Primate β-defensins - Structure, Function and Evolution

Current Protein and Peptide Science, 2005, 6, 7-21 7

1389-2037/05 $50.00+.00 © 2005 Bentham Science Publishers Ltd.

Primate β-defensins – Structure, Function and Evolution

Sergio Crovella1, Nikolinka Antcheva2, Igor Zelezetsky2, Michele Boniotto1, Sabrina Pacor3,Maria Vittoria Verga Falzacappa1 and Alessandro Tossi2,*

Departments of 1Reproduction and Development Sciences, of 2Biochemistry, Biophysics and Macromolecular Chemistryand of 3Biomedical Sciences, University of Trieste, Piazzale Europa 1, 34127 Trieste, Italy

Abstract: Host defense peptides (HDPs) are endogenous antibiotics that play a multifunctional role in the innateimmunity of mammals. Among these, β-defensins contribute to mucosal and epithelial defense, also acting as signalmolecules for cellular components of innate and adaptive immunity. Numerous members of this family have beenidentified in mammalian and avian species, and genomic studies in human and mouse indicate a considerable complexityin their gene organization. Recent reports on the evolution of primate and rodent members of this family indicate quite acomplex pattern of variation. In this review we briefly discuss the evolution of mammalian β-defensins in relation to othertypes of defensins, and then concentrate on the evolution of β-defensins 1 to 4 in primates. In particular, the surprisinglyvaried patterns of evolution, which range from neutral or weakly purifying, to positive selection to a high level ofconservation are analyzed in terms of possible genetics, structural or functional implications, as well as to observedvariations on the antimicrobial activity in vitro. The role of polymorphisms in the genes encoding for these host defensepeptides in determining susceptibility to human diseases are also briefly considered.

Keywords: β-defensin, antimicrobial peptide, host defense, innate immunity, molecular evolution.

HOST DEFENSE PEPTIDES - MULTIFUNCTIONALMOLECULES WITH AN ANCIENT LINEAGE

Small cationic peptides play an important role in hostdefense, as part of the innate immune system. Thesemolecules are phylogenetically ancient and ubiquitous,having been described in bacteria, moulds, plants,invertebrates and vertebrates, including mammals. In thehigher phyla, this defense system has not been superseded bythe adaptive responses, but rather serves to provide anefficient, permanent or rapidly activated first line of defense,that helps to mobilize other components of both innate andadaptive immunity. Like other polypeptides involved ininteractions between organisms, and particularly inimmunity, the host defense peptides (HDPs) areevolutionarily active and have continued to respond to thevarying challenges faced by mammalian species in theirradiation over the past 100 million years, just as they havedone over a longer time-scale in animals and otherorganisms that lack adaptive immunity, where they play aprimary defensive role.

Over a thousand different HDPs have likely beenidentified to date in plants and animals, and over 800 arecollected in the AMSDb database at the University of Trieste(http://www.bbcm.units.it/∼tossi/antimic.html - see also theissue preface for a list of online facilities). These peptidesshow a bewildering range of molecular shapes and sizes [1],as they have had ample time to diverge or converge during

*Address correspondence to this author at the Department of Biochemistry,Biophysics and Macromolecular Chemistry, University of Trieste, ViaGiorgieri 1, 34127 Trieste Italy; Tel: +39-040-5583673; Fax: +39-040-5583691; E-mail: [email protected]; Web: http://www.bbcm.units.it/~tossi/antimic.html

their long evolution, in response to changes in targetpathogens or the microbial biota faced by the host, or in thedevelopment of interactions with other components of theimmune system. They however show some common traits.In general, HDPs have the capacity to interact with microbialcell wall components, most often membrane lipids, and oftenact by compromising microbial membranes. This explainstheir cationicity, as it favours electrostatic interaction withanionic microbial surfaces, and the fact that they often showa spatial segregation of polar and hydrophobic residues,allowing interaction of their hydrophobic surfaces with themembrane bilayer. Furthermore, many have a dual capacityto act both as antibiotics and signal molecules. These traitsseem to be quite an ancient, antimicrobial peptides used bybacteria themselves to clear ecological niches from rivalstrains, such as type I and II bacteriocins, act both asmembranolytic antibiotics and pheromones in quorumsensing [2, 3] (see papers by Cox et al., and Cotter et al., thisissue). In animal species, many amphibian antimicrobialpeptides are closely related to opioid neuropeptides [4, 5],and several mammalian peptides have demonstratedchemokine and cytokine activity towards immune cells [6].

Among the many structural types shown by differentHDPs, two are preponderant: small (16-40 residue) linearpeptides that adopt an amphipathic α-helical conformationon contact with the microbial surface, found in bacteria,arthropods, amphibians and mammals [7], and peptides witha compact, disulphide bridge-stabilized β-sheet structure,found in moulds, plants, molluscs, arthropods and mammals,and collectively termed defensins [1]. Both types of scaffoldcan structurally tolerate a high degree of sequence variation,and neither conformation seems to be crucial forantimicrobial activity in vitro, but may be required for the invivo activity, and/or for receptor binding in signaling

8 Current Protein and Peptide Science, 2005, Vol. 6, No. 1 Crovella et al.

processes. The combined antibiotic and signaling functionsmay thus have served to maintain these conformationsduring evolutionary divergence of sequences from those ofremote common ancestors, explaining their presence indistantly related organisms. It may however also haveresulted from function-driven structural convergence, settinga trap for those attempting to untangle the phylogeneticrelationships of HDPs. This is particularly the case forpeptides from distantly related species, when structural andfunctional similarities of the mature peptides are the onlyelements of comparison that are available. In more closelyrelated species, and particularly when other components ofthe genes encoding HDPs are known, such as similarities insignal, propeptide or non-coding sequences, chromosomallinkage within a species or syntenic conservation amongspecies facilitate the assignment of relationships.

We are therefore quite far from a unified picture of thephylogenetic relationship linking HDPs throughoutevolution, but a growing body of work carried out on plant,insect and mollusc defensins [8-11], on amphibian helicalpeptides [12-14] and on mammalian defensins [15-17] andcathelicidins [18-20] (see also Tomasinsig and Zanetti thisissue), are helping to resolve some important pieces of thispuzzle. In this review, we will focus on the evolution of animportant group of mammalian HDPs, the β-defensins, andanalyze the surprising diversity in variation rates in terms oftheir structural and functional characteristics.

MAMMALIAN DEFENSINS: THE DUPLICATION -DIVERGENCE - SPECIALIZATION HYPOTHESIS

Three types of defensins are variously present inmammals, and are expressed either in hematopoietic orepithelial cells [21-23]. The α- and β-defensins have diffe-rent consensus sequences, different cysteine residue spacingand connectivity, and are expressed with different signal andpro-pieces. However the mature peptides have a similar sizes(~30-45 residues) and quite similar tertiary structures [1].The third group, the θ-defensins, have been identified asactive genes only in primates to date [24-27] and areproduced by the post-translational ligation of two truncatedα-defensins to form peptides with a circular peptidebackbone. The products of the defensin genes have a directantimicrobial action with varying spectra of activity thatinclude Gram-positive and Gram-negative bacteria, fungiand enveloped viruses, that is however generally quite salt-and medium-sensitive. The antibacterial action appears toinvolve permeabilization of target cells as a crucial step,whereas the antiviral activity of some defensins appears toinvolve binding to membrane glycoproteins [22, 23]. Somedefensins have also been shown to act as chemokines andcytokines towards host defense cells [6].

