Defensins

Pfiarmac.

Pergamon

0163-7258(94)00074-X

Thu. Vol. 66, pp. 191-205, 1995 Copyright 0 1995 Elwier Science Ltd Printed in Great Britain. All rights reserved 0163-7258/95 $29.00

Associate Editor: D. GRUNBERGER

DEFENSINS TOMAS GANZ* and ROBERT

I. LEHRER

Department of Medicine, University of California School of Medicine, Los Angeles, CA 90024-1736, U.S.A.

Abstract---Defensins are a family of small cationic, antibiotic peptides that contain six cysteines in disulfide linkage. The peptides are abundant in phagocytes and small intestinal mucosa of humans and other mammals and in the hemolymph of insects. They contribute to host defense against microbes and may participate in tissue inflammation and endocrine regulation during infection. Bioengineered defensins are potentially useful as prophylactic and therapeutic agents in infections. Keywords-Host defense, phagocytes, antibiotic peptides, mucosal immunity.

CONTENTS 191 193 196 196 197 198 198 198 200 200 200 200

1. Introduction 2. ‘Classical’Defensins 3. j?-Defensins 4. Insect Defensins 5. Biosynthesis of Defensins 6. Defensin Genes and Their Regulation 7. Disorders of Defensin Synthesis 8. Mechanisms of Action of Defensins 9. Defensin-binding Proteins 10. Clinical Applications 11. Summary References

1. INTRODUCTION Throughout their lifespans, multicellular organisms encounter a myriad of microbes, most often wi. out any adverse consequences. To establish residence on or in the host, the microbe must survive ins. le host cells or within the materials that cover or bathe the cells. Plants and animals have evolved a variety of antimicrobial substances that restrict the ability of microbes to take up residence in these environments. Some cell types specialized in host defense and developed heightened capacities for the detection of microbes, their physical confinement and destruction. Other host defense cells function predominantly as secretory cells that release antimicrobial substances into extracellular spaces. Yet others, cytotoxic lymphocytes, facilitate the clearance of infections by recognizing and killing host cells that harbor intracellular microbes. However, the more usual contribution of lymphocytes to host defense is to potentiate other microbicidal cells, either by paracrine signals (lymphokines) that activate microbicidal cells, or by secreting antibodies, adapter molecules that form bridges between microbial targets and receptors on microbicidal effector cells. These lymphocyte responses increase after repeated exposure to a particular microbial species or foreign substance, giving rise to ‘adaptive’ immunity that generates a more effective host defense response. *Corresponding author. adrenocorticotropic hormone; BNBD, bovine neutrophil /3-defensins; GAL, gallinacin; TAP, tracheal antimicrobial peptide. Abbreviations-ACTH,

191

T. Ganz and R. I. Lehrer

192

Adaptive immunity is a relatively recent evolutionary development that has reached prominence only in higher vertebrates. In contrast, the morphologic and functional properties of most microbicidal effector cells are surprisingly similar among molluscs, and insects and humans. A near-universal type of microbicidal effector cell, the ‘professional’ phagocyte, destroys microbes after ingesting them by a process called phagocytosis and sequestering them into vacuoles. The phagocytic vacuole allows this effector cell to target microbicidal substances onto the microbes and accumulate very high local concentrations of these endogenous antibiotics without perturbing the surrounding cells of the host. Antimicrobial substances employed by host cells range from simple inorganic chemicals (e.g. hydrogen peroxide, hypochlorous acid, nitric oxide) to relatively complex antimicrobial peptides and proteins. The proteins are widely distributed on mucosal epithelial surfaces, in body fluids and in the microbicidal organelles of phagocytic cells. They vary in size, structure and activity, but most are cationic, complementing the anionic charge of microbial cell surfaces. Although some antimicrobial proteins are enzymes (e.g. proteases or muramidases) that can digest the protective layers of microbes, the ability of many antimicrobial proteins to disrupt biological membranes may be their common mode of action. The generation of high local concentrations of microbicidal peptides on demand may be facilitated by specialized lysosome-like organelles, cytoplasmic ‘granules’, capable of storing large amounts of antimicrobial proteins in inactive or latent form. Antimicrobial proteins smaller than 100 amino acids will be arbitrarily referred to as antimicrobial ‘peptides’. Some antimicrobial peptides are stabilized by disulfide crosslinks, which may also increase their resistance to proteolysis or denaturation. Antimicrobial peptides that contain six cysteines have been classified as defensins. Defensins form at least three structural groups whose evolutionary relationship is uncertain: the originally described “classical” defensins and the subsequently ?luman HNPl

