Antimicrobial Proteins and Introductory article Peptides Artice Contents Tomas Ganz,University of Califomia,Los Angeles,Califomio,USA RobertLehrer,University of Califomio,Los Angeles,Califorio,USA Antimicrobial proteins and peptides are important effectors of the innate and adaptive arms of the immune system.They interfere with microbial integrity or metabolism by ing Protein (BPI targeting structures or nutrients specific to microbes. .Se ry Phospholipase Az Introduction ·Other Antin The production of polypeptides that disrupt microbial structures or interfere with microbial metabolism is a host defence in animals and plants tides.Concurrently,various microbial or host-derived differences between corresponding host and microbial molecules trigger an influx of mobile blood cells into the nfected area Structuresand some host peptideporease or on mic re targets.and thereby potentiate their antimicrobial effects Specific mechanisms to protect bystander host cells from Phagocytosis oetoor injury induces production of some anti- Two generaltypesof phagocytesexist:the potentially long microbial polypeptides.Fruit fly (Drosophila melanoga ived macrophages and the short-lived granulocytes ster)mutants unable to induce antimicr obal peptide Mature granulocy are smallerhan macrophages syn e ues that ar microbial polypeptides for host defence have been antimicrobial proteins and peptides and of hydrolytic achieved in plants,where transferring antimicrobial ece inery require on Certa faces directly,while others bind via intermediate adapter Mucosal Host Defence rom the s Initial encounters between microbes and multicellular Cshand Csi.and plasmalectinssuch as mannose-binding animals often take place on moist surfaces (mucosae). protein are particularly effective opsonins. with cmicrobial attag zlycoproteins (mucins thst ishvacre Opsonins bound to microbial surfaces interact with Fey Ep tute physica eptors.C3b echanically remove microbes by transporting the causing the plasma membrane of the blanket of mucus.Mucosal cells secrete antimicrobial around the target microbe peptides and proteins that damage microbes(Tabl and to form a some organs,such as the lungs. ceptors trigger s cyt rearrange e-adherent microbe containin granule tides to the ph multiply or breach the epithelial barrier,affected host vacuole.Concurrently,NADPH oxidase complexes are ENCYCLOPEDIA OF LIFE SCIENCES/2001 Na .els net 1
Antimicrobial Proteins and Peptides Tomas Ganz, University of California, Los Angeles, California, USA Robert I Lehrer, University of California, Los Angeles, California, USA Antimicrobial proteins and peptides are important effectors of the innate and adaptive arms of the immune system. They interfere with microbial integrity or metabolism by targeting structures or nutrients specific to microbes. Introduction The production of polypeptides that disrupt microbial structures or interfere with microbial metabolism is a common means of host defence in animals and plants. Although microbial and host structures share many features, antimicrobial polypeptides achieve specificity by targeting components that host cells lack, by exploiting differences between corresponding host and microbial structures, and by selectively concentrating polypeptides on microbial surfaces. Some host peptides release or activate latent lytic enzymes (autolysins) of their microbial targets, and thereby potentiate their antimicrobial effects. Specific mechanisms to protect bystander host cells from damage exist, and include cell-associated or soluble macromolecules that bind and detoxify antimicrobial polypeptides. Infection or injury induces production of some antimicrobial polypeptides. Fruit fly (Drosophila melanogaster) mutants unable to induce antimicrobial peptide synthesis succumb to bacterial or fungal infections. Even more direct demonstrations of the importance of antimicrobial polypeptides for host defence have been achieved in plants, where transferring antimicrobial peptide genes from one species to another can produce resistance to specific pathogens. Mucosal Host Defence Initial encounters between microbes and multicellular animals often take place on moist surfaces (mucosae). These are coated with glycoproteins (mucins) that interfere with microbial attachment. Epithelia constitute physical barriers to microbial penetration. When ciliated, they mechanically remove microbes by transporting them in a blanket of mucus. Mucosal cells secrete