In man, the genes encoding for these peptides present asimilar structure: two small exons divided by a large intron.The first exon encodes for the signal and the pro-piece, whilethe second exon encodes for the mature peptide. The genecluster for the α-defensins has been mapped to thechromosomal region 8p21-p23, where more recently, thefirst gene cluster for several β-defensins has been identified[28-31]. A cluster of α-defensin genes has been localized tothe syntenic region 8A4 in mice [15, 28-32]. The linkage

between α- and β-defensin genes, combined with structuraland functional similarities of their products, strongly indicatethey are phylogenetically related. Furthermore, given i) thatonly β-defensins have been identified in avian species to date[33-35] and in some mammalian species (bovids and pig)[23], ii) that structural homologues to the β-defensins arepresent in reptile venom [36], and iii) that based on themature peptide sequence, the β-defensins are suggested to bemore closely related to the insect defensins than α-defensins[17], it appears possible that the ancestral vertebrate defensinwas a β-defensin. If this were so, then at some stage inmammalian evolution duplication gave rise to the original α-defensin gene, and further duplication then occurred afterspecies divergence, as α-defensin genes cluster in a species-specific manner [17]. A model for the evolutionary history ofthe human genes has been proposed, also indicating how thegenes acquired different expression specificities [15]. Inprimate species, a subset of α-defensin genes apparentlyharnessed an unusual post-translational modificationmechanism to produce the θ-defensins, which are howeversilenced in humans [27, 37]. For β-defensins, the presence oforthologues in several different mammalian species suggestsan early, and possibly pre-mammalian, duplication for somegenes, whereas others seem to be species-specific,suggesting an ongoing evolutionary process subsequent toradiations.

For both α- and β-defensins the rate of synonymoussubstitutions is greater in the region coding for the signal(exon 1) and pro-pieces, and that of non-synonymoussubstitutions is greater in the region coding for the maturepeptides (exon 2), comparing paralogous genes. This patternof nucleotide substitution suggests duplication followed by aburst of divergence involving only the mature peptide, whichslows down after a new specialization is fixed [17, 30, 32].The process appears to be driven by positive selection with aspecial emphasis on residue charge, which is consistent withthe defensive role against pathogen infection. In α-defensins,charge variation in the mature peptide is often balanced by aneutralizing opposite variation in the pro-piece [16], whereasthis cannot occur in the β-defensins, in which the pro-pieceis small or absent.

EVOLUTION OF β-DEFENSINS - PEPTIDES INSYNTENY

The genes coding for sheep and cattle β-defensinslocalize onto equivalent chromosomes (26 and 27respectively) [18] with conserved synteny with the human 8pand mouse 8A4 chromosomal regions [15, 38]. Thechromosomal location of genes coding for characterized β-defensins thus appears to be conserved in mammals,consistent with an early and possibly pre-mammalian origin.Other β-defensin genes have been identified in humanchromosome 8 [39], and clusters of genes coding forpeptides with structural similarities to β-defensins have beenidentified also in other chromosomes in both man andmouse, but the role of their putative products in host defenseis not known [40, 41].

Comparison of both the gene and the mature peptidesequences indicate an uneven pattern of variations amongstthe different β-defensin orthologues in mammals. β-Defensin

Primate β-defensins – Structure, Function and Evolution Current Protein and Peptide Science, 2005, Vol. 6, No. 1 9

1 displays the same set of conserved residues in primate androdent species (Fig. 1), but finds no equivalent in bovids orpig. Variation observed amongst the defb1 gene in severalrodent species, [32] and the DEFB1 gene in numerousprimate species [42] (see below), indicate that evolution wasneutral rather than driven by positive selection. Also, unlikemost other defensins, the gene products are constitutivelyexpressed in both mouse and man [21]. The gene coding forprimate β-defensin 2 (DEFB4) is orthologous to that formouse β-defensin 4 (defb4) [30, 43], and similarities areapparent also with other rodent and bovid peptides. Thepattern of amino acid substitutions is more spread (Fig. 1)and it has been suggested that these peptides evolved underpositive selective pressure [30, 44]. The primate β-defensin 3gene (DEFB103 in humans) has a predicted orthologue inmouse (defb14), as well as the chinchilla [45], and in thiscase an impressive conservation of the gene productsequences is apparent (Fig. 1). A fourth human β-defensin,hBD4, has been described, and its gene (DEFB104) hasorthologues in other primates [31], but not in othermammalian species, although the mature peptide sequenceshows a tenuous similarity to mouse mBD2.

These patterns of variations would suggest that the genesorthologous to DEFB1 and DEFB4 arose by duplicationquite early, possibly prior to mammalian radiation.DEFB103 and its mouse defb14 orthologue arose later, butprior to the primate/rodent separation, while the DEFB104gene after primate radiation but prior to the human/baboonseparation, and this then duplicated to several more genes inprimates [30]. An analogous situation occurred in rodents,with respect to the defb2 gene, which arose after themouse/rat separation, and then duplicated into a number ofother genes in Mus musculus, although not all are present inrelated species. All this indicates that the β-defensins have along evolutionary history that remains quite active in presentmammalian species.

These hypotheses must however be weighed also in thelight of the relative lack of information that still exists withrespect to human and mouse genomic DNA, and in particularwith respect to the amplification of copy numbers forDEFB103 and DEFB104 in primates (see below), as thisprocess could lead to erroneous conclusions being drawn ongene evolution. Furthermore, we do not have enough dataabout other mammals genomic sequences, so orthologousgenes may exist but they have not yet been sequenced. Thefact that orthologues to the DEFB1 have not yet been foundin prosimians, even if it seems quite sure that this gene ispresent in these species, is a case in point.

PRIMATE β-DEFENSINS - EVOLUTION IN DIFFE-RENT GEARS

We have recently undertaken a systematic study of β-defensin genes in primates [42, 44, 46], concerningprincipally the four peptides whose expression and hostdefense activities have been well defined in man, and namelyhBD1, 2, 3 and 4. We reasoned that orthologues for all thesegenes should be present at least in catarrhine (Old WorldMonkeys, gibbons and Great Apes) and platyrrhine (NewWorld Monkey) species. The absence of genome projects formost of these species required amplification of the relevantgenes directly from genomic DNA preparations, rather thanfrom BAC clones, using appropriate primers based on thehuman sequences, as described briefly below. The speciesanalyzed, and the number of variations observed at both thenucleotide and amino acid levels, for the whole codingregion, for the signal peptide, the pro-piece and for themature peptides, are listed in (Table 1), whereas the aminoacid sequences of the gene products are compared in Fig. (2).

