θ-Defensins: Cyclic Peptides with Endless Potential
θ-Defensins, the only cyclic peptides of animal origin, have been isolated from the leukocytes of rhesus macaques and baboons. Their biogenesis is unusual because each peptide is an 18-residue chimera formed by the head-to-tail splicing of nonapeptides derived from two separate precursors. θ-Defensins have multiple arginines and a ladder-like tridisulfide array spanning their two antiparallel β-strands. Human θ-defensin genes contain a premature stop codon that prevents effective translation of the needed precursors; consequently, these peptides are not present in human leukocytes. Synthetic θ-defensins with sequences that correspond to those encoded within the human pseudogenes are called retrocyclins. Retrocyclin-1 inhibits the cellular entry of HIV-1, HSV, and influenza A virus. The rhesus θ-defensin RTD-1 protects mice from an experimental severe acute respiratory syndrome coronavirus infection, and retrocyclin-1 protects mice from infection by Bacillus anthracis spores. The small size, unique structure, and multiple host defense activities of θ-defensins make them intriguing potential therapeutic agents. θ-Defensins, the only cyclic peptides of animal origin, have been isolated from the leukocytes of rhesus macaques and baboons. Their biogenesis is unusual because each peptide is an 18-residue chimera formed by the head-to-tail splicing of nonapeptides derived from two separate precursors. θ-Defensins have multiple arginines and a ladder-like tridisulfide array spanning their two antiparallel β-strands. Human θ-defensin genes contain a premature stop codon that prevents effective translation of the needed precursors; consequently, these peptides are not present in human leukocytes. Synthetic θ-defensins with sequences that correspond to those encoded within the human pseudogenes are called retrocyclins. Retrocyclin-1 inhibits the cellular entry of HIV-1, HSV, and influenza A virus. The rhesus θ-defensin RTD-1 protects mice from an experimental severe acute respiratory syndrome coronavirus infection, and retrocyclin-1 protects mice from infection by Bacillus anthracis spores. The small size, unique structure, and multiple host defense activities of θ-defensins make them intriguing potential therapeutic agents. A complex immune system enables vertebrate animals to resist challenges from potential pathogens. Some immune system components, such as antibodies and T-cells, show exquisite specificity. Others, like antimicrobial peptides (AMPs), 2The abbreviations used are: AMPantimicrobial peptidePMNpolymorphonuclear neutrophilHNPhuman neutrophil peptideRTDrhesus θ-defensinPGprotegrinRCretrocyclinMMCminimal microbicidal concentrationIAVinfluenza A virus. act more broadly. Most AMPs are small (1–5 kDa), positively charged, and amphipathic. Two types of AMPs are pertinent to this minireview: cathelicidins and defensins. Both have ancient roots, with cathelicidins having been traced back to hagfish and β-defensins to bony fish. antimicrobial peptide polymorphonuclear neutrophil human neutrophil peptide rhesus θ-defensin protegrin retrocyclin minimal microbicidal concentration influenza A virus. These structurally diverse peptides share a conserved prodomain called “cathelin” (1Tomasinsig L. Zanetti M. The cathelicidins–structure, function, and evolution.Curr. Protein Pept. Sci. 2005; 6: 23-34Crossref PubMed Scopus (202) Google Scholar). Whereas cattle and pigs express 10 or more different cathelicidins, mice and humans express only one. Porcine protegrins are cathelicidins with many structural similarities to θ-defensins (Fig. 1). These include having 18 residues, a β-hairpin backbone, intramolecular disulfides, and multiple arginines. Unlike the θ-defensins, protegrins lack a cyclic backbone, unless one is imparted in the laboratory (2Tam J.P. Wu C. Yang J.L. Membranolytic selectivity of cystine-stabilized cyclic protegrins.Eur. J. Biochem. 