antimicrobial peptides and proteins that damage microbes (Table 1). In some organs, such as the lungs, resident host defence macrophages patrol the epithelial surfaces, ingesting microbial trespassers. When surface-adherent microbes multiply or breach the epithelial barrier, affected host tissues produce signalling molecules (cytokines) that may induce the synthesis of additional antimicrobial polypeptides. Concurrently, various microbial or host-derived molecules trigger an influx of mobile blood cells into the infected area. Phagocytosis Phagocytes are specialized host defence cells that can ingest microbes and expose them to antimicrobial substances. Two general types of phagocytes exist: the potentially longlived macrophages and the short-lived granulocytes. Mature granulocytes are smaller than macrophages, manifest considerably less protein synthesis, and contain abundant cytoplasmic granules that are a repository of antimicrobial proteins and peptides and of hydrolytic enzymes. Phagocytes recognize their targets by phagocytic receptors coupled to the cellular machinery required for motility and ingestion. Certain receptors, such as the mannose receptor on macrophages, bind microbial surfaces directly, while others bind via intermediate adapter molecules (opsonins) deposited onto microbial surfaces from the surrounding fluid. Specific antibodies (especially immunoglobulin G (IgG) class), complement fragments C3b and C3bi, and plasma lectins such as mannose-binding protein are particularly effective opsonins. Opsonins bound to microbial surfaces interact with Fcg receptors, C3b receptors (complement receptor 1, CR1), C3bi receptors (complement receptor 3, CR3), and other phagocytic receptors, causing the plasma membrane of the phagocyte to gradually creep around the target microbe and to form a vacuole called the phagosome. Engagement of phagocytic receptors triggers cytoskeletal rearrangements, and fusion of granules or transport vesicles containing antimicrobial polypeptides to the phagosomal vacuole. Concurrently, NADPH oxidase complexes are Article Contents Introductory article . Introduction . Mucosal Host Defence . Phagocytosis . Complement . Defensins . Bactericidal Permeability-increasing Protein (BPI) . Proteases . Secretory Phospholipase A2 . Lactoferrin . Cathelicidins . Other Antimicrobial Polypeptides ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 1
Antimicrobial Proteins and Peptides Table 1 Antimicrobial peptides and proteins secreted by Polypeptide Mass (kDa) Distribution Peroxidases 150 Myeloperoxidase Neutrophils,monocytes hi 80 Various epithelia Bactericidal permeability-increasing 55-60 Neutrophils protein (BP1) 25-30 Neutrophils 14 Neutrophils,monocytes,macrophages Varous epithelia 14 Many other tissues Secretory leucoprotease inhibitor(SLPI) 12 Macrophages Various epithelia Cathelicidins 1-18 Defensins Neutrophils Various epithelia omal mem 1.4-glycosidic linkage between Smate and undergo further reactions that g atehydrogen oxide,and (in concert with myeloperoxidase)more potent bacteriolytic properties.Because of its small size,abun antimicrobial oxidants. nsome species, a phagocyte and easy purification,its crystal structure and enzym e is also The me have been extensively tides destr ed microbes within minutes The sequestration of microbial targets in phagocytic vacuoles nd omoiseiance.Disruption of the peti ons of cytocidal doglycan layer suffices to kill some bacteria and rend stress.Enzym wit The most abundant granulocyte in human blood is the nimals and also in host defence settings.Chitinases ar polymorphonucleated neutrophil (called PMN,poly',or functionally and structurally related enzymes that degrade chitin,an exoskelet al polymer of fungi and insects eous,one igh avian egg deliver antimicrobial components to the p tions.granuloevtes and macrophas s.in human granulo Other granule subsets-called secondary (or spec fic)and cytes,lysozyme is highly abundant in both primary and rete their contents inte the extra secondary granules,and it is a principal secr eted protein of or into the pl a membrane.Th nacropha ges polypeptides man neutrophi sitive b and Bacils species).Gram-negative bacteria are more resis Lysozyme tant because their outer membrane access o ysozyme to At still rea activity that depends on the highly cationic nature of this ENCYCLOPEDIA OF LIFE SCIENCES/e 2001 Nature Publishing Group /www.els.net
