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A Cyclic Antimicrobial Peptide Produced in Primate Leukocytes by the Ligation of Two Truncated -Defensins

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A Cyclic Antimicrobial Peptide Produced in Primate Leukocytes by the Ligation of Two Truncated -Defensins R ESEARCH A RTICLES (arrow in Fig. 1A) was relatively abundant and possessed the greatest antibacterial activity of A Cyclic Antimicrobial Peptide any of the peptides isolated. The yield of RTD-1 was ϳ100 ␮g per 109 neutrophils. Produced in Primate Leukocytes Amino acid analysis of RTD-1 revealed that it was composed of 18 amino acids: 1 Thr, 1 by the Ligation of Two Val, 1 Leu, 1 Phe, 1 Ile, 2 Gly, 5 Arg, and 6 Cys. MALDI-TOF MS analysis (9) of the native ␣ (2082.0) and S-pyridylethylated (10) (2720.3) Truncated -Defensins peptides demonstrated that the six cysteines Yi-Quan Tang,1 Jun Yuan,1 George O¬ sapay,1 Klara O¬ sapay,1 exist as three intramolecular disulfides. Because the peptide’s apparent NH -terminus was Dat Tran,1 Christopher J. Miller,2 Andre J. Ouellette,1 2 1 blocked, the primary structure of RTD-1 was Michael E. Selsted * determined by sequencing overlapping frag- ments produced by methanolic-HCl treatment Analysis of rhesus macaque leukocytes disclosed the presence of an 18-residue (11) and digestion with chymotrypsin and tryp- macrocyclic, tridisulfide antibiotic peptide in granules of neutrophils and mono- sin (12) (Fig. 2A). These analyses revealed that cytes. The peptide, termed rhesus theta defensin-1 (RTD-1), is microbicidal for the RTD-1 backbone is cyclized through pep- bacteria and fungi at low micromolar concentrations. Antibacterial activity of tide bonds (Fig. 2B). The cyclic conformation the cyclic peptide was threefold greater than that of an open-chain analog, and accounts for the 18 atomic mass unit difference the cyclic conformation was required for antimicrobial activity in the presence (equal to one water molecule) between the mea- of 150 millimolar sodium chloride. Biosynthesis of RTD-1 involves the head- sured mass (2082.0; 2081.6 calculated) of to-tail ligation of two ␣-defensinÐrelated nonapeptides, requiring the forma- RTD-1 and that of a linear tridisulfide peptide tion of two new peptide bonds. Thus, host defense cells possess mechanisms (2099.6 calculated) of the same composition. for synthesis and granular packaging of macrocyclic antibiotic peptides that are The disulfide structure of RTD-1 was deter- components of the phagocyte antimicrobial armamentarium. mined by characterizing protease digestion fragments produced by sequential incubation of Antimicrobial peptides are evolutionarily an- Chromatographic peaks eluting between 20 and native peptide with trypsin and thermolysin cient elements of innate immunity (1). Mam- 50 min were purified to homogeneity and (13). Following trypsin digestion, a major prod- malian defensins, comprising genetically dis- screened for antibacterial activity against Esch- uct was purified by HPLC and its mass was tinct ␣ and ␤ subfamilies, are cationic, tridi- erichia coli ML35 and Staphylococcus aureus determined to be 1998.1. Comparison of the sulfide peptides of 29 to 42 amino acids 502a (6), and microbicidal peptides were char- mass and amino acid analysis of this peptide length that possess potent broad-spectrum acterized by amino acid analysis and automated revealed that it was produced by cleavage at the microbicidal activities in vitro (2). Many de- Edman degradation (7). Seven of the eight COOH side of all five arginine residues, thus fensins appear to be expressed constitutively, purified antimicrobial peptides were ␣-de- generating a 17-residue oligopeptide (or 17- whereas the biosynthesis of others is induced fensins that were similar in sequence and activ- mer) composed of four peptides linked by three by mediators of inflammation (3). Neutrophil ity to orthologous human peptides (8). RTD-1 disulfides (calculated mass ϭ 1997.5; Fig. 2C). ␣-defensins are packaged in azurophil gran- ules that are mobilized during phagocytosis for delivery to phagosomes containing ingest- Fig. 1. Purification of RTD-1. (A) RP- HPLC of peripheral blood leukocyte ed microbes. In vitro studies demonstrate that extracts. An ␣-defensin-enriched ex- defensins kill microorganisms by permeabi- tract of 6 ϫ 106 leukocytes (91% lizing one or more target cell membranes (4). PMNs) (8) was fractionated by RP- We undertook studies to characterize de- HPLC on a 0.46 ϫ 25 cm C-18 col- fensin expression in the leukocytes of rhesus umn equilibrated in 0.1% aqueous macaques. In addition to several ␣-defensins TFA and developed with a linear acetonitrile gradient (dotted line). isolated from monkey neutrophils, we isolat- RTD-1 eluted in the arrow-marked ed a tridisulfide-containing antimicrobial peak. (B) Analytical RP-HPLC of pu- peptide, termed rhesus theta defensin 1 rified RTD-1. The purity of RTD-1 (RTD-1), in which the peptide backbone is was assessed by RP-HPLC of RTD-1 naturally cyclized. obtained from the peak (arrow) in Isolation and characterization of RTD-1. (A) on