ACYCRIPA-CIAGERRYGTCIYQGRLWAFCC

HNP2

CYCRIPA-CIAGERRYGTCIYQGRLWAFCC

HNP3

DCYCRIPA-CIAGERRYGTCIYQGRLWAFCC VCSCRLVF-CRRTELRVGNCLIGGVSFTYCCTRV

HNP4 Guinea

GPNPl GPNP2

30 29 30 33

Pig RRCICTTRT-CRFPYRRLGTCIFQNRVYTFCC RRCICTTRT-CRFPYRRLGTCLFQNRVYTFCC

31 31

Rabbit

NPl NP2 NP3A NP3B NP4 NP5 NP6

WCACRRAL-CLPRERRAGFCRIRGRIHPLCCRR WCACRRAL-CLPLERRAGFCRIRGRIHPLCCRR GICACRRRF-CPNSERFSGYCRVNGARYVRCCSRR GRCVCRKQLLCSYRERRIGDCKIRGVRFPFCCPR VSCTCRRFS-CGFGERASGSCTVNGVRHTLCCRR VFCTCRGFL-CGSGERASGSCTINGVRHTLCCRR GICACRRRF-CLNFEQFSGYCRVNGARYVRCCSRR

33 33 34 34 33 33 34

Rat

RTNPl RTNPZ RTNP3 RTNPI Mouse CRYPT1 CRYPT2 CRYPT3 CRYPT4 CRYPT5

VTCYCRRTR-CGFRERLSGACGYRGRIYRLCCR VTCYCRSTR-CGFRERLSGACGYRGRIYRLCCR CSCRTSS-CRFGERLSGACRLNGRIYRLCC ACYCRIGA-CVSGERLTGACGLNGRIYRLCCR LRDLVCYCRSRG-CKGRERMNGTCRKGHLLYTLCCR LRDLVCYCRTRG-CKRRERMNGTCRKGHLMYTLCCR LRDLVCYCRKRG-CKRRERMNGTCRKGHLMYTLCCR GLLCYCRKRGHCKRGERVRGTCGIRFLY---CCPR LSAKKLICYCRIRG-CKRRERVFGTCRNLFLTFVFCC

32 32 29 31 35 35 35 31 36

Fig. 1. Amino acid sequences of classical defensins obtained by peptide sequencing. The conserved cysteine framework is highlighted. NP, neutrophil peptide; H, human; RT, rat; GP, guinea pig; CRYPT, cryptdin (murine intestinal defensin). For cryptdins, the numbering scheme of Selsted et al. (1992) is used.

Defensins PROPIECE

SIGNAL NPl NPS NP3A HNPl HNP4 HD5 HD6 GPDEF CRYPTA

MRTLALLAAILLVALQAQA MRTLALJJJJILLVTLQAQA MRTLILLAAILLAALQAQA MRTLAILAAILLVALQAQA MRIIALLAAILLVALQVRA MRTIAILAAILLVALQAQA MRTLTILTAVLLVALQAKA MRTVPLFAACLLLTLMAQA MKKLVLLFALVLLGFQVQA *. . . . * .* . . ..* PROPIECE

NPl NP5 NP3A HNPl HNP4 HD5 HD6 GPDEF CRYPTA

SSALEALGVKAG TSPLEVLGAKAG SSALQVPDTK SLAPKHPGSRKNM SSALQVSGSTRGM LSALRTSGSQARA SSSLRALGSTRAF STSLEDAGAGAG GTSLQE-ES

193

EHVSVSIDEW-------DQQPPQAEDQDVAIYVKEHE ELHSGMADDGV-------DQQQPRAQDLDVAWIKQDE ELFSVNVDEVL-------DQQQP-GSDQDLVIHLTGEE EPLQARADEVAA------APEQIAADIPEWVSL,AWDE GPLQARGDE-AP------GQEQRGPEDQDISISFAWDK ESLQERADE-AT------TQKQSGEDNQDLAISFAGNG EPLQAEDDPLQAKAYEADAQEQRGANDQDFAVSFAEDA EPLPRAADH-------SDTKMKGDREDHVAVISFWEEE DSIQNTDE-------ETKTEEQPGEEDQAVSVSFGDPE . . . ...

MATURE PEPTIDE 4 12 3 56 WCACRRALCLPRERRAGFCRIRGRIHPLCCRR VFCTCRGFLCGSGERASGSCTINGVRHTLCCRR GICACRRRFCPNSERFSGYCRVNGARYVRCCSRR ACYCRIPACIAGERRYGTCIYQGRLWAFCC VCSCRLVFCRRTELRVGNCLIGGVSFTYCCTRVD TCYCRTGRCATRESLSGVCEISGRLYRLCCR TCHCR-RSCYSTEYSYGTCTVMGINHRFCCL RRCICTTRTCRFPYRRLGTCIFQNRVYTFCC LRDLVCYCRSRGCKGRERMNGTCRKGHLLYTLCCR * * ** * *. * .

