Evolution and Function of Leukocyte RNase A Ribonucleases of the Avian Species, Gallus gallus

• Published in: The Journal of biological chemistry 2006-09, Vol.281 (35), p.25622-25634
• Main Authors: Nitto, Takeaki, Dyer, Kimberly D., Czapiga, Meggan, Rosenberg, Helene F.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Bone Marrow Cells - metabolism; Chickens; Evolution, Molecular; Gallus gallus; Humans; Kinetics; Leukocytes - enzymology; Leukocytes - metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Phylogeny; Protein Structure, Tertiary; Ribonuclease, Pancreatic - chemistry; Sequence Homology, Amino Acid
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M604313200

In this study, we explore the evolution and function of two closely related RNase A ribonucleases from the chicken, Gallus gallus. Separated by ∼10 kb on chromosome 6, the coding sequences of RNases A-1 and A-2 are diverging under positive selection pressure (dN >dS) but remain similar to one another (81% amino acid identity) and to the mammalian angiogenins. Immunoreactive RNases A-1 and A-2 (both ∼16 kDa) were detected in peripheral blood granulocytes and bone marrow. Recombinant proteins are ribonucleolytically active (kcat = 2.6 and 0.056 s-1, respectively), and surprisingly, both interact with human placental ribonuclease inhibitor. RNase A-2, the more cationic (pI 11.0), is both angiogenic and bactericidal; RNase A-1 (pI 10.2) has neither activity. We demonstrated via point mutation of the catalytic His110 that ablation of ribonuclease activity has no impact on the bactericidal activity of RNase A-2. We determined that the divergent domains II (amino acids 71-76) and III (amino acids 89-104) of RNase A-2 are both important for bactericidal activity. Furthermore, we demonstrated that these cationic domains can function as independent bactericidal peptides without the tertiary structure imposed by the RNase A backbone. These results suggest that ribonucleolytic activity may not be a crucial constraint limiting the ongoing evolution of this gene family and that the ribonuclease backbone may be merely serving as a scaffold to support the evolution of novel, nonribonucleolytic proteins.