assembled and activated in the phagosomal membrane, allowing copious production of superoxide anions. These undergo further reactions that generate hydrogen peroxide, and (in concert with myeloperoxidase) more potent antimicrobial oxidants. In some species, a phagocyte enzyme that generates nitric oxide is also induced. The combined action of oxidants and antimicrobial polypeptides destroys most ingested microbes within minutes. The sequestration of microbial targets in phagocytic vacuoles exposes microbes to high concentrations of cytocidal substances while minimizing collateral damage to the host cell. The most abundant granulocyte in human blood is the polymorphonucleated neutrophil (called PMN, ‘poly’, or neutrophil, for short). Although the cytoplasmic granules of human neutrophils are heterogeneous, one of its granule subsets (the primary or azurophilic granule) functions to deliver antimicrobial components to the phagosome. Other granule subsets – called secondary (or specific) and tertiary granules – secrete their contents into the extracellular fluid or into the plasma membrane.The primary and secondary granules of human neutrophils both contain antimicrobial polypeptides. Lysozyme Lysozyme (also called muramidase) is a widely distributed, cationic (i.e. positively charged) enzyme that cleaves the b 1,4-glycosidic linkage between N-acetyl muramate and Nacetyl glucosamine. It was first described by Sir Alexander Fleming in 1922, who called attention to its remarkable bacteriolytic properties. Because of its small size, abundance and easy purification, its crystal structure and catalytic mechanism have been extensively investigated. The principal target of lysozyme is peptidoglycan, a structural glycosidic polymer that gives bacteria shape stability and osmotic resistance. Disruption of the peptidoglycan layer suffices to kill some bacteria and renders others more susceptible to osmotic stress. Enzymes with lysozyme activity are found in the digestive organs of many animals and also in host defence settings. Chitinases are functionally and structurally related enzymes that degrade chitin, an exoskeletal polymer of fungi and insects. High concentrations of lysozyme occur in avian egg white, and in mammalian tears, milk, respiratory secretions, granulocytes and macrophages. In human granulocytes, lysozyme is highly abundant in both primary and secondary granules, and it is a principal secreted protein of macrophages. Organisms susceptible to low concentrations of lysozyme include certain nonpathogenic Grampositive bacteria (e.g. Micrococcus lysodeikticus and Bacillus species). Gram-negative bacteria are more resistant because their outer membranes hinder the access of lysozyme to peptidoglycan. At high but still realistic concentrations of lysozyme, nonenzymatic antimicrobial activity that depends on the highly cationic nature of this Table 1 Antimicrobial peptides and proteins secreted by mucosal cells Polypeptide Mass (kDa) Distribution Peroxidases 150 Myeloperoxidase Neutrophils, monocytes Eosinophil peroxidase Eosinophils Lactoperoxidase Epithelia (milk, saliva) Lactoferrin 80 Neutrophils Various epithelia Bactericidal permeability-increasing protein (BPI) 55–60 Neutrophils Serprocidins (cathepsin G, elastase, proteinase 3, azurocidin/CAP37) 25–30 Neutrophils Lysozyme 14 Neutrophils, monocytes, macrophages Various epithelia Phospholipase A2 14 Neutrophils, macrophages, platelets Paneth cells Many other tissues Secretory leucoprotease inhibitor (SLPI) 12 Macrophages Various epithelia Cathelicidins 1–18 Neutrophils (secondary granules) Inflamed skin Testes Defensins 4 Neutrophils Various epithelia Antimicrobial Proteins and Peptides 2 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
Antimicrobial Proteins and Peptides molecule is also observed.The abundance and wide cytes.Trace amounts ofa fourth defensin.HNP-4.are also distribution of lysozyme suggest that it is of considerable found in the same compartment.HNP-I and HNP-4 are importance encoded by genes designated D上rAI and DEFA4. gene on chrom some 12 encodes the huma of the DEE4 HNP-2 results from p removal of the N terminal residue from HNP-1 and/or HNP-3.The remain ing a-detensins HD- and HD-6 are expressed by human and are pro boto oreensins s in the small I intestine.Human B-defensin Complement rious epithelia,and The DEFBI.DEFB2 The direct microbicidal effect of blood plasma on many aned the microbial species is mediated by complement.The key luster are neighbours.occup region of about 