95 95 93 94 97 94 100 93 93

Fig. 2. Amino acid sequences of representative classical preprodefensins determined from cDNA or genomic DNA sequences. HNP, human neutrophil peptide; NP, (rabbit) neutrophil peptide; HD, human (Paneth cell) defensin; GPDEF, guinea pig (neutrophil) defensin; CRYPTA, cryptdin A (mouse Paneth cell defensin). Conserved residues are marked by asterisks, similar residues by dots. The cleavage sites for mature defensins in HD-5 and -6 are based on similarity to HNP-I, but have not been determined experimentally. In the mature peptide, cysteines are in bold and numbered 1-6.

discovered /I-defensins and insect defensins (Figs 14). The three defensin other in the spacing and connectivity of their six cysteine residues.

2. ‘CLASSICAL’

groups differ from each

DEFENSINS

These mammalian peptides are stored in high concentrations in granules, specialized organelles of phagocytes (granulocytes and some macrophages) and Paneth cells, secretory cells located at the base of ‘crypts’, recesses between intestinal villi. The context within which these peptides were discovered strongly suggested that host defense was their predominant function and inspired the name ‘defensins’ (Ganz et al., 1985a; Selsted et al., 1985a). Defensin peptides make up about 5% of the total protein of human granulocytes and are the principal proteins in their denser (azurophil) granules (Ganz, 1987; Rice et al., 1987). Like other azurophil granule constituents, defensins are delivered into phagocytic vacuoles by granule fusion, a process that is well documented by electron microscopy of phagocytosing granulocytes. Subcellular fractionation studies of granulocytes that had ingested bacteria showed that defensins are among the most abundant proteins of phagocytic vacuoles (Joiner et al., 1989). The lighter (specific) granules of granulocytes contain little or no defensin and are thought to function as the exocytic or secretory organelles of granulocytes. Indeed, granulocytes that were stimulated with secretagogs (e.g. phorbol myristate acetate) or phagocytic stimuli (serum-treated zymosan particles), released most of their secretory granule lactoferrin, but very little of their defensin (Ganz, 1987). In contrast, Paneth cell defensins are probably secreted by exocytosis of granules into crypts, tubular spaces between intestinal villi

T. Ganz and R. I. Lehrer

194

(Selsted et al., 1992). Since the crypts are very narrow, even this extracellular environment may favor relatively high local concentrations of defensins. The tissue distribution of defensins (Table 1) is species-dependent: murine granulocytes lack detectable defensins (Eisenhauer and Lehrer, 1992) although these are abundant in rat granulocytes (Eisenhauer et al., 1989, 1990). The diverse nondefensin antimicrobial substances apparently confer enough redundancy to ensure that the lack of defensins in murine granulocytes is well tolerated. However, in the murine intestine, more than 10 defensin (cryptdin) genes are transcribed (Huttner et al., 1994). Small amounts of defensins have apparently been detected in cell types other than phagocytes or Paneth cells but these cells have not yet been characterized in detail (Tominaga et al., 1992). The defensins of human, rabbit, guinea pig and rat granulocytes, rabbit alveolar macrophages and murine intestinal Paneth cells have been purified, sequenced and assayed for antimicrobial activity (Zeya and Spitznagel, 1966a,b; Patterson-Delafield et al., 1980; Selsted et al., 1983, 1985a, 1992; Lehrer et al., 1983; Ganz et al., 1985a; Loeffelholz and Modrzakowski, 1986; Selsted and Harwig, 1987; Eisenhauer et al., 1989, 1992; Ouellette et al., 1989, 1992; Wilde et al., 1989; Yamashita and Saito, 1989). In addition, the amino acid sequences of two human intestinal Paneth cell defensins have been deduced from cDNA or genomic DNA clones only (Jones and Bevins, 1992, 1993). The three-dimensional structure of human defensin HNP-3 was established by X-ray crystallography (Hill et al., 1991), and those of human HNP-1 and rabbit NP-1 and NP-5 were derived from 2D-NMR studies (Pardi et al., 1988, 1992; Bach et al., 1987; Levy et al., 1989; Kominos et al., 1990; Zhang et al., 1992). Although these structural studies sampled only a few of the more than 20 known defensin species, it is likely that all classical defensin molecules have a similar conformation consisting of a triple-stranded P-sheet structure and a connecting loop as a base from which a P-hairpin hydrophobic finger protrudes nearly orthogonally. In crystals, human defensins form dimeric structures that allow the two hairpins to appose in a four-stranded hydrophobic fi sheet that forms the narrow part of a funnel. The broader polar and cationic top of the funnel is formed by a six-stranded P-sheet and two connecting loops facing away from each other. Higher order multimers are likely to form in solution and in the lipid bilayer of target cells. The connectivity of the six cysteines has been established by direct chemical analysis (Selsted and Harwig, 1989) and confirmed by crystallographic and 2D-NMR data as l-6,24,3-5, so that defensins are effectively cyclic peptides. The conservation of peptide fold is contrasted with the tolerance of the structure for naturally occurring amino acid substitutions (Fig. 1). It is not yet clear whether the remarkable sequence variability of defensins reflects evolutionary drift or specific genetic mechanisms for generating peptide diversity. The chemical synthesis of several defensins in a biologically active form was reported recently (Rao et al., 1992; Fuse et al., 1993) and some synthetic defensins are available commercially. The production of defensins in genetically engineered bacteria has been problematic (Piers et al., 1993) but human prodefensin

