Defensins

Defensins Tomas Ganz* Medicine and Pathology, School of Medicine, University of California, Los Angeles, 10833 Le Conte Ave., Los Angeles, CA 90095-1690, USA * corresponding author tel: 310 825 6112, fax: 310 206 8766, e-mail: [email protected] DOI: 10.1006/rwcy.2000.12006.

SUMMARY Defensins are a family of variably cationic 3±5 kDa peptides with a conserved motif of six disulfide-linked cysteines. They are expressed in host defense settings (in phagocytes and epithelia) and display a broad spectrum of antimicrobial activity. The production of many defensins is constitutive but others are induced by infectious or inflammatory stimuli. Some defensins are chemoattractant for monocytes, lymphocytes, and dendritic cells.

BACKGROUND

Discovery Defensins were discovered during a search for antimicrobial substances in human and animal phagocytes. The class designation, purification, antimicrobial characteristics (Ganz et al., 1985) and amino acid sequences (Selsted et al., 1985a) of three human neutrophil defensins were reported in 1985. Although rabbit and guinea pig peptides that in retrospect belonged to the same peptide family were first identified more than two decades earlier (Zeya and Spitznagel, 1963, 1966) their structural characterization had to await improved technologies (Selsted et al., 1983, 1985c; Selsted and Harwig, 1987). Since then, additional defensins have been found in polymorphonuclear leukocytes of chickens (Harwig et al., 1994), turkeys (Evans et al., 1994), rats (Eisenhauer et al., 1989), and hamsters (Mak et al., 1996) but were absent from the polymorphonuclear leukocytes of mice (Eisenhauer and Lehrer, 1992). Defensins produced by Paneth cells, specialized intestinal epithelial cells, were first identified as a class

of abundant developmentally regulated mRNAs in the mouse small intestine (Ouellette and Lualdi, 1990). Human Paneth cell defensins were discovered by cDNA and gene cloning based on homology to the human neutrophil defensins (Jones and Bevins, 1992, 1993). The first -defensins were found by fractionation of bovine tracheal mucosa (Diamond et al., 1991) and bovine neutrophils (Selsted et al., 1993). The two known human epithelial -defensins were discovered by a systematic search for novel peptides in human hemofiltrate (Bensch et al., 1995) and in inflamed human skin (Harder et al., 1997). Most recently discovered epithelial defensins include -defensins in sheep (Huttner et al., 1998), pigs (Zhang et al., 1998), rhesus monkeys, rats, and mice (Huttner et al., 1997). The name defensin is also used for peptides of similar structure and function in plants (Broekaert et al., 1995) and invertebrates (Lambert et al., 1989; Charlet et al., 1996), but the evolutionary relationships between vertebrate, plant, and invertebrate defensins remain uncertain.

Alternative names Initial naming of several defensins put emphasis on their tissue of origin, e.g. human neutrophil peptides 1±3 (HNP-1 to HNP-3) (Ganz et al., 1985) also known as human defensins 1±3, macrophage cationic peptides 1 and 2 (Lehrer et al., 1983) also known as rabbit defensins 1 and 2, or skin antimicrobial peptide (Harder et al., 1997), now known as human defensin 2 (HBD-2). Other designations emphasized alternative activity, e.g. corticostatins, so named because of the inhibitory effect of some defensins on the production of cortisol by adrenal cells (Zhu et al., 1988).

1358 Tomas Ganz

Structure Vertebrate defensins are 29±47 amino acid sheetrich cationic and amphipathic peptides with a conserved three disulfide-linked structure. There are two subfamilies, -defensins (Selsted and Harwig, 1989; Hill et al., 1991), whose six cysteines are linked 1±6, 2±4, 3±5, and -defensins (Tang and Selsted, 1993; Zimmermann et al., 1995), linked 1±5, 2±4, 3±6. Since cysteines 5 and 6 are always adjacent to one another the two subfamilies are structurally very similar (Figure 1 and Figure 2).