2000; 267: 3289-3300Crossref PubMed Scopus (82) Google Scholar). In vertebrates, these peptides comprise three subfamilies called α-, β, and θ-defensins. All of these defensins have six conserved cysteines, three intramolecular disulfide bonds, a net positive charge, and β-sheet regions. The cysteines in α- and β-defensins differ in their spacing and pairing (3Tang Y.Q. Selsted M.E. Characterization of the disulfide motif in BNBD-12, an antimicrobial β-defensin peptide from bovine neutrophils.J. Biol. Chem. 1993; 268: 6649-6653Abstract Full Text PDF PubMed Google Scholar), and some β-defensins (but no α-defensins) contain a short α-helical region. Other peptides have also been called defensins based on their structural and functional similarities to those of vertebrates. Plectasin, from the saprophytic fungus Pseudoplectania nigrella, shows remarkable homology to the defensins of dragonflies (Aeschna cyanea) and mussels (Mytilus galloprovincialis), lineages that diverged over 1 billion years ago (4Mygind P.H. Fischer R.L. Schnorr K.M. Hansen M.T. Sönksen C.P. Ludvigsen S. Raventós D. Buskov S. Christensen B. De Maria L. Taboureau O. Yaver D. Elvig-Jørgensen S.G. Sørensen M.V. Christensen B.E. Kjaerulff S. Frimodt-Moller N. Lehrer R.I. Zasloff M. Kristensen H.H. Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus.Nature. 2005; 437: 975-980Crossref PubMed Scopus (527) Google Scholar). It is possible (5Zhu S. Discovery of six families of fungal defensin-like peptides provides insights into origin and evolution of the CSαβ defensins.Mol. Immunol. 2008; 45: 828-838Crossref PubMed Scopus (132) Google Scholar), but unproven, that fungal defensins are ancestral to vertebrate β-defensins. The human genome contains >30 different β-defensin genes, and mice have even more (6Schutte B.C. Mitros J.P. Bartlett J.A. Walters J.D. Jia H.P. Welsh M.J. Casavant T.L. McCray Jr., P.B. Discovery of five conserved β-defensin gene clusters using a computational search strategy.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 2129-2133Crossref PubMed Scopus (449) Google Scholar). However, research has focused mostly on human β-defensin-1–3, which are expressed by epithelial cells throughout the body. Certain other human and rodent β-defensins are expressed in localized regions of the male genitourinary tract and participate in events related to reproduction (7Tollner T.L. Venners S.A. Hollox E.J. Yudin A.I. Liu X. Tang G. Xing H. Kays R.J. Lau T. Overstreet J.W. Xu X. Bevins C.L. Cherr G.N. A common mutation in the defensin DEFB126 causes impaired sperm function and subfertility.Sci. Transl. Med. 2011; 3: 92ra65Crossref PubMed Scopus (122) Google Scholar). Polymorphonuclear neutrophils (PMNs) are white blood cells that can ingest and kill intruding pathogens. PMNs from cattle (8Selsted M.E. θ-Defensins: cyclic antimicrobial peptides produced by binary ligation of truncated α-defensins.Curr. Protein Pept. Sci. 2004; 5: 365-371Crossref PubMed Scopus (106) Google Scholar) and chickens (9Harwig S.S. Swiderek K.M. Kokryakov V.N. Tan L. Lee T.D. Panyutich E.A. Aleshina G.M. Shamova O.V. Lehrer R.I. Gallinacins: cysteine-rich antimicrobial peptides of chicken leukocytes.FEBS Lett. 1994; 342: 281-285Crossref PubMed Scopus (182) Google Scholar) contain multiple β-defensins, but primate PMNs contain α-defensins instead. The presence of α-defensins in basal mammals and marsupials indicates that these genes arose before the groups diverged, some 130 million years ago (10Lynn D.J. Bradley D.G. Discovery of α-defensins in basal mammals.Dev. Comp. Immunol. 