HNP-1 is readily engineered

SIGNAL

PROPIECE

MRLHHLLLALLFLVLSAWSGFTQGVG

TAP

MATURE 1

PEPTIDES 2

3

4

56

NPVSCVRNKGICVPIRCPGSMKQIGTCVGRAVKCCRKK

TAP

DFASCHTNGGICLPNRCPGHMIQIGICFRPRVKCCRSW

BNBD-1 BNBD-6

AND

into and expressed in baculovirus-infected

pEGVRNHVTCRIYGGFCVPIRCPGRTIQRGTCFGRPVKCCRRW GPLSCRRNGGVCIPIRCPGPMRQIGTCFGRPVKCCRSW

BNBD-11

GRKSDCFRKSGFCAFLKCPSLTLISGKCSRFYL-CCKRIW * * ** * * *

GAL-l

**

Fig. 3. Amino acid sequences of representative j?-defensins and a precursor peptide. Conserved residues are marked by asterisks. In the mature peptide, cysteines are in bold and numbered l-6. Posttranslationally

generated

pyroglutamyl

residues are noted as PE.

insect

195

Defensins

SIGNAL AND PROPIECE PHOTE

MKFFMVFWTFCLAVCFVSQSLAIPADAANDAHFVDGVQALKEIEPELHGRYKRA

54

SARPE

MKSFIVLAVTLCLAAFFMGQSVASPAAAAEESKFVDGLHALKTIEPELHGRYKRA

54

MATURE

PEPTIDES 1

2

3

4

PHOTE

ATCDLL----SGTGINHSACAAHCL-LRGNRGGYCNG--KGVCVCRN

SARPE

ATCDLL----SGTGINHSACAAHCL-LRGNRGGYCNG--KAVCVCRN

AESCY

5

6 94 94

GF---GCPLDQMQCHRHCQTITGRSGGYCSGPLKLTCTCYR

38

ZOPAT

FTCDVLGFEIAGTKLNSAACGAHCL-ALGRRGGYCNS--KSVCVCR

APIME

VTCDLLSFK---GQVNDSACCL-SLGKAGGHCE---KGVCICRKTSF~L~KYF * * * * * ** *

43 51 *

Fig. 4. Amino acid sequences of selected insect defensins and their prepropeptides. PHOTE, Phormia terranovae (black blowfly); SARPE, Sarcophaga peregrina (fleshfly); AESCY, Aeschna cyanea (dragonfly); ZOPAT, Zophobas stratus (a beetle); APIME, Apis mell~fera (honey bee). Conserved residues are marked by asterisks. In the mature peptide, cysteines are in bold and numbered l-6.