400- lethal event is the assembly of the membrane 6.9 neutrophils or Paneth cells.epithelial B-defensins tored granule of tare microbes.The insertion of th rimarily by their nth initiating C5b fragment is the end result of complement esis and secretion rates Other members of the defensin family of peptides hav nen t cascades. pathway as abundant uents of granulocytes in surfaces,thea nsters.guinea ternative pathway is triggered by the interaction of C3b with the microbial surface,while the and horses.Epithelial defensins have been found in Paneth contact of Clq-like mannose. ng和 repe disrupts the ionic homcostasis of the microbes.killing HBD.2 is susceptible microbes.Many pathogenic microbes have matory stimuli.Inducible antimicrobial cationic peptides evolved thick cell with six cysteines have been identified in the haemolymph apsu les that make and ot rates and icidal a ment is illustrated by the predisposition of pa tients with a ve tebrate def structure and composition are found in host defence roles deficiency of C6.C7 or C8 to recurrent infections with eisseria gonorrhoed and N.meningitid n plants where they plant anifest broac bial function ready in arthropods (e.g.horseshoe crabs). ions many hact ia.fung and ome enveloped viruses are inactivated by defensins.The nitial interaction of defensins with bac Defensins orces,and i into the target cell.Preferential interaction of defensins Defensins are 3-4-kDa cationic antimicrobial peptides es as opposed to host cells is probably due to with three intramolecular cystine disulfide the microbi men ane composition: largely B sheet struc cture The vere ate and Although similar in shape to-defensins.B-defensins nulocvte defensins.and suffer from rent bact erial slightly larger and differ in the placement and connectivity infections. of their six conserved cysteine residues.Human s produc at e st sD fensins and tw content of phagocytic granules of neutrophilic granulo- ENCYCLOPEDIA OF LIFE SCIENCES/ Publishing Group/www.els.net 3
molecule is also observed. The abundance and wide distribution of lysozyme suggest that it is of considerable importance. A single gene on chromosome 12 encodes the human lysozyme enzyme. The mouse has two lysozyme genes, one of which (lysozyme M) is expressed in macrophages and granulocytes, while the other (lysozyme P) is expressed by Paneth cells of the small intestine. In other animals, the number of lysozyme genes and their tissue-specific patterns of expression are highly variable. Complement The direct microbicidal effect of blood plasma on many microbial species is mediated by complement. The key lethal event is the assembly of the membrane attack complex (MAC) consisting of components C5b, C6, C7, C8 with two or more associated C9 molecules in the membranes of target microbes. The insertion of the initiating C5b fragment is the end result of complement activation via one of three complement cascades. The classical pathway is initiated by the binding of C1q molecules to specific antibody clustered on microbial surfaces, the alternative pathway is triggered by the interaction of C3b with the microbial surface, while the lectin pathway depends on contact of C1q-like mannosebinding protein with specific repeating glycans. The formation of the membrane attack complexes disrupts the ionic homeostasis of the microbes, killing susceptible microbes. Many pathogenic microbes have evolved thick cell walls or capsules that make them partially or completely resistant to complement. The importance of the direct microbicidal activity of complement is illustrated by the predisposition of patients with a deficiency of C6, C7 or C8 to recurrent infections with Neisseria gonorrhoeae and N. meningitidis. Complementlike proteolytic cascades appeared early in the evolution of multicellular animals and have a documented antimicrobial function already in arthropods (e.g. horseshoe crabs). Defensins Defensins are 3–4-kDa cationic antimicrobial peptides with three intramolecular cystine disulfide bonds and a largely b sheet structure. The vertebrate defensin family contains two branches, designated a- and b-defensins. Although similar in shape to a-defensins, b-defensins are slightly larger and differ in the placement and connectivity of their six conserved cysteine residues. Humans produce at least six different a-defensins and two b-defensins. Three human a-defensins (human neutrophil peptides: HNP-1, HNP-2 and HNP-3) constitute about 30% of the protein