Nitto Takeaki, Dyer Kimberly D, Czapiga Meggan, Rosenberg Helene F
Laboratory of Allergic Diseases and Research Technologies Branch, NIAID, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 2006 Sep 1;281(35):25622-34. doi: 10.1074/jbc.M604313200. Epub 2006 Jun 27.
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.
在本研究中，我们探究了来自家鸡（Gallus gallus）的两种密切相关的核糖核酸酶A核糖核酸酶的进化与功能。核糖核酸酶A-1和A-2的编码序列在6号染色体上相隔约10 kb，它们在正选择压力下（dN > dS）发生分化，但彼此仍相似（氨基酸同一性为81%），且与哺乳动物血管生成素相似。在外周血粒细胞和骨髓中检测到了免疫反应性核糖核酸酶A-1和A-2（两者均约为16 kDa）。重组蛋白具有核糖核酸酶活性（kcat分别为2.6和0.056 s(-1)），令人惊讶的是，两者都与人胎盘核糖核酸酶抑制剂相互作用。阳离子性更强的核糖核酸酶A-2（pI 11.0）具有血管生成和杀菌活性；核糖核酸酶A-1（pI 10.2）则没有这些活性。我们通过催化性组氨酸110的点突变证明，核糖核酸酶活性的缺失对核糖核酸酶A-2的杀菌活性没有影响。我们确定核糖核酸酶A-2不同的结构域II（氨基酸71 - 76）和结构域III（氨基酸89 - 104）对杀菌活性都很重要。此外，我们证明这些阳离子结构域可以作为独立的杀菌肽发挥作用，而无需核糖核酸酶A主链所赋予的三级结构。这些结果表明，核糖核酸酶活性可能不是限制该基因家族持续进化的关键限制因素，核糖核酸酶主链可能仅仅作为一个支架来支持新型非核糖核酸酶蛋白的进化。