cells (Valore quantitatively

et al.,

1994). ProHNP-

cleaved

with

cyanogen

1, purified bromide

from

media

to yield

conditioned

fully

active

by these cells, can be nearly peptide

HNP-1. and gram-negative bacteria, mycobacteria, fungi and enveloped viruses, even including human immunodeficiency virus (Patterson-Delafield er ul., 1980, 1981; Lehrer et al., 1983, 1985a,b, 1986, 1989; Selsted et al., 1984, 1985b, 1992; Ganz et al., 1985a; Segal et al., 1985; Daher et ai., 1986; Levitz ef ul., 1986; Greenwald and Ganz, 1987; Selsted and Harwig, 1987; Shafer et al., 1988; Eisenhauer et ul., 1989; Yamashita and Saito, 1989; Cullor et al., 1990, 1991; Miyasaki et al., 1990a,b; Borenstein et al., 1991; Kohashi et al., 1992; Ogata et al., 1992; Wu et al., 1992; Nakashima et al., 1993). The activity of granulocyte defensins against many bacteria and fungi is substantially inhibited by serum proteins and cations, such as sodium and calcium. The sodium effect probably reflects the inhibition of nonspecific electrostatic interactions between the cationic defensins and anionic microbial surfaces. The calcium effect, most evident in gram-negative bacteria, occurs at millimolar, concentrations and may result from competition for specific binding sites in the outer membrane of these organisms. Interventions that block the generation of metabolic energy by microbes or discharge microbial transmembrane potential diminish the activity of defensins (Lehrer et al., 1988b. 1989; Sawyer et al., 1988). Published comparisons of microbicidal activities of the various defensins and other antimicrobial peptides must be interpreted with caution since these activities depend critically on the selection of microbial targets, conditions of testing and the growth phase of the target organism. In general, the more cationic defensins are more active in microbicidal assays, but modifications that disrupt the conformation of defensins without altering their charge decrease or abolish the microbicidal activity of defensins. Thus, cationicity by itself is not sufficient for microbicidal activity. Pathogenic strains of Salmonella typhimurium are relatively resistant to defensins and other antimicrobial peptides, and this resistance involves the selective activation of a number of bacterial virulence genes (Fields et al., 1989; Groisman et al., 1989; Groisman and Saier, 1990; Miller et al., 1990; Miller, 1991). Conversely, mutations that decrease the resistance to defensins are frequently associated with diminished virulence of Salmonella in the mouse model (Groisman et al., 1992). These findings suggest that defensins or similar host substances play an important role in host defense and, in turn, induce adaptive microbial countermeasures. At somewhat higher concentrations, and in the absence of serum, the peptides can be cytotoxic to mammalian cells in vitro, and may contribute to neutrophil-mediated antibody-dependent lysis of tumor cells (Lichtenstein et al., 1986, 1988a,b; Okrent et al., 1990; Gera and Lichtenstein, 1991; Lichtenstein, 1991; Barker and Reisfeld, 1993). Defensins may also modulate inflammation and repair. The barrier permeability of Madin-Darby canine kidney monolayers to mannitol was increased up to 5-fold by human defensins HNPl-3 without evidence of cytotoxicity (Nygaard et al., 1993). Both human and rabbit defensins stimulated DNA synthesis and growth in Various

classical

defensins

are active at l-100 pg/ml against gram-positive

T. Ganz and R. I. Lehrer

196

Nakano mouse lens epithelial cells, with an optimum defensin concentration for HNP-l-3 at l-3 PM (3.5-10 pg/mL) and had similar effects on fibroblasts (Murphy et al., 1993). Defensins have been reported to be chemotactic for monocytes (Territo et al., 1989) and to inhibit the action of adrenocorticotropic hormone (ACTH) on adrenal cells by interacting with the ACTH receptor (Zhu et al., 1987, 1989; Hu et al., 1991; MacLeod et al., 1991; Solomon et al., 1991; Zhu and Solomon, 1992; Singh et al., 1988; Tominaga et al., 1990). The latter property has been referred to as corticostatic activity, and the defensins that mediate it have also been called “corticostatins”. In the aggregate, these noncytotoxic activities could blunt the release of immunosuppressive cortisol during stress from infection, and up-regulate tissue inflammation and repair. Based on the measured amounts of defensin in each granulocyte (e.g. 3-5 pg/million human granulocytes) and the known number of defensin-containing granules per granulocyte (about 1000 in humans), high concentrations of classical defensins (at least l-10 mg/mL) are likely to exist in phagocytic vacuoles containing ingested microbes. The three major human granulocyte defensins, HNP-l-3, account for almost 99% of the total defensin content of these cells. The concentrations of HNP4 in granulocytes are about lOO-fold lower (Gabay et al., 1989). The recent development of sensitive immunoassays facilitated measurements of extracellular concentrations of human defensins (Panyutich et al., 1991; Shiomi et al., 1993). The concentrations of HNP-1-3 in plasma in normal volunteers are about 40 ng/mL but rise to the > 1 pg/mL range during severe infections (Shiomi et al., 1993; Panyutich et al., 1993). Serum defensin concentrations may correlate with the severity of infection, perhaps by reflecting the number and intensity of granulocyte encounters with microbes or their products. The local concentrations of defensins in infected tissues are probably much higher, but have not yet been measured systematically. The local concentrations of mucosal defensins are of much interest, but as yet, completely unknown.

3. ,&DEFENSINS /I-Defensins contain 38-42 amino acid residues and have been found in bovine trachea (tracheal antimicrobial peptide, TAP) (Diamond et al., 1991) bovine granulocytes (bovine neutrophil P-defensins, BNBD-l-13) (Selsted et al., 1993) and in chicken leukocytes (gallinacin (GAL)-la, GAL- 1, GAL-2) (Harwig et al., 1994). The six cysteines of the bovine p-defensin BNBD- 12 are linked l-5,2-4 and 3-6 (Tang and Selsted, 1993). The bovine and avian peptides share a 9-residue consensus sequence, and are active against both gram-positive and gram-negative bacteria in vitro at peptide concentrations of lo-100 pg/mL. In situ hybridization studies indicate that the bovine tracheal /I-defensin TAP is produced by epithelial cells (Diamond et al., 1993). Unlike the intestinal Paneth cells that produce classical epithelial defensins, the TAP-producing cells lack visible granules.