With respect to genes orthologous to DEFB1, the rates ofsynonymous and non-synonymous (amino acid altering)nucleotide substitutions indicate that dS > dN throughout thegene, so that it appears to have evolved in primates with a

Fig. (1). Comparison of β-defensin congeners from man and other mammals. Residues conserved within each set of putative orthologuesfor hBD1, hBD2, hBD3 and hBD4 are shaded in grey. Highly conserved residues are shaded in black. Nomenclature follows that ofsequences in the AMSDb database. defb14_MOUSE is a partial sequence from a predicted gene [40].


pattern of random nucleotide substitutions, as predicted bythe neutral theory of molecular evolution [42]. This is similarto what has been observed for the orthologous gene in rodentspecies, defb1 [32]. Apparently, the function(s) of the geneproducts tolerates rates of variation which are consistent withneutral evolution or weak purifying selection in both humanand mouse.

In contrast to DEFB1, there is evidence for positiveselection in the evolution of the DEFB4 gene orthologues(encoding for hBD2 in humans) at least at two points on thephylogenetic tree relating this gene in primates, the firstbeing at the divergence of the platyrrhines (NWM) from thecatarrhines (OWM) and the second at the divergence of theCercopithecidae from the Hylobatidae, Great Apes andhumans [44]. Furthermore, sequence variation is conside-rably higher in the region coding for the mature peptide than

in the signal sequence (Table 1, Fig. 2). This positiveselection may possibly be due to interactions of the peptideswith targets that are also undergoing variation.

The DEFB103 gene orthologues in primates show yet adifferent pattern of variation; the gene is in fact very highlyconserved for all the primates, including the New WorldMonkeys (Table 1, Fig. 2). This is in line with a high level ofconservation that reaches back to rodents (Fig. 1). It wouldthus appear that the gene product fixed a function(s) quiteearly in mammalian evolution that is less tolerant ofsequence variations, placing the gene under strong purifyingselection.

We have recently also sequenced the orthologues for theDEFB104 gene in several primates [Crovella et al.,unpublished data]. The pattern of variations in this case ismore similar to that of the DEFB1 gene than to the other two

Table 1. Analysis of the Gene Sequence Variation for Primate β-defensins (Nucleotide/Amino Acid)

DEFB1 (hBD1) DEFB4 (hBD2) DEFB103 (hBD3) DEFB104 (hBD4)in

frao

rder

supe

rfam

ilyspecies

tot sig pro pep tot sig pep tot sig pep tot sig pep

P. troglodytes (PTR) 2/1 0/0 0/0 2/1 2/1 0/0 2/1 4/2 2/1 2/1 1/1 1/1 0/0

G. gorilla (GGO) 4/2 1/1 1/0 2/1 0/0 0/0 0/0 4/1 2/1 2/0 4/3 2/2 2/1

Pon

gida

e

P. pygmaeus (PPY) 0/0 0/0 0/0 0/0 0/0 0/0 0/0 3/0 0/0 3/0 4/2 2/2 2/0

H. concolor (HCO) 11/4 0/0 2/1 9/3 8/4 0/0 8/4 5/3 0/0 5/3 14/10 4/4 10/6

H. lar (HLA) 11/4 0/0 2/1 9/3 11/5 3/1 8/4 5/2 1/0 4/2 13/8 4/4 9/4

Hyl

obat

idae

H. moloch (HMO) 11/4 0/0 2/1 9/3 11/4 5/1 6/3 5/3 1/1 4/2 nd nd nd

P. cristata (PCR) 18/5 6/1 3/1 9/3 18/13 2/1 16/12 9/2 2/1 7/1 13/6 4/2 9/4

P. obscurus (POB) 18/5 6/1 3/1 9/3 18/12 2/1 16/11 7/1 0/0 7/1 14/6 4/2 10/4

P. melalophos (PME) 18/5 6/1 3/1 9/3 17/11 1/0 16/11 9/2 2/1 7/1 nd nd nd

C. aethiops (CAE) 17/4 5/0 3/1 9/3 20/14 4/3 16/11 7/0 1/0 6/0 9/5 4/2 5/3

C. preussi (CPR) 16/4 5/0 3/1 8/3 19/13 3/2 16/11 7/1 1/1 6/0 14/7 6/3 8/4

C. erythrogaster (CER) 17/5 4/0 3/1 10/4 18/13 3/2 15/11 7/0 2/0 5/0 10/5 4/2 6/3

P. anubis (PPA) 16/5 4/0 3/1 9/4 18/14 2/1 16/13 7/0 1/0 6/0 10/5 4/2 6/3

M. fascicularis (MFA) 16/5 4/0 3/1 9/4 19/14 3/1 16/13 7/0 1/0 6/0 nd nd nd

M. rhesus (MRH) nd nd nd nd nd nd nd 7/0 1/0 6/0 nd nd nd

M. mulatta (MMU) 15/5 3/0 3/1 9/4 19/14 3/1 16/13 nd nd nd nd nd nd

M. tonkeana (MTO) nd nd nd nd nd nd nd nd nd nd 10/5 4/2 6/3

M. fuscata (MFU) nd nd nd nd nd nd nd nd nd nd 10/5 4/2 6/3

Cat

arrh

ini

Cer

copi

thec

idae

(O

WM

)

M. arctoides (MAR) nd nd nd nd nd nd nd nd nd nd 10/5 4/2 6/3

S. oedipus (SOE) 22/12 8/5 4/2 10/5 nd nd nd 3/0 0/0 3/0 29/14 14/6 15/8

C. jacchus (CJA) nd nd nd nd 24/17 3/1 21/16 nd nd nd 30/14 13/6 17/8

Pla

tyrr

hini

(NW

M)

A. fusciseps (AFU) nd nd nd nd nd nd nd nd nd nd 28/13 13/6 15/7

Note - in each column the number of nucleotide changes in the indicated region is shown before the slash and the number of amino acid changes after the slash, nd = not determined,tot = total coding sequence, sig = signal sequence, pro = prosequence, pep = mature peptide sequence.


Fig. (2). Comparison of the amino acid sequences of primate β-defensins gene products, including signal sequence, propiece and maturepeptide.

(Table 1), and indicates that evolution is neutral rather thanaffected by positive or purifying selection. This gene appearsto be primate specific, and has been subject to duplicationgiving rise to a cluster of other genes, that have a specifictissue distribution (they are predominantly expressed in malereproductive tract) and properties (e.g. low cationicity)which may imply novel functions [32].

CYTOGENETIC LOCALIZATION OF β-DEFENSINSIN PRIMATES - FISHING IN THE GENOME

A FISH approach based on two BAC clones that alsocontain one or more of the four well-characterized human β-defensins (DEFB1, DEFB4, DEFB103 and DEFB104) hasbeen used to assess the degree of conservation of this genecluster in metaphasic chromosomes of non-human primate


and two prosimian species [47]. The RP11-287p18 BACclone contains the DEFB4-DEFB103-DEFB104 cluster,while the RP11-791n7 clone contains DEFB1.