content of phagocytic granules of neutrophilic granulocytes. Trace amounts of a fourth defensin, HNP-4, are also found in the same compartment. HNP-1 and HNP-4 are encoded by genes designated DEFA1 and DEFA4, respectively. HNP-3 arises from a single nucleotide substitution in one of the several DEFA1 gene copies. HNP-2 results from posttranslational removal of the Nterminal residue from HNP-1 and/or HNP-3. The remaining a-defensins HD-5 and HD-6 are expressed by human small-intestinal Paneth cells and are products of the DEFA5 and DEFA6 genes. Paneth cells are specialized, granule-laden secretory epithelial cells located at the bottom of crypts in the small intestine. Human b-defensins HBD-1 and HBD-2 are produced in various epithelia, and are encoded by the DEFB1 and DEFB2 genes, respectively. The DEFB1, DEFB2 gene and the human a-defensin gene cluster are neighbours, occupying a region of about 400– 600 kb on chromosome 8p23. Unlike the a-defensins of neutrophils or Paneth cells, epithelial b-defensins are not stored in cytoplasmic granules. Consequently, their local concentration may be governed primarily by their synthesis and secretion rates. Other members of the defensin family of peptides have been found as abundant constituents of granulocytes in rabbits, rats, hamsters, guinea pigs, cows, sheep, chickens and turkeys but are absent from granulocytes of mice, pigs and horses. Epithelial defensins have been found in Paneth cells of mice and rats, and in various epithelia of cows, pigs, sheep and mice. The production of tracheal and lingual antimicrobial peptides (TAP and LAP), as well as that of human epithelial defensin, HBD-2, is induced by inflammatory stimuli. Inducible antimicrobial cationic peptides with six cysteines have been identified in the haemolymph of many insects and other invertebrates and have been called ‘insect’ defensins but their evolutionary relationship to vertebrate defensins is uncertain. Peptides of similar structure and composition are found in host defence roles in plants where they are referred to as plant defensins. Defensins manifest broad-spectrum antimicrobial activity that is characteristically but variably inhibited by increasing salt concentrations. Many bacteria, fungi and some enveloped viruses are inactivated by defensins. The initial interaction of defensins with bacteria or fungi involves electrostatic forces, and is followed by permeabilization of microbial membranes and the entry of defensins into the target cell. Preferential interaction of defensins with microbes as opposed to host cells is probably due to the microbial membrane composition: rich in anionic phospholipids and lacking cholesterol. Patients with ‘specific granule deficiency’ also have a deficiency of granulocyte defensins, and suffer from recurrent bacterial infections. Antimicrobial Proteins and Peptides ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 3
Antimicrobial Proteins and Peptides Bactericidal Permeability-increasing another bacteric Protein (BPI) lastase ed by Bactericidal permeability-increasing protein (BPD)is a highly cationic 55-60-kDa prote Cathepsin G gene is located in another cluster of serine prote. n chromosom dim/CAP37 ompa to th and like LBP,binds to lipopolysaccharide with high affinity It kills many Gram-negative bacteria by a process that activity (serine and histidine).In granulocytes.serpr initially involves the perme dins are located in the phagocytic (primary,azurophil) granule Their collective abundancein human neutrophil BPI is ve ag recombinant BPI has promise as an e rmental treatment in meningococcal and other forms of Gram-negative sepsis terial properties of serprocidins can often be dissociated Human BPI is a proteolytic activity,and d by i An N of BPr a prolin easing ionic bicidal,it also converts inactive procathelicidins into quired for binding ated bac PI,LBP th re in cl ntraper are suscepu cteri some 20 (g11.23 sting a common and origin.De pite the Secretory Phospholipase A2 logic LBP does not kill bacteria and greatly enhances the pro mn groupⅡphospholipase A3s.The」 DI A nte an target bac ia differ in their preferred substrates.Human group II inv the ns of BPI and the phospholipase eA preterentially acts on the ester linkages near the highly conserved lipid A region of LPS.In the pnos ane t bacteria,the insertion of BP by other phospholipases even though their catalytic outer me PS mechanisms are very y similar.Group II PLA2 is found in and