Abstract
In this study, we explore the evolution and function of two closely related RNase A ribonucleases from the chicken, Gallus gallus. Separated by approximately 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 approximately 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.
Highlights
The RNase A ribonuclease gene family has been a tremendous source of information on unusual evolutionary constraints and their effects on protein structure and function at the molecular level
100 l Chicken, G. gallus—Three RNase A ribonucleases have been from a single bacterial colony of overnight culture grown in identified in the genome of the chicken, G. gallus [11]
Ten microliters of malian RNase A ribonucleases indicate the following: 1) these bacteria in buffer are incubated for 4 h at 37 °C with 10 l of three sequences are most closely related to one another; 2) they recombinant protein or peptide at the concentrations indicated are more closely related to the other known nonmammalian or diluent control and were diluted 10, 100, or 1000-fold RNase A ribonucleases than to any of the mammalian superprior to plating on LB agar for overnight growth for colony family members; and 3) together, the nonmammalian RNase counts
Summary
Isolation of Leukocytes from Bone Marrow—Bone marrow cells were collected from femurs and tibiae of White Leghorn chickens (Gallus gallus) by flushing sterile phosphate-buffered saline (PBS, pH 7.4) through opened bones. Red blood cells were sedimented, and the leukocyte-rich supernatant (buffy coat) was collected by centrifugation at 400 ϫ g, washed twice with PBS, and separated into phases via Lymphocyte Separation Medium (density,1.077–1.080 g/ml at 20 °C; Mediatech, Herndon, VA) at 400 ϫ g for 30 min at room temperature. Production of Recombinant Chicken Leukocyte RNases A-1 and A-2—Recombinant chicken RNase proteins were prepared in Escherichia coli BL21 strain using the pFLAG-CTS expression vector (Sigma), which utilizes an isopropyl 1-thio--Dgalactopyranoside-inducible promoter, an amino-terminal bacterial OmpA secretion piece and a carboxyl-terminal FLAG tag. This expression system has been used extensively for expression of recombinant RNase A ribonucleases (28 –32).
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