For these studies, chromosomal preparations from thethree Great Apes (chimpanzee, gorilla, and orangutan), threeOld World Monkeys (rhesus, Cercopithecus aethiops andPresbytis cristata), two New World Monkeys (Callithrixjacchus and Callicebus moloch) and two prosimians (Lemurcatta and Eulemur macaco) were used. Hybridization withboth the clones resulted in clear signals in correspondence to8p22-p23 in human preparations and in homologous bands inchromosomes from all the non-human primate species (seeFig. 3). However, neither BAC clone resulted in signals fromprosimian chromosomes (not shown).

The FISH approach indicates that chromosomal regionssyntenic to 8p22-p23 in the non-human primatechromosomes have a considerable degree of similarity at thelevel of their whole nucleotide sequence, including defensinintronic regions and other genes present, such as theepididymis specific HE/SPAG11 gene located in humansbetween the DEFB104 and DEFB103 genes [31, 41]. Thedegree of similarity is instead insufficient for hybridizationwith the prosimians chromosomes. It will be interesting todetermine if this difference involves the lack of genes suchas DEFB104 in these species, helping to pinpoint when thesearose, or is due to a general rearrangement of the regionexcessively reducing hybridization.

Schutte et al. [40], using a bioinformatics approach,reported the existence of three other β-defensin gene

Fig. (3). Comparative FISH analysis of the β-defensin genes. In situ hybridization of the BAC 287p18 probe, containing the DEFB104gene (A) and of the BAC 791n7 probe, containing DEFB1 (B) on elongated human chromosomes. Arrows indicate the four visible signals onchromosome 8 in the first case, and only two in the second. In situ hybridization of the BAC RP11-791n7 probe, containing the DEFB1 (C)and of the BAC RP11-287p18 probe, containing the DEFB4-DEFB103-DEFB104 cluster (D) onto metaphasic chromosome preparationsfrom Homo sapiens (HSA), the Great Apes: Pan troglodytes (PTR), Gorilla gorilla (GGO) and Pongo pygmaeus (PPY), the Old WorldMonkeys: Cercopithecus aethiops (CAE), Macaca mulatta (MMU) and Presbitis crystata (PCR), and the New World Monkeys: Callitrixjacchus (CJA) and Callicebus moloch (CMO). Chromosome numbering in both (C) and (D) are 8 for HSA, PTR GGO, PPY, CAE andMMU; 7 for PCR, 13 for CJA and 9 for CMO.


clusters, localized in humans on chromosomes 6p12,20q11.1 and 20p1, besides the 8p22-p23 cluster. They basedtheir method for identifying β-defensin genes only on exon2, hypothesizing that the characteristic six-cysteine motifwas always conserved. They were effective in finding suchmotifs, but their data are not supported by experimentalverification of gene expression and functionality. The FISHanalysis did not show hybridization of the BAC clones withchromosomal regions other than 8p22-p23, indicating a lowidentity of the other gene clusters to those containing knowndefensin genes.

STRUCTURAL IMPLICATIONS OF SEQUENCEVARIATIONS

β-Defensin sequences are highly variable, apart from the6 invariant cysteines and a highly conserved glycine residue(Fig. 1), but the determined tertiary structures (e.g. human β-defensins 1, 2 and 3, mouse β-defensins 7 and 8, and bovineβ-defensins 12) are quite similar [48-52]. They consist of atriple-stranded anti-parallel β-sheet with an N-terminal α-helical segment of different size and stability, present in allbut the bovine peptide. This conserved structural scaffoldlacks a hydrophobic core, and the fold thus appears to bestabilized mainly by the three disulfide bridges, so thatfolding requirements may not place too stringent a break onsequence variation. This variation can however result in

quite different surfaces, and consequently in differentdimerisation and oligomerisation patterns that are likely tobe functionally important (see Fig. 4).

In this respect, quite minor changes at the primarystructural level could cause marked changes at thequaternary level. In the case of hBD1, dimerization in thecrystal structure occurs longitudinally and involves asymmetric pair of salt bridges between Asp1 and Arg29 [51](Fig. 4A). The dimer forms a sickle-shaped structure, with ahydrophobic convex surface (seen face-on in the figure).String-like oligomers could then arise by hydrophobicinteraction involving these convex surfaces, and it has beensuggested that these could have a biological significance,occurring at the microbial surface [51]. Remarkablyhowever, Arg29 is only present in the human and gorillasequences (Fig. 2) and, in fact, only this position variessignificantly between the different primate groups. Thus, anapparently casual variation at this single residue may havehad a profound structural effect in the human molecule, buthow this reflects on the function remains to be determined.

Human β-defensin 2 dimerizes side-on in the crystalstructure, forming an extended six-stranded β-sheet [49](Fig. 4B). The amino acid sequences of the human and GreatApe peptides are effectively identical (Fig. 2), and those ofthe gibbon species (Hylobatidae) are very similar except atthe N-terminus, which instead resembles that of the Old

Fig. (4). Structures of human β-defensins. hBD1 (A) and hBD2 (B) dimers are shown as present in the crystal structures [49, 51] while theproposed dimeric structure of hBD3 (C) was adapted from ref. [52]. The position of residue variations in primate congeners with respect tothe human molecules is indicated in (D), (E) and (F).


World Monkey sequences. The latter differ considerablyfrom the human peptide also in the central region. Thisrelatively high degree of variation however does not appearto greatly affect the dimerization interface as seen in thehuman molecule. The characteristic N-terminal stretch in thehuman and Great Ape peptides, which corresponds to the α-helical segment (see Fig. 4B), does not quite reachsignificance for positive selection [44], but it includes aresidue (Asp4) that has been suggested to be relevant forsignaling functions, as the D4XXXC8L stretch, by analogywith a similar stretch in the mouse chemokine MIP3α, mayparticipate in the activation of the CCR6 receptor onimmature dendritic cells [53].

The remarkable conservation of the β-defensin 3sequences suggests a role that does not tolerate sequence orsurface variations. It has been shown that this highly cationicmolecule forms stable dimers or oligomers in solution, andthis has been proposed to involve a symmetric pair ofintermolecular salt bridges between Glu28 in one moleculeand Lys32 in another [52] (Fig. 4C). The dimeric structurewould be stabilized by a local reduction in cationicity due tointramolecular salt bridges formed between Glu27 and Arg17.It has further been suggested that these structuralcharacteristics could explain the fact that only hBD3, of allthe tested β-defensins, has an antimicrobial activity that isnot sensitive to salt concentration [54]. In this respect, theonly significant variation observed in the primate sequencesis that found in Hylobates concolor (HCO), where Arg17 issubstituted by Trp (Fig. 2), thus disrupting the intramolecularsalt bridge.

STRUCTURE, ACTIVITY AND EVOLUTION - WHATIS THE CONNECTION?

