structure–activity relationships in β-defensin peptides

ReviewStructure–Activity Relationships in b-Defensin Peptides

Karen Taylor,1 Perdita E. Barran,2 Julia R. Dorin11MRC Human Genetics Unit, Edinburgh EH4 2XU, Scotland, United Kingdom

2School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, United Kingdom

Received 29 October 2007; accepted 13 November 2007

Published online 26 November 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20900

This article was originally published online as an accepted

preprint. The ‘‘Published Online’’ date corresponds to the

preprint version. You can request a copy of the preprint by

emailing the Biopolymers editorial office at biopolymers@wiley.

com

INTRODUCTION

Defensins are diverse members of a large family of cat-

ionic host defence peptides (HDP), widely distrib-

uted throughout the plant and animal kingdoms.

These cysteine-rich peptides vary in their length, the

spacing of their cysteine residues, and their disulfide

connectivities.1 Plant defensins2 display eight cysteine resi-

dues, which have disulfide linkages CI��CVIII, CII��CV,

CIII��CVI, and CIV��CVII. In insect defensins there are six

cysteine residues which are connected CI��CIV, CII��CV, and

CIII��CVI. Defensin-like peptides from Platypus venom and

toxins from rattlesnake also contain disulfide stabilized struc-

tures similar to those of defensins. The mammalian defensins

are generally small (3–6 kDa), cationic peptides which have

only six conserved cysteines and were originally isolated

from rabbit neutrophil granules.3

DEFENSIN SUB-FAMILIESMammalian defensins can be classified into three subfamilies

based on the arrangement of the canonical six cysteine motif

and the disulfide bridges that stabilize the b-sheet structure.This defensin family consists of the originally isolated a-defensins (also termed classical defensins), the b-defensins,and the more recently identified h-defensins or retrocyclins.4

The connectivities of the six cysteine mammalian a-defensinsare CI��CVI, CII��CIV, and CIII��CV with the b-defensinsreported to have a CI��CV, CII��CIV, and CIII��CVI. The

h-defensin (found in rhesus monkey) displays the same

connectivity associated with that of the a-defensins but is notfunctionally present in humans.5

The mammalian defensin gene family is present at five

loci in the human and chimpanzee genomes with the main

locus on human chromosome 8p22-23. In rat, mouse, and

dog the five loci are contained in four chromosome clusters

with the largest cluster in mouse present on chromosome

8A3.6,7 The different subfamilies of defensins are though to

share a common ancestry8 despite their evolved differences,

with the b-defensins being the ancestral gene. The majority

of b-defensins are two exon genes with the signal sequence

being encoded by the first and part of the second exon

and the mature peptide being encoded by the second exon.

Figure 1 shows the mature peptide sequence and properties

ReviewStructure–Activity Relationships in b-Defensin Peptides

Correspondence to: Dr. Julia Dorin; e-mail: [email protected]

ABSTRACT:

The b-defensins comprise a large family of small cationic

antimicrobial peptides widely distributed in plants,

mammals and insects. These cysteine rich peptides

display multifunctional properties with implications as

potential therapeutic agents. Recent research has

highlighted their role in both the innate and adaptive

immune systems as well as being novel melanocortin

ligands. Studies investigating structure and function

provide an insight into the molecular basis of their

immunological properties. # 2007 Wiley Periodicals, Inc.

Biopolymers (Pept Sci) 90: 1–7, 2008.

Keywords: defensin; antimicrobial; structure/activity

Contract grant sponsors: EPSRC, Cystic Fibrosis Research Trust UK, MRC, the

University of Edinburgh

VVC 2007 Wiley Periodicals, Inc.

PeptideScience Volume 90 / Number 1 1

of five characterized b-defensins from the main human chro-

mosome 8 locus.

STRUCTURAL CHARACTERISTICSAlthough the predicted peptide sequence is available for over

40 human b-defensins, the native peptide has only been

isolated for three human b-defensins (HBDs 1, 2, and 3).

