergent surface loop of ANG were substituted for the analogous 15 residues of RNase. The value of kcat/Km for the cleavage of uridylyl(3'-->5')adenosine by this hybrid protein was 20-fold less than that of RNase but 10(5)-fold greater than that of ANG. The thermal stability of the hybrid protein was also less than that of RNase. Nevertheless, the RNase/ANG hybrid protein promotes angiogenesis in mice at least as extensively as does authentic ANG. Thus we present a protein endowed with a noncognate biological activity simply by replacing a single element of secondary structure. In addition, a 13-residue peptide corresponding to the surface loop of ANG inhibits endogenous angiogenesis in mice. These results support a model in which both a surface loop and a catalytic site are necessary for the promotion of blood vessel formation by ANG or RNase. The dissection of structure/function elements in ANG reveals a unique opportunity to develop new molecules that modulate neovascularization.
Raines
R T
RT
Department of Biochemistry, University of Wisconsin-Madison 53706, USA.
Toscano
M P
MP
Nierengarten
D M
DM
Ha
J H
JH
Auerbach
R
R
D000595:Amino Acid Sequence; D043925:Angiogenesis Inducing Agents / Q000737:chemistry*; D000818:Animals; D001483:Base Sequence; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000494:pharmacology; D013329:Structure-Activity Relationship
D043925:Angiogenesis Inducing Agents; D011506:Proteins; D011993:Recombinant Fusion Proteins; C046366:angiogenin; D012259:Ribonuclease, Pancreatic