Main activities and pathophysiological roles Defensins have a broad-spectrum antimicrobial activity in vitro (Ganz and Lehrer, 1995) against grampositive and gram-negative bacteria, yeasts, and fungi, and enveloped viruses. Their common mechanism of action is membrane permeabilization followed by interactions with additional as yet undefined intracellular targets. Their differential activity against

microbes may be dependent on differences in membrane composition between microbial membranes (rich in anionic phospholipids) and host membranes (rich in cholesterol and neutral phospholipids). Other reported activities include in vitro inhibition of cortisol production by blockade of ACTH receptors (Solomon et al., 1991), chemoattraction for mononuclear cells, including T lymphocytes (Chertov et al., 1996), observed in vitro, in immunocompetent mice and in chimeric huPBL-SCID mice, and stimulation of phagocyte accumulation at sites of infection and phagocytic antimicrobial activity in the mouse model (Welling et al., 1998). Some defensins are opsonic (Fleischmann et al., 1985) or increase adherence of bacteria to epithelial surfaces (Gorter et al., 1998).

GENE AND GENE REGULATION

Accession numbers Table 1 contains a list of accession numbers for known human and animal defensin genes and mRNAs.

Figure 1 The three-dimensional molecular structure of human neutrophil defensin HNP-3 (Hill et al., 1991). (Full colour figure may be viewed online.)

Defensins 1359 Figure 2 The three-dimensional molecular structure of bovine neutrophil -defensin-12 (Zimmermann et al., 1995). (Full colour figure may be viewed online.)

Chromosome location Human defensin genes are located in a single cluster on chromosome 8p23 spanning about 400 kb (Liu et al., 1997, 1998). The mouse (Ouellette et al., 1989b) defensin (cryptdin) gene cluster is also located on chromosome 8 in a location syntenic to that of the human defensin cluster, as is the bovine defensin cluster (Gallagher et al., 1995) on bovine chromosome 27.

and the promoter regions of these defensins contain NFB-binding sites (Russell et al., 1996). The synthesis of human epithelial defensin HBD-2 (but not HBD-1) is also inducible by bacteria, TNF and IL-1 (Harder et al., 1997; Singh et al., 1998) and the promoter of HBD-2, but not HBD-1, contains multiple NFB sites (Liu et al., 1998). Consistent with their myeloid-specific expression, the promoter regions of human neutrophil defensins contain the binding site for the myeloid factor PU.1 (Ma et al., 1998).

Regulatory sites and corresponding transcription factors

Cells and tissues that express the gene

The production of bovine epithelial defensins TAP and LAP is inducible by lipopolysaccharide and TNF,

The known defensins follow three patterns of expression: (1) granulocyte-specific, with synthesis occurring

1360 Tomas Ganz Table 1 Defensin genes, mRNAs, and their accession numbers Product

Gene(s)

mRNA(s)

HUGO

Human neutrophil defensins, HNP-1 to HNP-3

L12690, U10268

M21130, M21131, M23281, M26602, X52053, X13621

HDEFA1

Human neutrophil defensin, HNP-4

U18745

X65977

HDEFA4

Human Paneth cell defensin, HD-5

M97925

Human Paneth cell defensin, HD-6

U33317

M98331

Human -defensin-1

SEG_HSBDONE

Z50788, X92744, U73945

HDEFB1

Human -defensin-2

AF071216

Z71389

HDEFB2

HDEFA5

Rhesus (Macaca mulatta) -defensin

AF014016

Rabbit neutrophil and macrophage defensins

M28072, M28073, M64599

M28883, M28884, M64600, M64601, M64602, L10841, L10842

Mouse cryptdins

U03061, U12559, U12562, U12565,

S73391, U03028, U03030, U03032, U03033, U03034, U03035, U03036, U03037, U03064, U03065, U03066, U03067, U73623, U73624, X15617