NMR and X-ray crystallography data have determined the

three-dimensional structures of a number of synthetic

and recombinant defensins. The tertiary structures of these

defensins are remarkably similar despite low sequence con-

servation in their amino acids (Figure 2). These three human

b-defensins consist of three b-strands arranged in an antipar-

allel sheet and held together by three intramolecular disulfide

bonds. The b-sheet is flanked by a a-helical segment formed

by the N-terminal fragment of the molecule.10 This is not

present in the a-defensins or bovine b-defensins whose struc-tures have been solved to date.11 The a-helix orientation is

stabilised to the b-sheet by the disulfide bond (CI��CV). A

conserved motif Gly-X-CysIV is found in the second b-strandand forms a b-bulge.12 The b-bulge is thought to be respon-

sible for a twist in the b-sheets and to be necessary for the

formation of the structure and the correct folding.13 Kluver

et al. 200543 synthesized two HBD3 peptides and obtained

significantly different products. From the oxidative folding of

the 45 residue parent peptide a major product with the

desired CI��CV, CII��CIV, CIII��CVI disulfide connectivity

was found. The second, a 40 residue peptide with the first five

residues missing from the mature peptide sequence led to a

mixture of fully disulfide bonded isomers none of which con-

tained a product with the canonical b-defensin disulfides.

Since both oxidation reactions were performed under identical

conditions it is possible that the first five residues of the pep-

tide facilitate the folding of the b-defensin in a manner that

greatly favors the canonical disulfide bonded pattern and are

important sterically in the formation of a key intermediate.

However Wu et al. 200341 found that their synthetic 45 mer

HBD3 preparation did not fold preferentially into a native

conformation in vitro under various oxidative conditions.

FUNCTIONDespite b-defensins being classic antimicrobial peptides,

their functional role appears not to be limited to this solely.

Recent work has demonstrated that b-defensins have immu-

nomodulatory activity and potentially provide a link between

innate and adaptive immunity, being chemotactic for CD4+

T cells and immature dendritic cells.14 In addition b-defen-sins have been associated in the pathogenesis of numerous

diseases. HBD1 has been implicated in cancer as it is lost at

high frequencies in malignant prostatic tissue and has been

shown to induce cytolysis and apoptosis in prostate cancer

cell lines.15 HBD2 and HBD3 are highly expressed by proin-

flammatory cytokines and were originally purified from the

inflammatory skin disease, psoriasis.16 HBD2 has been dem-

onstrated to be subject to copy number variation and is

highly polymorphic within the healthy population.8,17 Feller-

man et al. 200618 found that individuals with three or less

copies of HBD2 have a significantly higher risk of developing

colonic Crohn’s disease than individuals with four of greater

copies. b-defensins have also been implicated in cell differen-

tiation and tissue remodeling in osteoarthritic joints.19

DEFB123 has been shown to bind lipopolysaccharide (LPS)

and to prevent LPS-mediated effects in vitro and in vivo but

HBD3 does not bind LPS.20 The b-defensins are highly

expressed in the reproductive tract and may provide antimi-

crobial protection of this potentially vulnerable system, but

FIGURE 1 b-defensin sequence and properties of human paralogues from the major b-defensinlocus. Five human b-defensin sequences either purified from tissue (HBD1-3) or predicted sequence

(HBD4, HBD7) with antimicrobial properties described. Canonical cysteine residues highlighted in

red, basic amino acids in blue, and conserved glycine in green. Net charges are calculated for pH 7.0.

FIGURE 2 Tertiary structures of b-defensins. Ribbon diagrams of

HBD1, 2, and 3 based on their NMR data9,36 demonstrating similar

tertiary structures despite low sequence conservation. The antiparal-

lel b-sheets are shown in yellow and alpha helices in magenta.