PS- granulo Paneth cells,epith al secre ions,and infam n hydrophobic molecules.increased susce tory I PI pe otibility of nhos pholipids to phospholipase A attack,and bacterial growth inhibition n ivit e cell wall),penetra many Gram-negative bacteria.Some defensins.cathelici- entrations of complement potentiate the ependent antibacterial activity of mammalian group I Gram-negativ eria with PLA2 requires millimolar concentrations and is augmented elatively resistant to rei y complemen Moreover,other host detence substar y-mcrea of the PLA by disrupting the bacteral outer membrane Proteases Human and other mammalian granulocytes contain Lactoferrin highly cationie rot a -30-kDa senne protea (serprocidins) is by a gene in the ENCYCLOPEDIA OF LIFE SCIENCES/e 2001 Nature Publishing Group /www.els.net
Bactericidal Permeability-increasing Protein (BPI) Bactericidal permeability-increasing protein (BPI) is a highly cationic 55–60-kDa protein, abundant in human and other mammalian neutrophils. It is structurally related to the lipopolysaccharide-binding protein (LBP) in plasma, and like LBP, binds to lipopolysaccharide with high affinity. It kills many Gram-negative bacteria by a process that initially involves the permeabilization of their outer membranes, but is inactive against Gram-positive bacteria. BPI is an effective scavenger of lipopolysaccharide and recombinant BPI has promise as an experimental treatment in meningococcal and other forms of Gram-negative sepsis. Human BPI is a highly elongated molecule formed by two domains of similar size connected by a proline-rich linker. An N-terminal domain of BPI expresses the bactericidal and endotoxin-neutralizing activities of BPI while the C-terminal domain may be required for binding events that facilitate phagocytosis of BPI-coated bacteria. The human genes encoding BPI, LBP and several other lipid-binding proteins are in close proximity on chromosome 20 (q11.23–q12), suggesting a common ancestral origin. Despite their similarity in overall structural and functional design, the biological activities of BPI and LBP are strikingly different: whereas BPI is bactericidal toward Gram-negative bacteria and inhibits endotoxin signalling, LBP does not kill bacteria and greatly enhances the proinflammatory action of low doses of LPS. Initial interactions between BPI and target bacteria probably involve electrostatic attraction between the cationic regions of BPI and the anionic moieties clustered near the highly conserved lipid A region of LPS. In the outer membrane of intact bacteria, the insertion of BPI triggers the rearrangement of outer membrane lipids (LPS and phospholipids) and displacement of LPS-boundMg2+ and Ca2+, resulting in increased permeability to small hydrophobic molecules, increased susceptibility of phospholipids to phospholipase A attack, and bacterial growth inhibition. In a rabbit model of peritoneal inflammation, extracellular BPI secreted by granulocytes into inflammatory exudates confers potent bactericidal activity against many Gram-negative bacteria. Some defensins, cathelicidins and low concentrations of complement potentiate the activity of BPI. Gram-negative bacteria with long lipopolysaccharide chains (‘Smooth’ colony type) tend to be relatively resistant to BPI. Proteases Human and other mammalian granulocytes contain elastase, cathepsin G and proteinase 3, all of which are highly cationic 25–30-kDa serine proteases with antibacterial properties (serprocidins). Azurocidin/CAP37 is another bactericidal, but enzymatically inactive, member of the same protein family. Azurocidin/CAP37, neutrophil elastase and proteinase 3 are encoded by a cluster of genes located at the tip of the short arm of chromosome 19. Cathepsin G gene is located in another cluster of serine protease genes on chromosome 14q11.2. Compared to the active serine proteases, azurocidin/CAP37 contains amino acid substitutions in two residues essential for catalytic activity (serine and histidine). In granulocytes, serprocidins are located in the phagocytic (primary, azurophil) granules. Their collective abundance in human neutrophils is similar to the other major granule proteins (lactoferrin, lysozyme, myeloperoxidase and defensins). The antibacterial properties of serprocidins can often be dissociated from their proteolytic activity, and are competitively inhibited by increasing ionic strength, indicating the importance of charge interactions in their mechanism of action. Although