We have chemically synthesized the human and Macacafascicularis β-defensin 2 (hBD2 and mfaBD2), that representthe two most different sequences in this set of congeners, totests the effect of sequence variations on structure andantimicrobial activity, as well as a des-Asp version of hBD2,[(-D)hBD2], in which the Asp4 residue was omitted, to probeits role in modulating the activity of the human peptide. Toexamine the role of dimerization in β-defensin 3, we haveprepared the human peptide (hBD3), the Hylobates concolorpeptide (hcBD3) to probe the role of the intramolecular saltbridge, and an artificial analogue of hBD3 in which theLys26-Glu27-Glu28 stretch was replaced with the equivalentstretch from hBD1 (Phe-Thr-Lys) (ftkBD3), to probe the roleof dimerization. Extensive studies have been carried out onthe human molecules, hBD1, hBD2 and hBD3, by otherworkers, using isolated, synthetic or recombinant molecules[54-56], but to our knowledge, this type of comparison hadnot been previously made.

Native PAGE (Fig. 5A) did not reveal the presence ofstable dimers for the human or macaque β-defensin 2variants under these conditions [57]. This does not howeverpreclude that a form of dimerization based on H-bonding,similar to that seen in the crystal structure might not occurfor the human molecule at the microbial membrane surface,where the peptide is likely to concentrate. Both synthetic andrecombinant hBD3 [46, 52] were instead found to migrateslowly in non reducing conditions, indicating a dimeric or

more probably an oligomeric form, although the effect ofincomplete shielding by SDS in this highly cationic molecule(+22/dimer) on mobility must also be taken into account.hcBD3 and ftkBD3, on the other hand, were mainly in themonomeric form, also in non reducing conditions. Thisconfirmed that both the intermolecular bridge involvingGlu28 and Lys32, and the intramolecular salt bridge betweenArg17 and Glu27, are required for the formation of thequaternary structure.

CD spectroscopy of the synthesized and folded peptidesindicated that hBD2 is well structured in aqueous buffer(Fig. 5B), and is largely unaffected by interaction withmembrane mimicking SDS micelles (Fig. 5C), corroboratingthe presence of a stable N-terminal α-helical stretch, asobserved in the crystal structure [49]. The spectra of the des-Asp variant of the human molecule, or of mfaBD2 do notsupport the presence of an N-terminal α-helical stretch sothat this may be a structural characteristic of the human andGreat Ape molecules, requiring Asp4. The CD spectrum ofhBD3 indicated the presence of the β-sheet core in aqueousbuffer (Fig. 5D, 5E), while the helical component wasevident only in the presence of SDS micelles. Furthermore,the structural stability appeared to decrease in the orderhBD3 > hcBD3 > ftkBD3, so that salt bridging may berelevant also in stabilizing the tertiary structure. Theseresults generally confirm the structural implications ofsequence variations discussed above. But how did this reflecton the peptides’ biological activities? The in vitroantimicrobial activity of the peptides on some referencemicro-organisms, suggest that one must be careful inextrapolating functional implications from structural data.

The peptides showed a differing spectrum ofantimicrobial activity, in terms of MIC, on the testedbacterial and fungal micro-organisms, when tested at lowsalt and medium concentration (Table 2). It was generallymarkedly decreased at higher medium concentrations, in linewith numerous other reports on the in vitro antimicrobialactivity of defensins in general. Only B. cepacia wasunaffected by all peptides but this micro-organism isresistant to most antimicrobial peptides [58]. The (-D)hBD2variant of hBD2 and the ftkBD3 variant of hBD3 weregenerally less effective in terms of the MIC values, underour low medium assay conditions, than the respective nativepeptides. Moreover, while mfaBD2 showed a significantlydifferent behaviour to hBD2, being more active towards theGram-positive bacteria and yeast, hcBD3 showed acomparable potency, spectrum of activity, and mediumdependence to the human peptide, despite a reduced capacityto dimerize. However, ftkBD3 was more active than thenative peptide in high concentration medium, and this wasconfirmed in bacterial inactivation experiments carried outon representative Gram-positive and Gram-negative bacteria(Fig. 6A), at increasing NaCl concentrations, for both hcBD3and ftkBD3. The supposition that the potent and salt-insensitive activity of hBD3 might derive from stabledimerization in solution is thus apparently unfounded.Rather, these characteristics may be imputed to the very highcationicity of these defensins.

Time-killing studies were also quite revealing (Fig. 6B,6C). Under low salt conditions E. coli was rapidly and


Table 2. In vitro Antimicrobial Activity (MIC, µM) of Selected Human and Primate β-defensins and their Analogs

MIC (µM)

B. cepaciaβ-defensin charge E. coli(ML-35)

P. aeruginosa(ATCC 27853)

(6981) (14273)

S. aureus(710A)

C. albicans(c.i.)

hBD2 +6 4 4 >32 >32 8-16 16

mfaBD2 +7 4-8 8 >32 >32 4 4

(-D)hBD2 +7 4 16-32 >32 >32 16-32 32

hBD3 +11 1 (32) 2 (32) >32 >32 1 2 (32)

hcBD3 +10 1 (16) 2 (32) >32 >32 2 1 (16)

ftkBD3 +13 8 (8) 4 >32 >32 4 4 (16)

MIC values were determined with a low medium concentration [5 % (v/v) MH broth for bacteria or 5 % (v/v) SAB medium for yeast] in 10 mM SPB using 105 CFU/ml micro-organisms at logarithmic phase, and are the mean of at least three experiments. Values in parentheses were determined at a high medium concentration [50 % (v/v) MH broth or SABmedium], and are furnished only if ≤32.

Fig. (5). Native PAGE and CD spectra of selected primate β-defensins. (A) PAGE of peptides in non-reducing (lanes 2, 4, 6, 8,10, 12) and reducing (3, 5, 7, 9, 11, 13) conditions. CD spectra ofhBD2 (), mfaBD2 (---) and (-D)hBD2 (•••) in 5 mM sodiumphosphate buffer pH 7 (B) and in 10 mM SDS (C) . CD spectra ofhBD3 (), hcBD3 (---) and ftkBD3 (•••) in 5 mM sodiumphosphate buffer pH 7 (D) and in 10 mM SDS (E).

completely eradicated by both hBD3 and hcBD3, as well asby hBD2. [46, 57]. The somewhat slower killing bymfaBD2, and by (-D)hBD2 in particular, may indicate asignificant role of the N-terminal helical stretch in this case.With respect to the Gram-positive S. aureus, mfaBD2 wasinstead the most efficient, and both hBD3 and hcBD3 werequite slow. In the latter case, the quite reasonable MICresults would seem to indicate that these more cationicpeptides act with a slow but effective mechanism.

Membrane permeabilization studies confirmed amembranolytic capacity for the β-defensins in general,although they display quite different membranepermeabilization kinetics (Fig. 6D, 6E) [46, 57]. hBD2seemed to be the most efficient in permeabilizing the E. colicytoplasmic membrane, after an initial lag period, whilemfaBD2 was by far the most active on the S. aureusmembrane, consistent with their respective killingefficiencies. As indicated by the slower permeabilizationkinetics observed in the presence of (-D)hBD2 than forhBD2, and for ftkBD3 than for hBD3, the capacity to rapidlypermeabilize the E. coli membrane seems to correlated withstructural integrity rather than just to the charge (ftkBD3 is+13). It was not possible to accurately assess the behaviourof the hBD3 or its analogues with respect to S. aureus, due toan interfering experimental artifact (see below), it washowever assessed to be quite slow, in line with killingexperiments.