2 Taylor, Barran, and Dorin

Biopolymers (Peptide Science) DOI 10.1002/bip

recently, these defensins have also been shown to be associ-

ated with specific sperm functions including initiation of

motility and capacitation.21,22 In addition DEFB126 has been

suggested to be involved in immunoprotection of sperm.23

EVOLUTION OF THE DEFENSINSREVEALS SITES OF POTENTIALFUNCTIONAL IMPORTANCEThe b-defensin peptides share common characteristics, their

cationic properties, small size, and cysteine residues. However

they are a rapidly evolving family with low sequence similarity

between paralogues (Figure 1). It is likely that their divergence

has arisen due to ‘‘birth and death’’ of paralogues arising by

unequal crossing over and tandem duplication.24,25

Nucleotide substitutions in recently duplicated mamma-

lian defensin genes show that the rate of nonsynonymous

substitutions exceeds that of synonymous substitution in the

region of the gene encoding the mature peptide. This is evi-

dence of positive selection driving the diversification of the

defensin genes26 This disproportionately favors alterations in

the charge of the peptide residues.26 However the evolution

of these genes is complex and we have examined the evolu-

tion of the genes at the major human b-defensin locus and

the orthologous loci in a range of other primates and mam-

mals. Using a combination of maximum-likelihood-based

tests and a maximum-parsimony-based sliding window

approach we find that during the divergence of primates,

variable selective pressures have acted on b-defensin genes in

different evolutionary lineages, with episodes of both nega-

tive and, more rarely, positive selection. Positive selection

appears to have been more common in the rodent lineage,

accompanying the birth of novel rodent-specific beta-defen-

sin gene clades. Sites in the mature peptide and intriguingly

some sites in the signal region have been subject to positive

selection and, by implication, are important in functional di-

versity.27 In the mature peptide region both primate and

rodent lineages show a large number of sites subject to posi-

tive selection within the N-terminal portion. Two sites in

particular were located within a region of the primary se-

quence which forms an alpha helix (Figure 3). Since regions

of proteins within membranes are often helical, with surfaces

covered with hydrophobic residues, we speculate that the

alpha helical section may be involved in anchoring the b-defensin to a bacterial cell wall. This also is supported by the

fact that many antimicrobial peptides form alpha helices.28

Thus the sites within the alpha helix under positive selection

may be significant in the specificity of the functions of b-defensins, either with respect to their antimicrobial or che-

moattractant properties. The longest loop region of these

peptides (between b-sheet 1 and 2) also contains sites of pos-

itive selection. In the murine lineages this loop is almost

exclusively subject to high selection, which suggests that this

part of the structure has a key functional role in these small

peptides. Further sites found to be subject to positive selec-

tion in rodents were at the end of the signal peptide. This

contrasts with studies of a-defensins which have found an

absence of positively selected sites in the prepropeptide

region.29 In primate lineages, sites within the prepeptide

region have undergone negative selection. These observations

strongly imply that the signal peptide is more important to

b-defensin function than has previously been appreciated.

Antcheva et al.30 synthesized variant molecules based on

their observation that DEFB4 (formerly DEFB2) has been

subject to positive selection during the divergence of various

primate lineages. They synthesized the M. fascicularis DEFB4

orthologue (‘‘mfaBD2’’) and a variant of the human peptide

lacking Asp4, which is characteristic only of the human/great

ape peptides. HBD2 and mfaBD2 showed a significant differ-

ence in specificity, the former being more active toward

Escherichia coli and the latter towards Staphylococcus aureus

and Candida albicans. Asp4 in the human peptide appears

to be important, as Asp4 hBD2 deletion was less structured

and had a markedly lower antimicrobial activity.