Mouse -defensins

SEG_MMDEFEN0

U03062, U03063, U12560, U12561, U12563, U12564, U12566

Rat neutrophil defensins

U16683, U16684, U16685, U16686

Rat -defensins

AF093536, AF068860, AF068861

Bovine neutrophil -defensins

HDEFA6

AF008307

Bovine enteric -defensins

AF016539

Sheep -defensins

U75250, U75251

AF000362

Porcine -defensins

AF031666

Chicken and turkey -defensins

AF033335, AF033336, AF033337, AF033338

in promyelocytic bone marrow precursors (neutrophil defensins of mammalian and avian species) (Daher et al., 1988; Ganz et al., 1989; Nagaoka et al., 1993; Yount et al., 1995; Brockus et al., 1998); (2) Paneth cellspecific (human defensins 5 and 6, mouse cryptdins) (Ouellette et al., 1989a; Ouellette and Lualdi, 1990; Jones and Bevins, 1992, 1993; Ouellette and Selsted, 1996); (3) epithelial cells with predominant expression in the tongue (porcine -defensin 1, bovine lingual antimicrobial peptide) (Schonwetter et al., 1995; Zhang et al., 1998), trachea (bovine tracheal antimicrobial peptide) (Diamond et al., 1993), small intestine and colon (bovine enteric -defensin) (Tarver et al., 1998), kidney (human -defensin 1, rabbit defensins RK-1 and RK-2) (Bateman et al., 1996; Valore et al., 1998; Wu et al., 1998) and skin (human -defensin 2) (Harder et al., 1997).

PROTEIN

Accession numbers Accession numbers of defensin peptide sequences in the Entrez (NCBI/NLM) database are listed in Table 2.

Sequence The canonical sequence of -defensins is xxCxCxxxxx CxxxxxxxGxCxxxxxxxxxCCxx, where x represents any amino acid and cysteines 1±6, 2±4, and 3±5 are linked. For -defensins the canonical sequence is CxxxxxxCxxxxCPxxxxxxxxCxxxxx(x)CCxx, where cysteines 1±5, 2±4, and 3±6 are linked. In general,

Defensins 1361 Table 2 Defensin peptides and their accession numbersa Species and name

Tissue

Accession numbers

Human neutrophil peptides 1±3, 4

Granulocytes, bone marrow

B40499, P11479, P12838, 30501, S65412, S65413, S65414, A47365, A40499, 292365, 29735, 665927, 181535, 181529, 181527, 292363, 553252

Human defensins 5, 6

Paneth cells

A44454, S27016, Q01523, Q01524, 1200182, 181547, 181533

Human -defensins 1±2

Epithelia

949876, 1755148, S66282, Q09573, 1293651, 1617088, O15263, 2239128, 3818537, 3510600

Rhesus -defensin 1

Epithelia

O18794, 2317750

Rabbit macrophage cationic peptides, neutrophil peptides

Macrophages (1 and 2), granulocytes (1, 2, 3, 3A, 4, 5, 6)

WTRBM1, WTRBM2, P01376, P01377, S32553, JC1462, P07466, P07467, P07468, P07469, P80223, 415517, 415518, 1912193A, 1912193B, 1904312A, 1904312B, 1904312C, 165561, 165559, 165478, 165476, 165474, 165472, 164999, 164997, 164995, 164993

Rabbit kidney peptides

Kidney

1839443

Mouse cryptdins

Paneth cells

437220, 437224, 437228, 437232, 437236, 437242, 437244, 437246, 437248, 437250, 437252, 437254, 437256, 437258, 437260, 437262, 437264, 437266, 437268, 437270, 497034, 497032, 531839, 531841, 531843, 531845, I48226, I49102, I49103, I49104, B44800, P11477, P28309, P28310, P28311, P28312, P50704, P50705, P50706, P50707, P50708, P50709, P50711, P50712, P50713, P50714, Q64016, 1245979, 1245980, 1245981, 1657995, 1657997, 1813205A, 50578, 192791