Taken together, these studies indicate that the amino acidsequence variations observed in primate β-defensin 2,resulting from positive selection, do in fact reflect on the invitro antimicrobial activity of these peptides. The macaquepeptide seems more efficient in inactivating Gram-positivebacteria and yeast, while the human peptide is more activeon Gram-negative bacteria. This would seem to indicatediffering modes of action that depend on the structuralcharacteristics of the peptides, such as the stable N-terminalstretch in the human congener. For the BD3 variants, thepotent and broad spectrum of activity seems instead to berelated more to the charge than to the capacity to dimerize.Also, bacterial inactivation by hBD3, although quite


effective in the long run, would appear to proceed by aslower mechanism than the less charged BD2 variants. Wehave found an analogous potency/killing-kinetics/cationicityrelationship in short α-helical lytic peptides [7, 59], and inthat case could explain it in terms of a decreased stability andlifetime of toroidal pores formed in the bacterial membranesat increased cationicity, as suggested by Matsuzaki et al., formagainin analogues [60]. However, it is not yet known if thedefensins act on bacterial membranes via this type of poreformation.

It is thus conceivable that the evolution of primate β-defensins may have occurred, at least in part, in response todifferent environmental pressures, so as to modulate thedirect antimicrobial activity. It has been suggested that thedifferent primate β-defensin genes have arisen by positiveselection which favoured alterations in the charge [30, 31].In the case of β-defensin 3, this has resulted in anexceptionally high cationicity which seems to have benefitedthe antimicrobial activity. It is possible that the requirementsfor packing such a high charge into a stable structure haveimposed restrictions on variation that underlie theremarkable conservation of this gene in mammals. Curiouslyhowever, this may have resulted in a lower reliance of thisactivity on structural integrity. We have seen that it does not

require the stable dimerization displayed by the humanpeptide, and others have indicated that it can even survivebridging isomerization, linearization or fragmentation, undercertain conditions [61, 62]. The β-defensin 2 peptides insteadretained a versatile scaffold that has allowed positiveselection to continue, possibly also in response to exposureto different microbial biotas, where structural integrity seemsrelevant. In both cases, however, the importance of the directantimicrobial activity in determining their evolution willrequire a more extensive analysis of the peptides’ activity ona wider panel of micro-organisms, and should also take intoaccount conditions likely to be present in vivo (see review byBowdish et al., this issue).

Other activities in immunity are also likely to have had arelevant, and possibly a fundamental role, in the evolution ofthese peptides. The β-defensins in fact have the capacity tomodulate both innate and adaptive responses to infection andinflammation with a variety of interdependent mechanismsthat seem to vary in a complementary manner, frommolecule to molecule. hBD1 and 2 have been reported toinduce chemotaxis of immature dendritic cells (iDC) andresting memory T-cells and by interacting specifically withthe CCR6 receptor [63], and the same receptor has recentlybeen reported to mediate chemotaxis of neutrophils primed

Fig. (6). Antimicrobial activity of selected β-defensins. (A) Bacterial killing for β-defensin 3 variants at increasing concentrations of NaCl,as determined after exposure of 105 CFU/ml of S. aureus (¨), E. coli (¡), P. aeruginosa (◊) to 8 µM peptide; (B) time killing plots for E.coli ML-35 (107 CFU/ml, 8 µM peptide in 10 mM SPB); (C) time killing plots for S. aureus 710A (107 CFU/ml, 8 µM peptide in 10 mMSPB); hBD2 (n), mfaBD2 (l), (-D)hBD2 (s), hBD3 (o) and hcBD3 (¡); (D) inner membrane permeabilizaton kinetics, assessed via thehydrolysis of ONPG, for E. coli ML-35 pYC; (E) cytoplasmic membrane permeabilizaton kinetics assessed via the hydrolysis of ONPG-6Pfor S. aureus 710A, (107 CFU/ml bacteria in the presence of 5 µM peptide in 10 mM SPB for both); hBD2 (), mfaBD2 (− −), (-D)hBD2 (−• −), hBD3 (••••••), hcBD3 (− •• −) and ftkBD3 (……..).


with TNF-α by hBD2, but not hBD1 [64]. hBD2 has alsobeen reported to act as a chemoattractant for mast cells, viaother receptors, and also to induce their maturation anddegranulation, resulting in the release of histamine andprostaglandin [65-67]. In this respect, mouse BD2, which isnot a homologue of hBD2 (see Fig. 1) has been reported toact both as a chemoattractant for iDCs through the CCR6receptor, and to induce their maturation via a Toll-likereceptor 4 [68] (see review by Biragyn, this issue). hBD3and 4, on the other hand, have been reported to act aschemoattractants for monocytes [69, 70].

We have started to investigate how the structuralcharacteristics of the β-defensins affect their capacity tointeract with host cells involved in defense. We havepreliminary evidence that hBD2 activates monocytes in astructure-dependent manner, while the chemotaxis ofimmature dendritic cells appeared to be more tolerant ofstructural variations, particularly with respect to the N-terminal stretch that contains the supposedly MIP3α-likemotif [Pacor et al., unpublished data].

β-DEFENSINS IN HOST DEFENSE AND THEPOSSIBLE CONSEQUENCES OF POLYMORPHISMS

It is difficult to determine the exact protective role ofhost defense peptides in vivo, due to their complexexpression patterns, to the simultaneous presence of differentpeptides that can have an additive or synergic activity, and tothe fact that these peptides can act both as antibiotics andsignaling molecules that modulate other immune responses.Several in vivo studies have been carried out to determine theeffect of loss-of-function or gain-of-function for defensins inanimal models, but results are not of straightforwardinterpretation. Thus, mice in which matrilysin, the enzymeresponsible for α-defensin maturation, has been knocked out,inactivating the whole family, show an increasedsusceptibility to some infections, while knocking out singlepeptides, such as hBD1 had a less quantifiable effect,possibly because it is just one of several host defensepeptides present in this animal [71, 72] (see [23] for review).On the other hand, expression of the α-defensin HD-5, intransgenic mice has a protective role [73].

There is evidence of a correlation between defensinexpression and incidence of infection in man. For example,in patients affected by psoriasis, epithelial β-defensins areover expressed and skin lesions are relatively free frominfection, while in atopic dermatitis their expression issuppressed, and lesions are infection prone [74, 75].Impaired β-defensin function [76], or their increaseddegradation by cysteine proteases [77] have both beenassociated with an increased colonization by bacteria in theupper-airways of CF patients. Recently, a possible role fordefensins in the etiology of Crohn’s Disease has beendescribed [78] and an absence of these molecules, as well asthe cathelicidin LL-37, has been demonstrated in patientssuffering from Morbus Kostmann [79]. Lastly, β-defensins 2and 3 have been associated with protection from HIVinfection [80]. All this implies that defensins may beimportant in determining susceptibility to and the severity ofhuman disease. It further implies that polymorphisms in theirgenes might increase or decrease the predisposition towards

specific diseases, by affecting expression, delivery, function,or copy number.