STRUCTURE ACTIVITY RELATIONSHIPS FORANTIMICROBIAL FUNCTIONThe main function described for b-defensins is their antimi-

crobial activity. b-defensins antimicrobial activity has been

demonstrated against both Gram positive and negative bacte-

FIGURE 3 Structural implications of primate sites under signifi-

cant positive selection. Sites subject to positive selection were

mapped to the structure of the human DEFB1 mature peptide. Sites

subject to selection are depicted as inflated regions of the structures

colored red to indicate positive selection and the particular residues

are named and arrowed. Ala (marked with an asterisk) is subject to

negative and positive selection in different primate lineages.

Structure–Activity Relationships in �-Defensin Peptides 3


ria in vitro as well as viruses, unicellular parasites, and yeast.

The mechanism of action of defensins is not completely

understood, although it certainly involves permeabilisation

of the cell membrane. Two general models of permeabiliza-

tion of cells by AMPs have been suggested.31

1. Carpet model: Where several molecules sit on the sur-

face of the cell bringing about necrosis.

2. Pore model: Here the peptide oligomerises and forms a

multimeric pore in the cell membrane causing leakage

of the cell contents.

It is not yet established which of these mechanisms

describes the mode of action of a typical b-defensin and is

possible that some defensins form pores and others demon-

strate a less concerted structural attack on cell walls.

Defb1 (orthologue to human DEFB1) gene knockouts in

mice demonstrate a phenotype of increased bacteria of the

Staphylococcus species in the bladder, suggesting a role for

these peptides in resistance to urinary tract infection.32 How-

ever, despite the Defb1 synthetic peptide showing a salt- sensi-

tive antimicrobial activity that was stronger against S. aureus

than against E. coli or P. aeruginosa in vitro, the Defb1(�/�)

mice are effective in the clearance of S. aureus from the airways

after nebulization. Moser et al.33 found that deletion of Defb1

results in delayed clearance ofH. influenzae from the lung.

Given the conservation of the cysteine spacing motif

throughout evolution and its maintenance between structur-

ally diverse paralogues, it was assumed that these residues

would be essential for the antimicrobial function (Figure 1).

Within humans an intragenic polymorphism of the DEFB1

gene changes one of the highly conserved cysteine residues to

a serine residue.34 As a result of this change, one cysteine

remains unpaired and HBD1 is unable to form three intra-

molecular disulfide bonds. A synthetic HBD1 with the vari-

ant serine in place of the cysteineVI has been synthesized and

tested for antimicrobial properties. The variant HBD1 still

retains antimicrobial properties against P. aeruginosa and

C.neoformans comparable with that of the parent HBD1.

A further human b-defensin DEFB107, has been described

that is highly expressed in the testes26 and contains only five

cysteines whereby the first cysteine of the canonical six cysteines

is replaced with a serine. This peptide has poor antimicrobial

activity but this could be because it has its free cysteine capped

with a glutathione hindering its ability to form intermolecular

dimers, it also has relatively low net charge (Figure 1).35

A murine b-defensin gene present in C57B16 mice has

been described that encodes a peptide with only five cysteine

residues. This gene named defensin related peptide (Defr1)

because of its lack of six cysteines. Defr1 contains a tyrosine

in place of cysteineI and yet retains potent antimicrobial

activity against both Gram positive and negative organisms

including multiresistant strains.37 This gene is a variant allele

of Defb8 that encodes six cysteines and is present in all other

inbred murine strains tested (unpublished data). Defb8 has

very high levels of identity (98%) with Defr1 differing in

only three amino acid residues (Figure 4).36

The relationship between the structure and activity of

Defr1 and a six cysteine analogue (Defr1-Y5C) has been

studied.37 The peptide Defr1-Y5C analogue replaces the tyro-

sine in Defr1 with a cysteineI, thus reinstating the canonical

six cysteines. Both Defr1 and Defr1-Y5C were shown to be

defensin dimers, Defr1 mediated by a covalent intermolecu-

lar bond and Defr1-Y5C by strong noncovalent interac-

tions.37 The cysteine connectivities of both these peptides

were assigned and although Defr1-Y5C presented the recog-

nized canonical b-defensin disulfide bond connectivity,

oxidized Defr1 is a complex mixture of different dimeric

isoforms. Defr1 and Defr1-Y5C possess a gross difference

in their ability to kill Gram negative and positive bacteria.