neutrophil elastase is modestly microbicidal, it also converts inactive procathelicidins into highly microbicidal cathelicidins. Transgenic mice lacking neutrophil elastase are susceptible to sepsis and death from intraperitoneal infections with Gram-negative bacteria. Secretory Phospholipase A2 Human group II phospholipase A2 (PLA2) is a potent 14- kDa enzyme encoded by a gene on chromosome 1p35. The enzyme belongs to a large family of secretory PLA2 enzymes that share similar structure and catalytic machinery, but differ in their preferred substrates. Human group II phospholipase A2 preferentially acts on the ester linkages of phospholipids found in bacterial membranes. The antimicrobial activity of this phospholipase is not shared by other phospholipases even though their catalytic mechanisms are very similar. Group II PLA2 is found in granulocytes, Paneth cells, epithelial secretions, and inflammatory fluids. Against many strains and species of Grampositive bacteria, purified group II PLA2 is active at nanomolar concentrations. Bacterial killing by PLA2 depends on binding to the bacterial surface (Ca2+- independent, presumably to sites in the cell wall), penetration of the cell wall and Ca2+-dependent degradation of membrane phospholipids. Against Gram-negative bacteria, independent antibacterial activity of mammalian group II PLA2 requires millimolar concentrations and is augmented by complement. Moreover, other host defence substances (e.g. bactericidal permeability-increasing protein, BPI) potentiate the action of nanogram per millilitre amounts of the PLA2 by disrupting the bacterial outer membrane. Lactoferrin Similar to the iron-binding plasma protein, transferrin, and encoded by a gene in the same region of chromosome Antimicrobial Proteins and Peptides 4 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
Antimicrobial Proteins and Peptides 3g21-23.lactoferrin is an iron-chelating 80-kDa protein members of the cathelicidin family have been identified in found in the secretory(specific or secondary)granules of bovine neutrophils (indolicidin,Bac5,Bac7,cyclic dode 9. have also bind a ferric cation. reported in mouse,guinea pig,sheep and horse neutro Lactoferrin is bacteriostatic by sequestering essential phils.In the pig and rabbit,neutrophil-derived cathelici iron from many bacterial species but other species (e.g dins have been shown to fur tion as antimicrobial peptide proteolysis of lactoferrin yields a cationic bactericidal ability-increasing protein (pl5a and plsb in the rabbit peptide,lactofe ieh ascitic fluid). lack with specific-granu The disorder is complex and several other granulocyte Other Antimicrobial Polypeptides proteins important in host defence are also deficient.The patients suffer from recurrent bacterial infections. cons It isa Cathelicidins and reproductive epithelia.The N-terminal domain o Cathelicidins terminal domn the proteere domain,cathelin,at the N-terminus of arouid glands and found in the precursor bunotallcathcticidinsiheacties albicans and Streptococcus mutans Calprotectin,a comp of 14-and 8-kDa proteins, secretion.Well-described x d ke It may cathelicidins pl5a and pl5b,which are microbicidal in the ment of professional phagocytes'by inhibiting the containing form 2 proliferation of any organisms that escape confinemen CAP18.is 1 n phagocyt cuoles.Its ability to exert The C.terminal 37 amino acid segment LL37)is a anifnealao uptured gran ratin cysteine-free peptide that readily forms an mphipathic o helix. bactene is Bacs and Bac? and the are exam prol /argini Further Reading the bovine indolicidin is unusually tryptophan-rich while protcin. 6191-205 Lepithet onuclear leukocyte precursors y56:263-27. are likely to b ivage and The human cathelicidin (LL-37/hCAP18)has beer found in neutrophils,testis and inflamed skin.Multiple antimicrobial domain.F Leer. Publishing Group www.els.ne 5