The discovery of new SNPs in these genes andassessment of their allelic distribution in various ethnicpopulations is the first important step that allows the designof association studies to understand the role of thesemolecules in both infectious and autoimmune diseases inman. To date, there are several reports describing a numberof novel SNPs in the human β-defensin genes, [81-83]. Anon-synonymous SNP [Val6àIle, (see Fig. 2) occurs in themature peptide region of hBD1, with functionalconsequences that are discussed below, while a non-synonymous LeuàHis variation in the signal sequence ofhBD2 could affect secretion or proteolytic cleavage.

The constitutive nature of hBD1, in particular, hassuggested that it could have a permanent protective role, andthe suggestion that the high salt conditions in CF could resultin its inactivation and correlate with the increased incidenceof infection in this condition, supports this role. Tocorroborate this hypothesis, the Val6àIle variant has beenassociated with Chronic Obstructive Pulmonary Disease in aJapanese population [84]. Recently two reports described adirect association between a SNP in the DEFB1 5’-UTR andinfections; a C→G transversion at position –44 of the gene,has been associated with Candida sp. carriage in the mouth[85], and an augmented risk of HIV-1 infection [86].However, it must be considered that the β-defensin genefamily in man reveals a very complex and redundantorganization, so that the impaired function of only one oreven a few peptides could be compensated for by expressionof the others.

Taken together, these and other recent observations onthe role of different defensins in important human diseases,suggest the importance of extending the mapping ofpolymorphisms to the genes for other defensin peptides.This, however, must take into account the presence of copynumber polymorphisms. Recent reports by Hollox et al., [87]and by Boniotto et al. [88], based both on cytogeneticmethods and whole-genome search, indicate that the copynumber of the DEFB4, DEFB103 and DEFB104 genes,varies in the human population, ranging from 2 to 12 copiesof genes per diploid genome, while only two copies ofDEFB1 are present. The existence of multiple copies with avery high sequence homology suggests that the directsequencing of PCR products is ineffective to assess SNPfrequencies, and that new methods for SNP detection shouldbe considered to map new polymorphisms in these genes.Furthermore, while the presence of multiple copies of a genewould likely decrease the likeliness of SNPs affectingfunction, a variable copy number could clearly correlate withthe expression levels of the corresponding peptides, whichmay also be associated with different predisposition to someinfections.

A final consideration is that the copy numberpolymorphism could provide a mechanism for the ongoingevolution of these molecules, as SNPs at the level of anysingle copy in multi-copy defensins may be less subject topurifying selection than those for a single-copy ones, andthis could bring out new functions. Were these to provideindividuals with a significant increase in protection from


infection, they could eventually become fixed in thepopulation. In this respect, this may have occurred quiterecently for the mouse defb7 gene, identified in the C57Bl/6inbred mouse strain, that codes for a novel defensin in whichone of the highly conserved cysteines has been substituted.The putative gene product was synthesized, and was found toform a covalent dimer, presumably via the unpartneredcysteine [89], which had potent activity in vitro against S.aureus and E. coli, and unusually also against the otherwisehighly resistant pathogen B. cepacia. Furthermore, activityagainst P. aeruginosa was not salt sensitive. This variationcould thus potentially provide increased protection also invivo and it is significant that it has become fixed in theinbred mouse population.

CONCLUDING REMARKS

The β-defensins family of peptides is an importantcomponent of innate immunity in mammals, contributing totheir protection from microbial infection. Effectively allpeptides of this family that have been tested havedemonstrated a direct antimicrobial activity in vitro, whichvaries from peptide to peptide, and which is generally, butnot invariably, salt and medium sensitive. They also displaya signaling activity, in vitro, on various types of host cellsconnected with host defense. While it has been demonstratedthat these peptides play a protective role in vivo, it is notclear whether one or the other of the above activitiespredominates in a real biological setting, or whether theirintegration leads to a multi-functional response to infection.The comparison of β-defensins from different mammalianspecies has revealed a pattern of evolution in which differentcongeners appear to pursue different evolutionary pathways,in some cases according to the neutral theory of molecularevolution, in others under the spur of positive selection, yetin others under a strong purifying pressure leading toconservation. A few invariant residues in these peptidesseem to ensure folding to a conserved tertiary structuralscaffold that tolerates a high level of sequence variation, sothat they have been free to sample different surfacecharacteristics with respect to cationic and hydrophobicresidues, resulting in different oligomerization patterns. Itremains to be determined how these are related to theirvarious biological functions.

The identification and comparison of β-defensincongeners from closely related primate species confirms thepattern of evolution seen in the broader mammalian context,and has provided molecules with defined sets of variations tobegin to probe the functional effects of their molecularevolution. Peptide-specific variations in the in vitroantimicrobial activity suggest that direct interactions withbacteria and bacterial biotas may have played some role inshaping their evolution, but the question as to what shapedtheir peculiarly varied patterns of evolution still remainswide open.

APPENDIX - WORKING WITH β-DEFENSINS

β-Defensins are not easy peptides to work with. To beginwith, they are not so easy to obtain. Studies of their activityhave been carried out on isolated natural peptides,

recombinant peptides and chemically synthesized molecules.The first method has made use of human plasma and urinefor isolating hBD1 [90, 91], and of psoriatic skin scales forisolating hBD2 and 3 [54, 56], in which these peptides areabundantly expressed. Recombinant peptides have generallybeen used for structure determination [49, 51, 52]. We havepreferred to use chemical synthesis as the most rapid methodto obtain several analogues in reasonable quantities. A majorproblem with both recombinant and synthetic defensins isfolding. These molecules seem to have a strong intrinsictendency to fold correctly, by forming the right disulfideconnectivities, which seems to be independent ofhydrophobic collapse, as they lack a hydrophobic core [50].However, the correct conditions have to be provided.Different folding conditions have been described, sometimesleading to several different isomers with different bridgingconnectivities [61]. To make matters worse, several reportsindicate that antimicrobial activity can be displayed bydefensins regardless of bridging isomerization, linearizationor fragmentation, under the low salt and medium conditionsgenerally used with these peptides [61, 62, 92, 93]. In ourexperience, it is thus useful to i) carry out folding on the bestpossible quality of crude peptide, which reduces the numberof isomers with S-S different connectivities; ii) use asufficiently robust medium to ensure microbial vitality and agreater resolution in antimicrobial activities; iii) use asufficiently wide panel of micro organisms and iv) use anumber of different assays, including MIC determination,time-killing and membrane permeabilization, so as to ensurea more significant evaluation of the in vitro activity.

Another problem with defensins is the salt and mediumsensitivity of their antimicrobial action. This may seem oddfor host defense peptides that are supposed to function inphysiological conditions (see paper by Bowdish et al., thisissue). However, they may in some cases be produced atsufficiently high concentrations in vivo to overcome theselimitations, and also to be expressed in physiological settingswith relatively hypotonic conditions. These milieus are notso facile to reproduce in the lab, and care must be taken tobalance in vitro conditions so that the target micro-organismscan grow relatively healthily while at the same time allowingthe peptides to function at accessible concentrations.