Defr1 is a strong antimicrobial, with minimum bactericidal

concentration (MBC) values between 3 and 10 lg/ml against

a panel of organisms including clinical isolates with multire-

sistant phenotypes.38 Defr1-Y5C with the canonical cysteine

motif and disulfide connectivities was a very poor antimicro-

bial. The activity of Defr1 was reduced to that of Defr1-Y5C

following reduction with DTT and this leads us to postulate

that the covalent nature of the Defr1 dimer rather than the

cysteine motif is important for its antimicrobial ability. This

is further supported by the synthesis of a Defr1 analogue

which has every cysteine except cysteineV replaced with an al-

anine. This peptide in its oxidized form is a dimer and is a

strong antimicrobial equivalent to Defr1 but in a reduced

form it has very poor activity with MBC values greater than

50 lg/ml.38 This work suggests that cysteine residues are not

essential for the antimicrobial activity of Defr1/Defb8. This is

strongly supported by the work on HBD3 and bovine pep-

tides BNBD-2 and BNBD-12.39–41 Wu et al.41 demonstrated

that a linear peptide of HBD3 where the cysteine residues

had been replaced with a-aminobutyric (Abu) acid is still

antimicrobial. This peptide does not contain a sulphur atom

and hence cannot create inter or intra disulfide bonds.

FIGURE 4 Defr1 and its analogues. Amino acid sequence of

Defr1 and its analogues and the net charge of monomer and dimers

given in brackets. The positions of the canonical six cysteines are

indicated by asterix.



The primary structures of HBD1-3 indicate that there are

considerable variations in the net charge between these

defensins. HBD1-3 display net positive charges of +4, +6,

and +11, respectively (Figure 1). Cationic residues occur pre-

dominantly after the third cysteine in HBD1 and 2. In HBD3

nine of the thirteen cationic residues are present after CysIII.

The antimicrobial activity of these peptides varies. HBD1 is

active only against Gram-negative bacteria. HBD2 displays

considerably greater activity compared to HBD1 and HBD3

has the strongest activity and the highest positive charge.

Alterations in the defensin peptide sequence in an effort

to relate structure to function have been studied extensively.

Single amino acid substitutions and N-terminal deletions

which do not alter the charge or substantially change the

hydrophobicity still retain antimicrobial activity. However

these changes can alter the bacterial susceptibility and the

overall rate of killing.42–44 Kluver et al.43 discuss that increas-

ing the net charge and the hydrophobicity increases the anti-

microbial activity. Peptides with lower numbers of cationic

residues and moderate hydrophobicity are virtually inactive

whereas peptides with a high net charge and significant

hydrophobic character are active. HBD3 has been subject to

several derivative studies41–43 with various regions of the

peptide being of interest. Synthetic peptides have been gener-

ated at both the N-terminus and C-terminus. The N-termi-

nus sequence of HBD3 has been synthesized containing 17

residues and possessing a net charge of only +4.42 Despite the

low charge, this peptide has displayed antimicrobial activity

against both Gram positive and negative organisms. N-termi-

nal deletion mutants of HBD3 have highlighted the impor-

tance of the initial seven residues for activity against Gram

positive organisms.42 Interestingly this N terminus is of vari-

able length between paralogues (Figure 1) and N-terminal

deletion isomers of HBD1 have been recorded in urine.45

Active C-terminus analogues of HBD1, HBD2, and HBD344

displayed activity against E.coli (MIC of 15–17 lM) and

S.aureus (MIC of 17–20 lM).