3q21–23, lactoferrin is an iron-chelating 80-kDa protein found in the secretory (specific or secondary) granules of neutrophilic granulocytes and in epithelial secretions such as airway fluid, cervical mucus and breast milk. The molecule contains two similar lobes, each of which can bind a ferric cation. Lactoferrin is bacteriostatic by sequestering essential iron from many bacterial species but other species (e.g. Neisseria gonorrhoeae) have developed mechanisms for pilfering iron from lactoferrin. Naturally occurring limited proteolysis of lactoferrin yields a cationic bactericidal peptide, lactoferricin, whose action is not dependent on iron sequestration. Patients with specific-granule defi- ciency lack lactoferrin in their granulocytes but have normal amounts of lactoferrin in their epithelial secretions. The disorder is complex and several other granulocyte proteins important in host defence are also deficient. The patients suffer from recurrent bacterial infections. Cathelicidins Cathelicidins are a family of antimicrobial peptides and proteins that contain a conserved 100 amino acid domain, cathelin, at the N-terminus of the precursor polypeptide. In most but not all cathelicidins, the active moieties are highly variable cationic C-terminal peptides that are cleaved from the precursor during or after secretion. Well-described exceptions include the rabbit cathelicidins p15a and p15b, which are microbicidal in the uncleaved, cathelin-containing form. The single human member of this family, variously named FALL-39, LL-37 or hCAP18, is encoded by a gene on chromosome 3p21.3. The C-terminal 37 amino acid segment (LL-37) is a cysteine-free peptide that readily forms an amphipathic a helix. The bovine bactenecins Bac5 and Bac7 and the porcine PR-39 are examples of proline/arginine-rich peptides with repetitive segments, porcine prophenins contain repetitive arginine/phenylalanine-rich segments, the bovine indolicidin is unusually tryptophan-rich while porcine protegrins are four-cysteine, arginine-rich peptides that resemble defensins and the horseshoe crab peptides, tachyplesins. For several bovine and porcine cathelicidins, neutrophil elastase has been shown to be the activating enzyme that releases cathelicidin from its inactive precursor. Many other cathelicidin precursors contain a conserved consensus sequence for elastase cleavage and are likely to be activated by the same mechanism. The human cathelicidin (LL-37/hCAP18) has been found in neutrophils, testis and inflamed skin. Multiple members of the cathelicidin family have been identified in bovine neutrophils (indolicidin, Bac5, Bac7, cyclic dodecapeptide and others), in rabbit neutrophils (p15a, p15b and CAP18), and in porcine neutrophils (protegrins, PR- 39, prophenins, and others). Cathelicidins have also been reported in mouse, guinea pig, sheep and horse neutrophils. In the pig and rabbit, neutrophil-derived cathelicidins have been shown to function as antimicrobial peptides in inflammatory fluids, either alone (protegrins in pig abscess fluid) or synergistically with bactericidal permeability-increasing protein (p15a and p15b in the rabbit ascitic fluid). Other Antimicrobial Polypeptides Secretory leucoprotease inhibitor (SLPI, antileucoprotease) is a 12-kDa cationic protein consisting of two similar subdomains. It is abundant in the secretions of respiratory and reproductive epithelia. The N-terminal domain of SLPI is weakly antibacterial and antifungal whereas its Cterminal domain contains the protease inhibitory activity. Histatins are a family of 24–38 amino acid, histidine-rich peptides produced in the parotid glands and found in saliva. Their antimicrobial spectrum includes Candida albicans and Streptococcus mutans. Calprotectin, a complex of 14- and 8-kDa proteins, is remarkably abundant in the cytoplasm of granulocytes and keratinocytes. It may protect the cytosolic compartment of ‘professional phagocytes’ by inhibiting the proliferation of any organisms that escape confinement in phagocytic vacuoles. Its ability to exert antifungal and antibacterial activity after release from ruptured granulocytes or keratinocytes is inhibited by zinc. Further Reading Elsbach P, Weiss J and Levy O (1994) Integration of antimicrobial host defenses: role of the bactericidal/permeability-increasing protein. Trends in Microbiology 2: 324–328. Ganz T and Lehrer RI (1995) Defensins. Pharmacology and Therapeutics 66: 191–205. Ganz T and Weiss J (1997) Antimicrobial peptides of phagocytes and epithelia. Seminars in Hematology 34: 343–354. Levy O (1996) Antibiotic proteins of polymorphonuclear leukocytes. European Journal of Haematology 56: 263–277. Marsh J and Goode JA (eds) (1994) Antimicrobial Peptides. CIBA Foundation Symposium 186. Chichester: John Wiley & Sons. Zanetti M, Gennaro R and Romeo D (1995) Cathelicidins: a novel protein family with a common proregion and a variable C-terminal antimicrobial domain. FEBS Letters 374: 1–5. Antimicrobial Proteins and Peptides ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 5