4. INSECT DEFENSINS The hemolymph of insects acquires antimicrobial activity after the insects are challenged by injury or microbial penetration (Hoffmann and Hoffmann, 1990). Insect defensins are a large family of peptides found in the hemolymph of several insect orders (Matsuyama and Natori, 1988; Lambert et al., 1989; Dimarcq et al., 1990; Bulet et al., 1991, 1992; Hoffmann and Hetru, 1992; Cociancich et al., 1993b), and account for most of the antimicrobial activity of hemolymph in dragonfly larvae Table 1. Tissue Distribution of Defensins and B-Defensins in Mammals Species Human Rabbit Guinea pig Mouse Rat Cow (B-defensins)

Granulocytes

Macrophages

HNP-14 NP-1-5

NP 1,2 (lung)

GPNP none RtNP-1-4 BNBD-I-13

Paneth cells

Tracheal epithelium

HD5,6 CRYPTDINS TAP

197

Defensins

PRE

MRTLAILAAILLVALQAQA

PRO BOTH

MAT

41 ACYCRIPACIAGERRYGTCIYQGRLWAFCC I

I I

I

I 1

Fig. 5. Posttranslational processing of human preprodefensins HNP-1 and HNP-2. The arrows indicate sites of proteolytic cleavage as documented in polymorphonuclear leukocytes (PMN), HL-60 cells or both (Valore and Ganz, 1992; Harwig et al., 1992). PRE, PRO and MAT designate the signal sequence, propiece and mature peptide, respectively. (Hoffmann and Hoffmann, 1990). The peptides are produced by fat body cells and thrombocytoids, a blood cell type. They are 38-45 amino acids long, and consist of an a-helix linked by a loop to an antiparallel B-sheet (Bonmatin et al., 1992; Hanzawa et al., 1990). The connectivities of the six cysteines are 14, 2-5, 3-6 (Lepage et al., 1991). Under the conditions of the reported assays, the

peptides were predominantly

active against gram-positive organisms.

5. BIOSYNTHESIS

OF DEFENSINS

The cDNAs for representative members of all three classes of defensins have been cloned (Daher et al., 1988; Ganz et al., 1989; Ouellette and Lualdi, 1990; Diamond et al., 1991; Nagaoka et al., 1991; Jones and Bevins, 1992; Michaelson et al., 1992; Lin et al., 1992; Palfree et al., 1993; Dimarcq et al., 1990). All three types of defensins are synthesized initially as preprodefensins, consisting of a characteristic amino terminal signal sequence for insertion into the endoplasmic reticulum, a propiece, and the mature peptide at the carboxy terminal end of the prepropeptide. The posttranslational processing and sorting pathway for human defensins HNP- l-3 has been explored in detail both in the naturally defensin-producing myeloid cell line HL-60 (Valore and Ganz, 1992) and in the reconstituted system consisting of various murine cell lines transduced with human defensin cDNA (Ganz et al., 1993). All cell lines rapidly (probably cotranslationally) remove the 19 amino acid signal sequence and yield proHNP-1 as the first detectable HNP-1 precursor. The proteolytic pathway required to produce mature HNP-1 from proHNP-1 is active only in myeloid or closely related cell types, such as human HL-60 or murine 32D c 13 cells. These cell types process most newly synthesized prodefensin to mature defensin and store the mature peptide in cytoplasmic granules, and constitutively secrete a smaller amount of unprocessed prodefensin. Nonmyeloid murine AtT-20 pituitary adenoma cells store unprocessed prodefensin in their cytoplasmic granules and secrete the prodefensin when stimulated with secretagogues. Murine fibroblasts (NIH 3T3 cells) constitutively secrete almost all prodefensin. Analysis of the amino acid sequences of most known prodefensins (deduced from their cDNA sequences) reveals an unusual amino acid distribution, such that the anionic charge of the propiece neutralizes the cationic charge of the mature peptide at or near neutral pH. This has led to suggestions that the propiece interacts with the mature peptide during defensin biosynthesis to facilitate defensin folding and/or to prevent spurious interactions with other proteins or lipid membranes (Michaelson et al., 1992). To test this hypothesis, human prodefensin proHNP-1 was prepared in milligram quantities in recombinant-baculovirus-infected insect cells. As anticipated, proHNP-1 lacked microbicidal activity, but could be activated by the removal of the propiece by cyanogen bromide cleavage at a naturally occurring methionine (Valore et al., 1994). Studies in murine cells transduced with wild-type or altered HNP-1 cDNA suggested that the propiece of proHNP-1 is also important

T. Ganz and R. I. Lehrer

198

for normal subcellular trafficking during defensin biosynthesis (Liu and Ganz, 1994). Deletions of a conserved region in the carboxyterminal half of the propiece severely diminished the normal constitutive secretion of proHNP- 1 in both 3T3 fibroblasts and 32D myeloid cells and ablated the posttranslational proteolytic processing of proHNP-1 that normally takes place in 32D myeloid cells.