2007; 31: 963-967Crossref PubMed Scopus (43) Google Scholar). Humans express six different α-defensin peptides. Three of these, human neutrophil peptides (HNPs) 1–3, constitute 5–7% of total PMN protein. The DEFA1 and DEFA3 genes for HNP-1 (Fig. 1a) and HNP-3 have duplicated, and extensive copy number polymorphism exists. Consequently, some people carry four copies of both genes (i.e. two on each chromosome), whereas others have 11 copies of both (11Aldred P.M. Hollox E.J. Armour J.A. Copy number polymorphism and expression level variation of the human α-defensin genes DEFA1 and DEFA3.Hum. Mol. Genet. 2005; 14: 2045-2052Crossref PubMed Scopus (164) Google Scholar). Human PMNs also have small amounts of another α-defensin, HNP-4, which is identical to HNP-1–3 in only 11 of 29–30 residues. Human α-defensin-5 and -6 are secreted primarily by Paneth cells in the small intestine. HNP-1 prepropeptides contain a 19-residue signal sequence, a 45-residue anionic propiece, and a 30-residue defensin domain (Fig. 1). Removing the N-terminal residue from either HNP-1 or HNP-3 creates HNP-2, whose first and last residues are both cysteines that are joined by a disulfide bond, a common mode of cyclization. One more evolutionary event led to the backbone cyclic peptides described below. Much of our knowledge about these peptides appeared in the report describing rhesus θ-defensin-1 (RTD-1) (20Tang Y.Q. Yuan J. Osapay G. Osapay K. Tran D. Miller C.J. Ouellette A.J. Selsted M.E. A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated α-defensins.Science. 1999; 286: 498-502Crossref PubMed Scopus (640) Google Scholar). The authors purified an extract of rhesus macaque PMNs and tested its components for bactericidal activity against Escherichia coli, a common urinary tract pathogen, and the clinically temperate 502A strain of Staphylococcus aureus. They found eight AMPs, seven α-defensins, and one smaller (2082 Da) peptide. Biochemical characterization of the small AMP led to the cyclic structure illustrated in Fig. 1. Further studies demonstrated that this 18-residue peptide was heterodimeric, formed by the fusion of two nine-residue peptide fragments. Each of its initial monomers was the product of a mutated α-defensin gene containing a premature stop codon (Fig. 1b, red arrow) in its defensin domain. Because of the codon's position, the initial defensin domain products had only 12 residues and required proteolytic removal of a C-terminal tripeptide to create a nonapeptide “building block” with three cysteines, including one at its C terminus (Fig. 1b). Two such nonapeptides were converted into a θ-defensin by two peptide bonds that spliced them in a head-to-tail manner. Rhesus macaques have three θ-defensin (DEFT) genes that encode different nonapeptides. For simplicity, we will call the different nonapeptides A, B, and C. Because the nonapeptides in a θ-defensin can be identical (AA, BB, and CC) or different (AB, AC, and BC) (12Leonova L. Kokryakov V.N. Aleshina G. Hong T. Nguyen T. Zhao C. Waring A.J. Lehrer R.I. Circular minidefensins and post-translational generation of molecular diversity.J. Leukoc. Biol. 2001; 70: 461-464PubMed Google Scholar, 13Tran D. Tran P.A. Tang Y.Q. Yuan J. Cole T. Selsted M.E. Homodimeric θ-defensins from rhesus macaque leukocytes: isolation, synthesis, antimicrobial activities, and bacterial binding properties of the cyclic peptides.J. Biol. Chem. 2002; 277: 3079-3084Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), two different DEFT genes can produce three different peptides (AA, AB, and AC), and three different DEFT genes can produce six (AA, BB, CC, AB, AC, and BC). All six potential θ-defensin