Biological characterization of a novel mammalian antimicrobial peptide Renato Gennaro a , Marco Scocchi b; c , Laura Merluzzi c , Margherita Zanetti b; c ; * a Dipartimento di Biochimica, Bio¢sica e Chimica delle Macromolecole, Universita © di Trieste, I-34127 Trieste, Italy b Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie, AREA Science Park, Padriciano, 99 I-34012 Trieste, Italy c Dipartimento di Scienze e Tecnologie Biomediche, Universita © di Udine, I-33100 Udine, Italy Received 2 June 1998; revised 13 July 1998; accepted 4 August 1998 Abstract A putative antimicrobial peptide of 34 residues was recently deduced from a bovine cathelicidin gene sequence and named BMAP-34. A peptide based on the deduced sequence was chemically synthesized and used to study the localization, structure and biological activities of BMAP-34. A Western blot analysis using antibodies raised to the synthetic peptide showed that BMAP-34 is stored as proform in the cytoplasmic granules of bovine neutrophils. CD spectroscopy indicates that the peptide assumes an amphipathic K-helical conformation, as also predicted by secondary structure analysis. The peptide exerts a broad spectrum antimicrobial activity against both Gram-negative and Gram-positive organisms, and is not active against eukaryotic cells. When tested on Escherichia coli ML-35, the kinetics of bacterial killing and of inner membrane permeabilization are slower than those observed for other K-helical peptides derived from cathelicidins. ß 1998 Elsevier Science B.V. All rights reserved. Keywords : Cathelicidin ; Antimicrobial peptide ; K-Helical peptide ; Membrane permeabilization ; (Cow) 1. Introduction Antimicrobial peptides are small and amphipathic molecules produced by animals and plants as part of the immune defense system [1]. In general, these pep- tides are cationic and exert their antibiotic activity by permeabilizing bacterial membranes [1,2]. A variety of these peptides have been identi¢ed in leukocytes as derived from prepropeptides of the cathelicidin fam- ily [3]. Members of this family show a conserved N- terminal `cathelin' domain (propiece) of about 100 residues and a C-terminal cationic antimicrobial do- main of varied length (12^100 residues). Most often, a proteolytic cleavage site for elastase is placed be- tween the propiece and the C-terminal peptide [3], allowing for release of the active peptide [4^6]. Cath- elicidin-derived peptides show a remarkable structur- al diversity and exhibit a wide spectrum antimicro- bial activity in vitro at Wmolar concentrations. According to common structural features, these pep- tides have been grouped into K-helical, Cys-rich, Pro- and Arg-rich, and Trp-rich peptides [3]. We have recently reported the sequence of a novel bovine cathelicidin, as deduced from the gene sequence [7]. The predicted polypeptide includes a prepropiece of 131 residues and a C-terminal putative antimicrobial domain of 34 residues. The peptide was named BMAP-34 (bovine myeloid antimicrobial peptide of 34 residues) after the putative antimicrobial domain. In this report, a synthetic peptide based on the 0304-4165 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII:S0304-4165(98)00087-7 * Corresponding author. Fax: +39 (40) 398990; E-mail : zanetti@icgeb.trieste.it Biochimica et Biophysica Acta 1425 (1998) 361^368