RNase A ribonucleases and host defense: an evolving story

Abstract

RNase A (bovine pancreatic RNase) is the founding member an extensive family of divergent proteins that share specific elements of sequence homology, a unique disulfide-bonded tertiary structure, and the ability to hydrolyze polymeric RNA. Among the more intriguing and perhaps counterintuitive findings, at the current state of the art, the connection between RNase activity and characterized host defense functions is quite weak; whether this is a scientific reality or more a reflection of what has been chosen for study remains to be determined. Several of the RNase A family RNases are highly cationic and have cytotoxic and bactericidal properties that are clearly (eosinophil cationic protein, leukocyte RNase A-2) or are probably (RNase 7) unrelated to their enzymatic activity. Interestingly, peptides derived from the leukocyte RNase A-2 sequence are nearly as bactericidal as the entire protein, suggesting that among other functions, the RNase A superfamily may be serving as a source of gene scaffolds for the generation of novel cytotoxic peptides. Other RNase A ribonucleases are somewhat less cationic (mouse angiogenin 4, zebrafish RNases) and have moderate bactericidal activities that have not yet been explored mechanistically. Additional host defense functions characterized specifically for the RNase eosinophil-derived neurotoxin include reducing infectivity of RNA viruses for target cells in culture, which does require RNase activity, chemoattraction of immature human dendritic cells via a G-protein-coupled receptor-dependent mechanism, and activation of TLR2. The properties of individual RNase A ribonucleases, recent experimental findings, and important questions for the near and distant future will be reviewed.

Figure 1. Neighbor-joining phylogenetic tree featuring the relationships among the major mammalian and non-mammalian RNase A lineages

Amino acid sequences were aligned using ClustalW (http://clustalw.genome.jp/) and an unrooted tree was created using Mega 4.0 Evolutionary Genetics software [; http://www.megasoftware.net/]. Bootstrap values over 50 (1000 replicates) are shown. Sequences are from GenBank and NCBI Protein databases (http://www.ncbi.nlm.nih.gov/sites/entrez); accession numbers are available from the author on request.

Figure 2. Structure-function analysis of the bactericidal activity of ECP

(A) Spacefilling diagram documenting the location of various amino acids evaluated in a mutational analysis by Carreras et al. [61], reprinted with permission, followed by a demonstration of the importance of specific cationic arginines (R) and aromatic tryptophans (W) to the overall activity of ECP against (B) E. coli and (C) S. aureus. Overall, data suggest distinct mechanisms of action against gram negative vs. gram positive bacterial species.

Figure 3. (A) Isolated human eosinophils and (B) recombinant, ribonucleolytically active EDN reduce the infectivity of the pathogen, respiratory syncytial virus (RSV) for target epithelial cells in vitro

As shown, ribonucleolytically inactivated EDN (catalytic lysine K38 converted to arginine) has no antiviral activity, although the mechanism via which EDN reduces virus infectivity remains otherwise unclear. Reprinted with permission from Domachowske et al. [4].

Figure 4. EDN as a chemoattractant

(A) Migration of immature human dendritic cells to EDN was similar in magnitude to that seen to the chemokine SDF-1α and (B) was sensitive to inhibition by pertussis toxin (PTX). Reprinted with permission from Yang et al., [2].

Figure 5. Selective anti-pathogen activity of mouse angiogenin 4

Although the basis for this activity remains unclear, mouse angiogenin 4 displays activity against E. faecalis and L. monocytogenes, but not against the other pathogens shown. Reprinted with permission from Hooper et al., [21].

Figure 6. Zebrafish RNase A ribonucleases

(A) Tissue distribution determined by RT-PCR including (E) eye, (H) heart, (B) brain, (L) liver, (G) gut, (T) testis, (O) ovary, (S) skin, and (M) skeletal muscle. (B) Comparative ribonuclease activity against yeast tRNA substrate. Reprinted with permission from Cho and Zhang, [26].

Figure 7. Bactericidal activities of chicken leukocyte RNase A-2 and domain III peptide

(A) Leukocyte RNases A-1 and A-2 are found in chicken heterophils and bone marrow progenitors. (B) RNase A-2 is profoundly bactericidal, reducing the colony count of E. coli from 10⁷ to near zero and (C) the 17- amino acid domain III peptide, taken from sequence near the C-terminus, is nearly equally effective. Reprinted with permission from Nitto et al., [22].