6. DEFENSIN

GENES AND THEIR

REGULATION

The genes encoding classical defensins are characterized by extraordinary multiplicity and rapid evolution of the sequences that encode the mature protein (Ganz et al., 1989; Jones and Bevins, 1992; Nagaoka et al., 1992a; Linzmeier et al., 1993; Huttner et al., 1994). For reasons that are not understood, portions of the S-untranslated region and the signal sequence are highly conserved, a feature that has facilitated the cloning of previously unknown defensin genes. Myeloid defensin genes contain three exons, as compared with only two exons in enteric defensin genes. In each, the preproand mature parts of defensins are encoded by separate exons, an arrangement that may favor the recombination of the prepro- segment of defensins with an apparently unrelated mature peptide. Such recombination could target dissimilar mature peptides to the same subcellular compartment. In addition to genetic events, peptide diversity may be generated by differential posttranslational processing that produces peptides with differing N-termini (Ganz et al., 1985a; Daher et al., 1988; Selsted et al., 1992; Valore and Ganz, 1992). The production of some classical defensins (murine intestinal cryptdins and rabbit macrophage cationic peptides) is induced around the time of birth (Ganz et al., 1985b; Ouellette et al., 1989), while other defensins (rabbit macrophage cationic peptides, tracheal antimicrobial peptide and insect defensins) are synthesized in increased amounts after host exposure to microbial substances (Lehrer et al., 1981; Diamond et al., 1993; Lambert et al., 1989; Dimarcq et al., 1990). The acute phase transcriptional element NF-kB was found in the promoter region of the tracheal antimicrobial peptide gene (Diamond et al., 1993) and of several insect antimicrobial peptides and proteins known to be induced by microbial invasion (Hultmark, 1993). This element may prove to be important in the induction of many antimicrobial peptides. A different regulatory system may operate in granulocyte defensin genes (both classical and j?) that are synthesized in the bone marrow precursors of blood granulocytes. Defensin gene transcription in granulocyte precursors is likely to be relatively brief and massive, peaking probably in the late promyelocyte and terminating in the myelocyte. This timing of defensin gene transcription is supported by the detection of defensin mRNA by in situ hybridization in promyelocytes and myelocytes (Nagaoka et al., 1992b), defensin expression in cell lines with promyelocytic characteristics (Daher et al., 1988), and the lack of defensin mRNA in mature granulocytes (Daher et al., 1988) and in the human granulocyte maturation disorder ‘specific granule deficiency’, that predominantly involves proteins synthesized during the myelocyte stage (Johnston et al., 1992).

7. DISORDERS

OF DEFENSIN

SYNTHESIS

Defensin concentrations of 10% of normal are seen in patients with ‘specific granule deficiency’, a rare disorder of granulocyte development (Ganz et al., 1988; Johnston et al., 1992). In this congenital disease, synthesis of late (myelocyte) granule proteins is decreased or absent, due to the lack of the corresponding mRNAs, and affected patients suffer from frequent infections caused by common bacteria (Breton Gorius et al., 1980; Boxer et al., 1982; Gallin et al., 1982; Ambruso et al., 1984). Acquired defensin deficiency is seen occasionally in patients with chronic myelogenous leukemia, but its clinical consequences have not been explored (Borregaard et al., 1993).

8. MECHANISMS

OF ACTION

OF DEFENSINS

Classical and insect defensins form channels in microbial, mammalian and artificial lipid membranes, increasing the membrane permeability in a charge- or voltage-dependent