peptides exist in rhesus PMNs (14Tongaonkar P. Tran P. Roberts K. Schaal J. Osapay G. Tran D. Ouellette A.J. Selsted M.E. Rhesus macaque θ-defensin isoforms: expression, antimicrobial activities, and demonstration of a prominent role in neutrophil granule microbicidal activities.J. Leukoc. Biol. 2011; 89: 283-290Crossref PubMed Scopus (44) Google Scholar), but their relative amounts differ greatly, with RTD-1 being the most abundant. Because n different DEFT genes could produce (n/2)(n + 1) peptides (12Leonova L. Kokryakov V.N. Aleshina G. Hong T. Nguyen T. Zhao C. Waring A.J. Lehrer R.I. Circular minidefensins and post-translational generation of molecular diversity.J. Leukoc. Biol. 2001; 70: 461-464PubMed Google Scholar), the four DEFT genes of olive baboons (Papio anubis) could produce 10 different peptides. Five of these were identified at the peptide level (15Garcia A.E. Osapay G. Tran P.A. Yuan J. Selsted M.E. Isolation, synthesis, and antimicrobial activities of naturally occurring θ-defensin isoforms from baboon leukocytes.Infect. Immun. 2008; 76: 5883-5891Crossref PubMed Scopus (81) Google Scholar). These humanized θ-defensins resulted from a combination of cloning, peptide synthesis, and molecular archeology. One decade before RTD-1 was described, Vladimir Kokryakov and colleagues at the Institute for Experimental Medicine in St. Petersburg, Russia, and UCLA discovered protegrins (Fig. in of PMNs V.N. S.S. Panyutich E.A. Aleshina G.M. Shamova O.V. Lehrer R.I. antimicrobial peptides that of defensins and Lett. 1993; PubMed Scopus Google Scholar). The peptide had and antimicrobial and an J. Liu H. D.J. P.A. C. L. of protegrins for the and of of protegrin 2000; PubMed Scopus Google Scholar) human as a to with A of D. E.J. M. M. P. A of for the of in 2004; PubMed Scopus Google Scholar), to the a that have been by its backbone (2Tam J.P. Wu C. Yang J.L. Membranolytic selectivity of cystine-stabilized cyclic protegrins.Eur. J. Biochem. 2000; 267: 3289-3300Crossref PubMed Scopus (82) Google Scholar), as in Fig. The found a small AMP in rhesus macaque Because its and those of studies were to Rhesus expressed only one but as was and cysteines, was to the rhesus peptide C. Nguyen T. Hong T. C. D. Waring Lehrer R.I. an α-helical antimicrobial peptide of the rhesus 2001; 45: PubMed Scopus Google Scholar). six rhesus genes were including three whose C-terminal defensin have only 12 residues had been The were called but the were to 12 + 12 the (20Tang Y.Q. Yuan J. Osapay G. Osapay K. Tran D. Miller C.J. Ouellette A.J. Selsted M.E. A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated α-defensins.Science. 1999; 286: 498-502Crossref PubMed Scopus (640) Google Scholar), the the of a as as from the human PMNs θ-defensin human that the of rhesus θ-defensin precursors. However, the human an stop codon in its signal sequence, its from an expressed this for in θ-defensin that the to the θ-defensin by peptide They the peptide (Fig. from in and to its cyclic Retrocyclin-1 and RTD-1 D. Tran P. Roberts K. Osapay G. Schaal J. Ouellette Selsted M.E. properties and selectivity of rhesus macaque 2008; PubMed Scopus Google Scholar) in the as human α-defensins R.I. S.S. T. Selsted M.E. of human defensins with Escherichia of bactericidal PubMed Scopus Google Scholar), by its Rhesus θ-defensins were expressed in the PMNs and of macaques and but more in the V.N. S.S. Panyutich E.A. Aleshina G.M. Shamova O.V. Lehrer R.I. antimicrobial peptides that of defensins and Lett. 1993; PubMed Scopus Google Scholar, D. Tran P. Roberts K. Osapay G. Schaal J. Ouellette Selsted M.E. properties and selectivity of rhesus macaque 2008; PubMed Scopus Google Scholar). 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