STRUCTURE ACTIVITY RELATIONSHIPSFOR CHEMOTACTIC ACTIVITYThe b-defensins have been shown to maintain a role in both

the innate and adaptive immune response. Many defensins act

as chemoattractants at nanomolar concentrations with activity

against a variety of cell types. HBD3 induces the migration of

monocytes with a maximal response at 100 ng/ml41 although

no migration was evident with neutrophils46 unless acti-

vated.47 Migration has been observed with CD4+ T cells and

immature dendritic cells but not shown with mature dendritic

cells. Chemokine receptor 6 (CCR6) appears to be the chemo-

kine receptor through which the b-defensins are acting

through.14 In Yang et al. (1999)14 migration was observed with

HEK293 cells transfected with CCR6 at an optimal concentra-

tion of 10 ng/ml. In addition chemotaxis blocking experi-

ments were performed whereby CCR6 antibodies inhibited

the migration of the transfected cells suggesting this as the

receptor for the b-defensins tested. However a recent study48

appears to show that CCR6 is not a functional receptor for

b-defensins as the authors failed to show migration of HBD2

and HBD3 with cell lines stably transfecting CCR6. The differ-

ent results are most likely due to differences in the synthetic

peptide preparations used. HBD3-induce migration has been

observed with cells not expressing CCR6 indicating an addi-

tional unidentified receptor present on monocytes.41 This was

also evident in the study conducted by Soruri et al.48 The che-

motactic activity of HBD3 to monocytes must use a receptor

other that CCR6 because monocytes do not express functional

CCR6. Other antimicrobial peptides utilize different receptors

to that of the b-defensins. LL-37 has been shown to act

through the Formyl Peptide Receptor-Like 1 (FPRL1) as a re-

ceptor to chemoattract human peripheral blood neutrophils,

monocytes, and T cells.49

Comparison of the crystal structure of CCL20/MIP3a (the

known ligand for CCR6) and HBD1 and HBD2 has revealed

some structural similarities despite no sequence similar-

ity.10,12,50 An Asp4-Leu9 motif in HBD2, which resembles the

Asp5-Asp8 motif of CCL20/MIP3a is considered to be respon-

sible for specific interaction with CCR6, providing a structural

basis for the capacity of b-defensins and CCL20/MIP3a to

interact with the same receptor. This region flanks the first

cysteine. Work by41 with HBD3 revealed that the cysteine resi-

dues in their canonical disulfide connectivities impact on che-

moattractant activity. Six topological analogues of HBD3 were

prepared were different disulfide connectivities. The HBD3

with the canonical cysteine connectivity (CI��CV, CII��CIV,

CIII��CVI) displayed migration at 10 ng/ml with HEK293

CCR6 expressing cells. Noncanonical connectivities ranged

from 100–1000 ng/ml. The HBD3 analogue where the cysteine

residues are replaced with a-aminobutyric acid41 gave no

migration for both monocytes and HEK293 cells expressing

CCR6 even at concentrations greater than 10,000 ng/ml.

These results imply that the cysteine residues are essential

for activity and/or the peptide must be stabilized in a partic-

ular conformation to facilitate functional interaction with

the two receptors. We have data to support the requirement

for particular cysteine residues even without disulfide bond-

ing allowing functional chemoattraction of cells expressing

CCR6 (unpublished data). It is of interest that the HBD3

conformers that give optimal chemoattraction activity for

cells expressing the receptor on monocytes or CCR6 are



different supporting the necessity of a different structure

when interacting with one versus the other.41

The biological properties of HBD1 have been studied by

introducing single site mutations and noting their effect on

structure and function.51 Twenty-six single site mutations of

HBD1 in 17 positions were performed resulting in 10 high

resolution structures being generated. Mutations in the

sequence did not induce significant changes in the structure

but differences in biological properties were attributed to the

changes in the respective side chains. Cationic residues in the

C-terminus are implicated in the antimicrobial killing of

E. coli. Regarding chemotaxis and interactions with CCR6

the residues of importance are located at the N-terminal

a-helical region and a few adjacent residues.