Mouse -defensins 1, 2

Epithelia

2197075, P56386

Rat neutrophil peptides 1±4

Granulocytes, bone marrow

A61014, B61014, D61014, E61014, I46703, I46704, I46705, I46706, S14314, S36843, S36844, Q62713, Q62714, Q62715, Q62716, 1041805, 1041807, 1041809, 1041811

Rat -defensins 1, 2

Epithelia

3366932, 3668412, 3366934

Guinea pig neutrophil peptides

Granulocytes, bone marrow

P11478, P49112

Hamster neutrophil peptides 1±4

Granulocytes, bone marrow

P81465, P81466, P81467, P81468, 1911754, 1911755, 1911756, 1911757

Bovine neutrophil -defensins

Granulocytes, bone marrow

2367667, 2360981, 2360983, 2360985, 2145044, 225845, 298766, 298767, 298768, 298769, 298770, 298771, 298772, 298773, 298774, 298775, 298776, 298777, 298778, A45495, C45495, D45495, E45495, F45495, G45495, I45495, A47753, B47753, C47753, D47753, O02775, P46159, P46160, P46161, P46162, P46163, P46164, P46165, P46166, P46167, P46168, P46169, P46170, P25068, 1BNB, Q28880

Tracheal antimicrobial peptide, lingual antimicrobial peptide, enteric -defensin

Epithelia of the trachea, tongue and the digestive tract

P25068, Q28880, O02775, 2367667, 2145444

Sheep -defensins 1, 2

Epithelia

O19038, O19039, 2231305, 2231307

Pig -defensin 1

Tongue

2978564, O62697

1362 Tomas Ganz Table 2 (Continued ) Species and name

Tissue

Acession numbers

Gallinacins, chicken heterophil peptides

Granulocytes, bone marrow

P46156, P46157, P46158, 3617829, 3617831

Turkey heterophil peptides 1, 2

Granulocytes, bone marrow

3617833, 3617835

a Note that the Entrez database (National Library of Medicine) contains many partially and completely redundant entries. We made no effort to sort through the redundancies and did not include fragmentary or patent sequences.

defensins are rich in cationic amino acids, lysine, and arginine.

Description of protein Defensins are sheet-rich amphipathic peptides with a conserved disulfide-stabilized structure containing 29±47 amino acids.

Discussion of crystal structure In crystals and in solutions human neutrophil defensins HNP-1 to HNP-3 form homodimers (Hill et al., 1991) but other - and -defensins are monomeric in solution (Bach et al., 1987; Pardi et al., 1988; Levy et al., 1989; Kominos et al., 1990; Pardi et al., 1992; Zhang et al., 1992; Skalicky et al., 1994; Zimmermann et al., 1995). The molecular structures of representative - and -defensins are shown in Figure 1 and Figure 2 (PDB format). Studies in model membranes suggest that defensins form multimeric pores. A model of the pore consistent with the available data has been proposed (Wimley et al., 1994; White et al., 1995). The pore is formed by a ring of six defensin dimers whose arginines associate with phosphate head groups or point into the channel, while the hydrophobic side chains are immersed in the hydrophobic interior of the membrane.

Important homologies There is significant similarity of -defensins to snake (crotalid) myotoxins, indicating a possible common evolutionary origin. Defensin folds exhibit similarity to insect defensins, scorpion toxins, and sea anemone toxins (Mas et al., 1998).

Posttranslational modifications Defensins are synthesized as larger precursors, typically 64±100 amino acids long, with a 19 amino acid

signal sequence and a variable, typically anionic propeptide segment (Michaelson et al., 1992). Posttranslation modifications include the proteolytic removal of the signal sequence followed by further proteolytic cleavages in the Golgi and the nascent neutrophil granules to remove segments of the Nterminal propiece (Valore and Ganz, 1992). Compared to -defensin propieces, which consist of around 40± 45 amino acids, -defensin propieces are generally very short. The processing of epithelial -defensins generates multiple forms differing in N-terminal truncation, a mechanism which could serve to increase the diversity of antimicrobial peptides (Valore et al., 1998).