Defensins

199

manner (Lehrer et al., 1988a; Sawyer et al., 1988; Kagan et al., 1990; Fujii et al., 1993; Cociancich et al., 1993a). Permeabilization can be inhibited by agents or interventions that interfere with the generation of metabolic energy or decrease the electromotive force across the membrane. The interaction of classical defensins with permease-deficient gram-negative bacteria (Escherichia cofi ML-35 containing the B-lactamase plasmid pBR 322) was analyzed (Lehrer et al., 1988a, 1989) by measuring the conversion of externally applied colorigenic substrates by fi-lactamase and P-galactosidase. enzymes confined, respectively, to the periplasmic and cytoplasmic compartments of the bacterial target. In these bacteria, the extracellular substrates reach their respective enzymes at a rate that is proportional to the permeability of intervening membranes. Defensins induced sequential permeabilization of the outer and inner membranes of the target. Human defensins permeabilized only metabolically active bacteria. In comparison, permeabilization by the more cationic rabbit defensin NP-1 was both faster and less dependent on bacterial energy metabolism, suggesting that cationicity is one of the determinants of defensin activity. Studies with classical defensins and mammalian cell targets indicate that permeabilization of the membrane may be necessary, but not sufficient, for defensin-induced cell death, since there is a limited period of time during which cells can be rescued after they have become permeable to trypan blue dye (Lichtenstein et al., 1988a; Lichtenstein, 1991). The second, irreversible phase of defensin-mediated toxicity to mammalian cells can be inhibited by excess calcium, or the combination of azide and 2-deoxyglucose, which prevents the production of metabolic energy. Agents that interfere with protein synthesis (cycloheximide or actinomycin D) potentiate the second phase of defensin-mediated injury, presumably by interfering with repair processes. Artificial phospholipid membranes were permeabilized by classical defensins only when an electromotive force was applied across the membrane, with a polarity that would cause the cationic defensin molecule to insert into the phospholipid bilayer (Kagan et al., 1990). Membrane conductance increased as the second to fourth power of the defensin concentration. suggesting that two to four defensin molecules interacted to form a channel. Studies of defensin structure by X-ray crystallography and two-dimensional NMR led to models of channel formation by defensins (Hill et al.. 1991) but these have not been experimentally verified yet. We envision that defensin effects on microbes and mammalian cells take place in two phases. In the first phase, the (energized) cell membrane is permeabilized by defensin molecules that enter the membrane under the influence of electromotive forces to form multimeric complexes. Inhibition of permeabilization by cytochalasin B in mammalian cells may indicate that cytoskeletal rearrangements occur that act to enlarge or stabilize the defensin-induced pores. Unless these lesions are repaired or the defensins are removed, a second phase results in irreversible target cell injury. The second phase may depend on the entry of defensins into the cells causing lethal events, the nature of which remains to be ascertained. Recent studies of insect defensins acting on Micrococcus luteus indicate that insect defensins act by a similar mechanism, forming membrane channels that leak potassium and induce limited membrane depolarization (Cociancich et al., 1993a). Depletion of ATP and inhibition of cellular respiration occur subsequently and are not directly due to depolarization. Classical defensins permeabilize both the outer and inner membranes of gram-negative bacteria. The former activity probably is not shared by most insect defensins, since they are reported to be selectively active against gram-positive organisms. Although the relevant data are not yet available, b-defensins would be expected to act by the same mechanism as classical defensins. Another defensin bioactivity that has been examined in some detail is the ‘corticostatic’ effect of defensins. The production of corticosterone by rat adrenal cells is competitively inhibited by some defensins (‘corticostatins’) that interact with the ACTH receptor (Tominaga et al., 1990; Zhu and Solomon, 1992). The prototype peptide, rabbit defensin NP-3A, exerted this activity at concentrations of 5-500 nM (20-2000 ng/mL). Defensins may act by mimicking a cationic segment of ACTH that participates in ACTH binding to the receptor, but does not activate it. However, the report that rabbit NP-6-des-gly is a 16-fold less potent corticostatin than NP-6 is not readily explained by this hypothesis (Fuse et al., 1993).

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9. DEFENSIN-BINDING

PROTEINS

cc-ZMacroglobulin and especially its activated form (Panyutich and Ganz, 1991), plasma serpins a-1-antitrypsin, a-l-antichymotrypsin and antithrombin III (Panyutich and Ganz, 1994; unpublished), and Cl-complement (Panyutich and Ganz, 1994) are the principal binding proteins for human defensin HNP-1 in plasma. The defensin-binding proteins may confine the activity of granulocyte defensins to the phagosome and prevent cytotoxicity to surrounding tissues. It is likely that one or more of these proteins are active defensin carriers that facilitate clearance of defensins from tissues and blood. Each of these defensin-binding proteins interacts with receptors on macrophages or hepatocytes and undergoes receptor-mediated internalization. Further studies are required to determine to what extent defensins are cointernalized with their binding proteins, and whether they are degraded or reused by macrophages as microbicidal proteins.

10. CLINICAL

APPLICATIONS

Plasma levels of human neutrophil defensins HNP-l-3 may serve as specific markers of neutrophil-mediated inflammation (Panyutich et al., 1991; Shiomi et al., 1993). Monoclonal and polyclonal antibodies to HNP-1-3 specifically label neutrophils in tissue sections and could be useful in viva for imaging of abscesses or other neutrophil accumulations. The broad antimicrobial properties of defensins, their chemical resistance and their lack of antigenicity suggests that they may also be useful as topical or systemic agents for the prophylaxis and treatment of infections, assuming that procedures are developed to produce them in bulk economically. The combination of growth-promoting and antimicrobial activity has led to suggestions that defensins be developed as additives in cornea1 storage media for cornea1 transplantation (Schwab et al., 1992) or topical antibiotics that promote healing.

11. SUMMARY The diverse antimicrobial arsenal of many animals includes an array of small cationic antibiotic peptides that contain six cysteines in disulfide linkage. These peptides, called defensins, may also participate in tissue inflammation and endocrine regulation during infection. Their role in innate immunity and their use as prophylactic and therapeutic agents warrants further study.

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Panyutich, A. and Ganz, T. (1991) Activated alpha 2-macroglobulin

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