STRUCTURE ACTIVITY RELATIONSHIPS ASA MELANOCORTIN RECEPTOR LIGANDVery recently a mutation in the dog orthologue of HBD3

(DEFB103) was identified as being responsible for dominant

black pigmentation at the K locus.52 Black dogs carrying this

mutant allele have a three base pair deletion that results in

deletion of glycine 23 of the protein. This results in a mature

peptide without the first amino acid. The consequence of

this deletion is an increase in the amount of mature peptide

that is secreted into the medium of cultured cells carrying

the mutation. The black hair color of the dogs as the conse-

quence of this mutation is due to the ability of the mutant

peptide to bind to the melanocortin receptor (MC1R) in

dermal pigment cells. Normally dark pigment (black) is

produced by these cells due to MC1R activation and lighter

pigment (red/yellow) is produced when the inverse agonist

agouti signaling protein (ASIP) binds to MC1R and reduces

its activation. Clearly in dogs the interaction of the b-defen-sin with MC1R is functional and transgenic mice overex-

pressing either mutant or wildtype forms have black rather

than agouti fur. In vitro Candille et al. could demonstrate

that the mutant dog b-defensin 3 could bind dog MC1R

with 5-fold increased efficiency compared to the wildtype

peptide but 5-fold less affinity than its natural ligand a-Mela-

nocyte stimulating hormone (a-MSH). The antimicrobial ac-

tivity of this mutant peptide nor its chemoattractant activity

has been described as yet. Over expression of the dog gene in

transgenic mice resulted in animals with reduced size and it

maybe that this is due to interaction with other melanocortin

receptors which have pleiotropic functions including weight

and immune control. Both HBD1 and 3 could interact with

human MC1R, with HBD3 having 3 fold better affinity than

the dog peptide (but 5 fold less affinity than a-MSH) for the

human receptor. High level expression of HBD3 has been

observed in psoriatic lesions but no evidence of increased

pigmentation is observed.

STRUCTURE ACTIVITY RELATIONSHIPS FORMAMMALIAN MEMBRANE INTERACTIONThe interaction of b-defensins with eukaryotic membranes

is not well understood. Eukaryotic cell membranes consist

mainly of zwitteronic phosphatidylcholine and phosphatidy-

lethanlamine susceptible to hydrophobic interactions.53

Expression of HBD1 in prostate cell lines appeared to induce

cytolysis.54 Several studies have been performed addressing

the haemolytic and cytotoxic properties of HBD3.16,43 HBD3

has been shown not to exhibit significant lytic activity on

human erythrocytes16 between 0 and 500 ug/ml. In our stud-

ies we find that Defb14 (the mouse orthologue of HBD3)

and an analogue with only one cysteine, both gave similar

profiles against human erythrocytes with no significant lytic

activity observed (data unpublished). Generally the larger the

number of hydrophobic amino acids leads to an increased

haemolytic activity with erythrocytes. Kluver et al. 200543

demonstrated that differences in haemolytic and cytotoxic

activity of HBD3 peptides are due to the sequence rather

than the disulfides. Peptides where the cysteine residues were

replaced with alanine were less cytotxic than those containing

tryptophan residues. The alanine peptides created a slight

reduction in hydrophobicity55 whereby the tryptophan pep-

tides caused a marked increase in hydrophobicity.

CONCLUSIONAnalysis of the structural and functional characteristics of

the b-defensins highlights the ability to engineer these pep-

tides to gain a better understanding of their function. Altera-

tions in overall charge, length, sequence, and connectivities

encourages the exploration of b-defensins and focuses on

changing activity profiles. Expansion of their functional rep-

ertoire from antimicrobial peptides to important modulators

of the immune system is exciting. Further research will ad-

vance knowledge within the field and highlight their poten-

tial as therapeutic agents.

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structure–activity relationships in β-defensin peptides

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