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce Defensin peptides are generally expressed in the same cells that express the genes, i.e. promyelocytic bone marrow precursors, Paneth cells, and epithelial cells. The mature granulocytes of many animal species contain abundant neutrophil defensins accumulated during development of precursor cells in the bone marrow. However, they no longer contain defensin mRNA and do not actively synthesize defensin, only storing previously synthesized defensins in their granules.

IN VITRO ACTIVITIES

In vitro findings Most defensins manifest broad-spectrum antimicrobial activity at 1±10 mM concentrations, including activity against gram-positive and gram-negative bacteria, yeast, and fungi (Patterson Delafield et al., 1980; Lehrer et al., 1983, 1985a, 1986, 1988, 1989; Selsted et al., 1984, 1985b; Ganz et al., 1985; Segal

Defensins 1363 et al., 1985; Levitz et al., 1986; Miyasaki et al., 1990; Ogata et al., 1992; Couto et al., 1994), Giardia (Aley et al., 1994), and enveloped viruses (Lehrer et al., 1985b; Daher et al., 1986). Individual molecular species differ in their antimicrobial spectra but systematic understanding of these differences has not yet been achieved. At higher concentrations (15±30 mM) cytotoxic activity against cells grown in vitro has been observed (Lichtenstein et al., 1986, 1988a, 1988b; Okrent et al., 1990; Lichtenstein, 1991). Chemotactic activity for mononuclear cells (Territo et al., 1989), later identified as T lymphocytes (Chertov et al., 1996), has been shown at nanomolar concentrations. At  100 nM concentrations, some defensins bind to ACTH receptors on cortisol-producing adrenal cells and inhibit ACTH-mediated activation (corticostatic activity) (Tominaga et al., 1990; Zhu and Solomon, 1992). Other reported activities include opsonization (Fleischmann et al., 1985; Ichinose et al., 1996) by some defensins and inhibition of phagocytosis by others (Ichinose and Sawada, 1995). Defensins bind to complement components (Panyutich et al., 1994; van den Berg et al., 1998) and inhibit the activation of the classical complement pathway. Mitogenic effects on fibroblasts and other cells have been reported and these could have a role in wound healing (Murphy et al., 1993). Most recently, human neutrophil defensins have been found to inhibit fibrinolysis (Higazi et al., 1995, 1996) and promote the uptake of lipoprotein (a) by endothelial cells (Barnathan et al., 1997; Higazi et al., 1997), activities that could accelerate the development of atherosclerosis and its complications.

Regulatory molecules: Inhibitors and enhancers The antibacterial and antifungal activity of defensins is competitively inhibited by increasing concentrations of salt, divalent cations, and serum but the magnitude of inhibition depends on the target (Lehrer et al., 1985a, 1988, 1989; Ganz and Lehrer, 1995; Goldman et al., 1997; Porter et al., 1997; Bals et al., 1998a, 1998b; Singh et al., 1998; Valore et al., 1998). Antiviral effects of defensins are not affected by salt concentrations (Lehrer et al., 1985b; Daher et al., 1986). Defensins bind avidly to serum proteins, including 2 -macroglobulin, components of complement, and 1 -proteinase inhibitor (1 -antitrypsin) (Panyutich and Ganz, 1991; Panyutich et al., 1994, 1995; van den Berg et al., 1998). The ionic and protein composition of plasma may restrict defensin antimicrobial activity predominantly to sequestered environments where the salt and serum concentrations are low (e.g. epithelial

surfaces) or where defensin concentrations are so high that the inhibitors are overcome (e.g. the phagosomes of neutrophils). At lower concentrations of defensins in nonpermissive environments the regulatory effects of defensins may predominate.

