Biochem. J. (2013) 456, 99–108 (Printed in Great Britain) doi:10.1042/BJ20130123 99 Ribonucleases as a host-defence family: evidence of evolutionarily conserved antimicrobial activity at the N-terminus Marc TORRENT*† 1,2 , David PULIDO* 1 , Javier VALLE†, M. Vict` oria NOGU ´ ES*, David ANDREU† 2 and Ester BOIX* 2 *Department of Biochemistry and Molecular Biology, Universitat Aut` onoma de Barcelona, Cerdanyola del Vall` es, 08193 Barcelona, Spain, and †Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Dr. Aiguader 88, 08003 Barcelona, Spain Vertebrate secreted RNases (ribonucleases) are small proteins that play important roles in RNA metabolism, angiogenesis or host defence. In the present study we describe the antimicrobial properties of the N-terminal domain of the hcRNases (human canonical RNases) and show that their antimicrobial activity is well conserved among their lineage. Furthermore, all domains display a similar antimicrobial mechanism, characterized by bacteria agglutination followed by membrane permeabilization. The results of the present study show that, for all antimicrobial hcRNases, (i) activity is retained at the N-terminus and (ii) the antimicrobial mechanism is conserved. Moreover, using computational analysis we show that antimicrobial propensity may be conserved at the N-terminus for all vertebrate RNases, thereby suggesting that a defence mechanism could be a primary function in vertebrate RNases and that the N-terminus was selected to ensure this property. In a broader context, from the overall comparison of the peptides’ physicochemical and biological properties, general correlation rules could be drawn to assist in the structure-based development of antimicrobial agents. Key words: antimicrobial peptide, drug discovery, evolution, innate immunity, ribonuclease. INTRODUCTION RNase (ribonuclease) A-homologues comprise a vertebrate- specific superfamily that includes a wide network of diverse gene lineages [1]. Eight human members (known as canonical RNases) belong to the RNase A-like family [2,3]. They are all small secreted proteins (approximately 15 kDa), sharing a typical fold, with six or eight cysteine residues arranged in three or four disulfide bonds [4–9]. All RNases include a conserved catalytic triad comprising two histidine residues and one lysine residue, the latter located inside the characteristic RNase signature (CKXXNTF) [2] (Supplementary Figure S1 at http://www.biochemj.org/bj/456/bj4560099add.htm). All hcRNases (human canonical RNases) are catalytically active in variable degrees [10] and some of them share relevant antimicrobial properties [11]. However, antimicrobial and catalytic activities of hcRNases are apparently unrelated [9,11,12]. In particular, studies in zebrafish [13] and chicken RNases [14] show they display antimicrobial properties independent of a catalytic activity. Interestingly, vertebrate RNases have high isoelectric points, a common feature in antimicrobial proteins, required for interaction with the negatively charged membranes of pathogens [11,15]. In summary, the presence of antimicrobial activity in diverse lineages of vertebrate RNases suggests that host defence might be a relevant physiological role of the superfamily [16]. However, a sequential or structural evolutionarily selected pattern that embodies the antimicrobial activity has not yet been reported. In this context, we previously described that, for RNase 3, a functional antimicrobial domain is located at the N-terminus of the protein [17–20]. This domain can be further dissected into two active regions: (i) residues 24–45 are essential for the antimicrobial action, and (ii) residues 8–16 contribute to agglutination and membrane destabilization [17]. Given that many RNases display antimicrobial properties unrelated to their ribonuclease activity, it is appealing to hypothesize that an N-terminal antimicrobial domain could be preserved and/or refined along evolution to embody the antimicrobial activity. To test this hypothesis, we have synthesized the N-terminal domains of all hcRNases and studied their antimicrobial properties. The results of the present study reveal that (i) antimicrobial activity is largely confined at the N-terminus, and that (ii) the mechanism of action of the N-terminal domains is similar to that of the original proteins. Moreover, complementary computational analyses suggest that the N-terminal domain in the protein might have been selected by evolution to provide a host-defence function. MATERIALS AND METHODS Materials and strains DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DOPG (1,2-dioleoyl-sn-glycero-3-phosphoglycerol) were from Avanti Polar Lipids. LPS (lipopolysaccharide) from Escherichia coli was purchased from Sigma–Aldrich. ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid), DPX (p- xylenebispyridinium bromide), BC [BODIPY ® cadaverine, where BODIPY is boron dipyrromethene (4,4-difluoro-4-bora- 3a,4a-diaza-s-indacene)] and SYTOX were purchased from Abbreviations used: ACN, acetonitrile; ANTS, 8-aminonaphthalene-1,3,6-trisulfonic acid; BC, BODIPY ® cadaverine; DIEA, N,N-di-isopropylethylamine; DLS, dynamic light scattering; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPG, 1,2-dioleoyl-sn-glycero-3-phosphoglycerol; DPX, p- xylenebispyridinium bromide; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; hcRNase, human canonical ribonuclease; hRNase, human ribonuclease; LPS, lipopolysaccharide; LUV, large unilamellar vesicle; MAC, minimal agglutination concentration; MIC, minimal inhibitory concentration; RBC, red blood cell; RNase, ribonuclease; TFA, trifluoroacetic acid. 1 These authors contributed equally to this work. 2 Correspondence may be addressed to any of these authors (email marc.torrent@uab.cat, david.andreu@upf.edu or ester.boix@uab.cat). c  The Authors Journal compilation c  2013 Biochemical Society