an analytical C-18 column equilibrated in aqueous 0.13% HFBA RTD-1 was isolated from rhesus macaque Ͼ and developed with a linear (20 to leukocytes [ 90% polymorphonuclear (PMN)] 60%) acetonitrile gradient in 20 by reversed-phase (RP) high-performance liq- min. (C) Acid-urea PAGE. Samples of uid chromatography (HPLC) of a whole-cell 30% acetic acid granulocyte extract extract enriched for ␣-defensins (Fig. 1) (5). (2 ϫ 106 cell equivalents; lane 1), the subsequent methanol:water ex- tract (1 ϫ 107 cell equivalents; lane ␮ 1 2), and 0.4 g of RTD-1 were re- Department of Pathology, College of Medicine, Uni- solved on a 12.5% acid-urea poly- versity of California, Irvine, CA 92697, USA. 2Califor- nia Regional Primate Research Center and Center for acrylamide gel and stained with for- Comparative Medicine, School of Veterinary Medi- malin-Coomassie blue. cine, University of California, Davis, CA 95616, USA. *To whom correspondence should be addressed. E- mail: [email protected] 498 15 OCTOBER 1999 VOL 286 SCIENCE www.sciencemag.org R ESEARCH A RTICLES To distinguish between the eight possible disul- bone. The only other macrocyclic peptides that yielded two sequences that separately corre- fide pairings in the 17-mer, the oligopeptide we are aware of lack disulfides. One, AS-48, is sponded to different portions of RTD-1: R8-R9- was digested with thermolysin and the resulting a plasmid-encoded peptide expressed by En- G10-V11-C12-[Q-L-L-stop] and T17-R18-G1-F2- fragments were analyzed by MALDI-TOF MS terococcus faecalis (23); the second is J25, a C3-[R-L-L-stop] (Fig. 2D) (25). The 3Ј RACE (9). The m/z values of the thermolysin frag- microcin from E. coli (24). products were then used to probe a rhesus ments were consistent with only one cystine Cloning of RTD-1 precursors. To eluci- macaque bone marrow cDNA library (26). Fif- motif (Fig. 2C), revealing that the peptide ring date the RTD-1 biosynthetic pathway, we teen positive clones were isolated and se- is stabilized by three disulfides in a picket sought to characterize the peptide precursor by quenced, revealing two highly similar cDNAs fence–like array that crosslinks two hypotheti- analysis of one or more of the corresponding (92% identity) termed RTD1a and RTD1b cal ␤-strands connected by turns at both ends cDNAs from rhesus macaque bone marrow (Genbank accession numbers AF191100 and (Fig. 2, D and E). Schematically, RTD-1 resem- mRNA. Because the NH2-terminus of RTD-1 AF191101, respectively). Surprisingly, none of bles the Greek letter theta (Fig. 2D), hence the could not be predicted from its cyclic sequence, the clones analyzed contained a sequence that selection of ␪-defensin to describe this molec- we conducted 3Ј RACE (rapid amplification of coded for all 18 amino acids in RTD-1. ular motif. The peptide is highly cationic, pos- cDNA ends) experiments using degenerate oli- RTD1a and RTD1b each encode 76–ami- sessing a net charge of ϩ5 at pH 7 (calculated gonucleotide primers corresponding to six to no acid pre-propeptides containing a 20-res- pI Ͼ 12), and the dense cystine motif is distinct seven amino acid segments of the peptide se- idue signal peptide and a 44–amino acid from that determined for ␣-or␤-defensins quence (25). These amplifications consistently prosegment, which are identical in size and (14). The structure and activities of RTD-1 were confirmed by the preparation and char- A acterization of synthetic RTD-1. The purified synthetic product was identical to natural RTD-1 as determined by RP-HPLC, acid- urea polyacrylamide gel electrophoresis (PAGE), circular dichroism spectroscopy, di- sulfide motif, and quantitative microbicidal activity [supplemental figures 1 and 2 (15)]. Together these data confirm the structure and biological activity of native RTD-1. Fig. 2. Peptide backbone B Searches for amino acid sequence similarity structure of RTD-1. (A) to RTD-1 were carried out using all 18 possible The amino acid sequence linearized peptides as query sequences (16). of the peptide chain was determined by Edman se- Taking into consideration the cysteine spacing quencing or MALDI-TOF and disulfide connectivities of RTD-1, the most MS, or both, of fragments similar polypeptide sequence identified was produced by methanolic- C that of protegrin-3 (PG-3), an antimicrobial HCl treatment or diges- peptide from pig neutrophils (17). Protegrins tion with chymotrypsin are 17- to 18–amino acid, bis-disulfide–con- (CT) and trypsin (T). Di- sulfides were reduced with taining peptides that are members of the cathe- dithiothreitol and alky- licidin family of antimicrobial peptides (18). lated with 4-vinyl pyri- Like protegrins, RTD-1 is predicted to be pre- dine before digestion so dominantly composed of two disulfide-stabi- that cysteines were ana- lized ␤-strands connected by turns. A model of lyzed as S-pyridylethyl RTD-1, derived from molecular dynamics sim- cysteine. Residues in pa- rentheses were assigned ulations (Fig. 2E) (19), is remarkably similar to based on MALDI-TOF MS the solution structure of protegrin-1 (PG-1), an data.
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