Bioassays used None of the bioassays are sufficiently specific for defensins to be useful in their quantification or identification.

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles The evidence for the normal physiologic role of defensins as antimicrobial effectors is indirect. Neutrophil defensins reach high, almost certainly microbicidal concentrations in the phagolysosome (Ganz, 1987; Joiner et al., 1989), the site of phagocytic killing of microorganisms. The local concentrations of epithelial defensins have not yet been reported. Subcutaneous administration of human neutrophil defensins (Chertov et al., 1996) to BALB/c mice resulted in a moderate neutrophil and mononuclear cell infiltrate at the site of injection by 4 hours, which was further increased by 24 hours. Additionally, subcutaneous injection of defensins into chimeric huPBL-SCID mice caused significant infiltration by human CD3+ cells within 4 hours. Potentiation of microbicidal activity of neutrophils in mice by very small intravenous doses of human defensin HNP-1 was recently reported (Welling et al., 1998) and may be mediated by increased accumulation of neutrophils at the sites of infection in mice primed by intravenous injections of HNP-1.

Species differences Although defensins have been detected in many mammalian and avian species, the tissue distribution is variable. Thus mice lack neutrophil defensins altogether (Eisenhauer and Lehrer, 1992), while rats have several neutrophil defensins (Eisenhauer et al., 1989, 1990; Yount et al., 1995). Mice express more than 20 defensin genes in Paneth cells of the small intestine (Huttner et al., 1994; Ouellette and Selsted, 1996) but humans have only two (Mallow et al., 1996). Systematic understanding of the evolution and

1364 Tomas Ganz patterns of expression in different animal species has not yet been achieved.

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects Human neutrophils contain 4±5 mg of HNP-1 to HNP-3 per million cells. Plasma from healthy donors contains less than 100 ng/mL of HNP-1 to HNP-3 but these concentrations rise many-fold during infections, reaching over 100 mg/mL in some severely septicemic patients (Panyutich et al., 1993; Shiomi et al., 1993; Ihi et al., 1997).

Role in experiments of nature and disease states Patients with a disorder of neutrophil maturation, specific granule deficiency, have about 10% of the normal defensin content in their neutrophils (Ganz et al., 1988), and suffer from frequent and severe infections. Because multiple neutrophil proteins are affected in this disorder, the clinical picture cannot be attributed solely to defensin deficiency. Defensins accumulate in atherosclerotic plaques and have been implicated in the pathogenesis of atherosclerosis and its thrombotic complications (Higazi et al., 1995, 1996, 1997; Barnathan et al., 1997). The colonization of airways by bacteria in cystic fibrosis has been attributed to the inactivation of epithelial defensins by increased salt concentrations in respiratory secretions (Smith et al., 1996; Goldman et al., 1997; Bals et al., 1998b).

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LICENSED PRODUCTS The following manufacturers sell defensin peptides or antibodies. The licensing status of the products has not been ascertained.

Human neutrophil defensin HNP-1

Phoenix Pharmaceuticals, Inc., Mountain View, CA, USA Tel: 650 988 9220, Fax: 650 988 9221, Email: [email protected]

Human neutrophil defensins HNP-1 and HNP-2

Sigma Chemical Company/Sigma-Aldrich Corp, St Louis, MO, USA Tel: 314 771 5765, Fax: 314 286 8304

Human neutrophil defensins HNP-1 and HNP-2

American Peptide Company, Inc., 777 East Evelyn Ave., Sunnyvale, CA 94086, USA Tel: 408 733 7604 or 1 800 926 8272, Fax: 408 733 7603 or 1 888 670 0070, E-mail: [email protected]

Monoclonal antibody to human neutrophil defensins HNP-1 to HNP-3

Accurate Chemical & Scientific Corp., Westbury, NY, USA Tel: 516 333 2221, Toll Free: 800 645 6264, Fax: 516 997 4948