Regarding genomic aspects, most phylogenetic studies onβ-defensins have been carried out in human and mouse,where sequence information and BAC clones were alreadyavailable from genome projects. This is limiting at present,and we have carried out our studies on genomic DNAobtained from hair or tissue extracts of several primatespecies. Furthermore, we have always attempted to obtainsequence information from DNA extracted from severalindividuals per species, to avoid errors coming fromcontamination or polymorphisms. However, this approachdepends on a sufficient conservation of genes at a nucleotidelevel to allow primers designed on the basis of the knowngene sequence from one species (in our case human) toamplify orthologous genes from another. It seems to workwell with all the primates tested, but was not applicable tothe prosimians. In the next few sections, a brief descriptionof the methods used in obtaining genetic information,preparing peptides and determining their activities isprovided.


DNA Extraction, PCR Design, DNA Sequencing andAlgorithms for Evolutionary Studies

Both exons of the DEFB1, DEFB4, DEFB103 andDEFB104 genes and short portions of their flanking,untranslated and intronic regions have been investigated in22 primate species for interspecific variations. The primatesample analyzed included the Great Apes (chimpanzee,gorilla, and orangutan), Hylobatidae (gibbons and lesserapes), Cercopithecidae (Old World Monkeys such asmacaques and baboons), and platyrrhine species (New WorldMonkeys). Hairs, muscle, bone or liver samples werecollected from live or deceased specimens at zoos, wherepossible from several individuals per species. GenomicDNAs was extracted from hairs using Chelex 100 extraction[94], and from tissues using the phenol/chloroform protocol[95]. Genomic DNA amplification of primate homologs tothe human genes was performed using appropriate primersdesigned on the basis of the published human sequences(NM_005218 for DEFB1; NM_004942 for DEFB4;NM_018661 for DEFB103; NM_080389 for DEFB104).Primer sequences and PCR experiments were designed usingthe Primer Express 2.0 software (Applied Biosystems).Species specific PCR amplification products were thensequenced by using the Big Dye Terminator 3.0 kit (AppliedBiosystems). Multiple alignments of the nucleotide andamino acid sequences were performed using the programCLUSTAL X [96]. Pair wise comparisons betweennucleotide sequences were performed to investigate theextent of evolutionary constraint at the amino acid level indifferent regions of the molecule. The number ofsynonymous nucleotide substitutions per synonymous site(dS) and the number of non-synonymous nucleotidesubstitutions per non-synonymous site (dN) betweenorthologous sequences were computed following the methodof Nei and Gojobori [97]. The neutral theory of molecularevolution [98] predicts that in most functional genes dS>dNas a consequence of the fact that synonymous positions aresubject to far fewer functional constraints than non-synonymous sites. On the other hand, if dN>dS then it isassumed to be evidence of positive selection acting topromote diversity at the amino acid level [99].

Comparative Cytogenetics

Cell lines. Fibroblast cells from PTR, GGO, PPY, MMU,CAE, PCR, CJA (see Table 1), Callicebus moloch, Lemurcatta and Eulemur macaco macaco were grown inDulbecco’s minimal essential medium (Gibco-BRLRockville, MD) supplemented with 10 % fetal calf serum, 2mM L-glutamine and penicillin-streptomycin sulfate andcultured at 37 ºC.

BAC clones. The BAC clones RP11-791n7, RP11-287p18, and RP11-351d16 were obtained from UCSCdatabase, April 2003, queering with the 4 gene sequenceslisted in the previous section. The former two are in the8p22-p23 region (http://genome.ucsc.edu/, April 2003), thelatter clone is a test probe containing the conserved RETgene.

FISH experiments. Stretched chromosome preparationswere obtained following standard protocols [100]. Slideswere hybridized in situ as described by Lichter et al. [101],

with minor modifications. Digital images were obtainedusing a Leica DMRXA epifluorescence microscopeequipped with a cooled CCD camera (Princeton Instruments,NJ). DAPI (4,6-diaminido-2-phenylindole dihydrochloride)was used to counterstain the chromosomes in order torecognize them on the basis of the DAPI banding pattern.Cy3 (Texas Red) and DAPI fluorescence signals, detectedwith specific filters, were recorded separately as grey scaleimages. Pseudocolouring and merging of images wereperformed using Adobe Photoshop™ software. The RP11-351d16 BAC clone, which contains the evolutionarilyconserved RET gene [102] was used as positive control.

Peptide Synthesis and Characterization

Peptides were prepared by an optimized solid phasepeptide synthesis protocol, using the Fmoc chemistry, andchlorotrityl chloride resin appropriately functionalized withthe C-terminal amino acid [46, 57]. Double coupling at allresidues, using activators TBTU or HATU (45 min) for thefirst cycle, and PyBOP, (120-180 min) for the second,generally ensured a reasonable final yield of good qualitycrude peptide. After cleavage, peptides were treated withtris(carboxyethyl)phosphine.HCl (TCEP) as reducing agentand purified by preparative HPLC, and the fractioncontaining the correct peptide, as determined by electrosprayionization mass spectrometry, was allowed to fold in N2-saturated aqueous buffer in the presence of cysteine andcystine (10/1). After completion of folding, as determined bymass spectrometry and analytical HPLC, the folding solutionwas directly purified on a preparative RP-HPLC column.The peptide with the correct folding and connectivitiesgenerally eluted as the principle band and was normally wellseparated from the other bands containing peptides withother connectivities [46]. Partial limited proteolysis and/orCD helped individuate the correct peptide.

The secondary structure can be qualitatively assessedusing CD spectroscopy on 20 µM peptide in 5 mM sodiumphosphate buffer pH 7.0, in the presence or absence of 50 %(v/v) trifluoroethanol, or 10 mM SDS. Spectra of peptideswith the correct connectivity and folding generally indicate ahigher conformational stability.

Antimicrobial Activity

An estimate of antimicrobial potency can be obtainedrelatively quickly and without excessive peptide usage, interms of the minimum inhibitory concentrations (MIC), byusing a version of the microdilution susceptibility testsuitable for β-defensins, which are generally salt andmedium sensitive [46]. Assays in 1 % (v/v) tryptic soy broth(TSB) are often reported in the literature, but we favour amore robust 5 % (v/v) concentration of MH or TSB broths,in 10 mM sodium phosphate buffer, inoculated with ~ 105

CFU/ml micro-organisms at logarithmic phase. 5 % (v/v)Sabouraud/dextrose medium can be used for yeast. Time-killing studies were carried out in sodium phosphate buffer,testing the salt sensitivity by varying the NaCl concentrationup to 150 mM.

Membrane permeabilization kinetics were determinedspectrophotometrically by following the unmasking ofcytoplasmic β-galactosidase or 6-phospho-β-galactosidases,


that were respectively constitutively produced in specificstrains of E. coli (ML-35 pYC) and S. aureus (710A), to theextracellular chromogenic substrates ONPG or ONPG-6P, asdescribed [103]. However, hBD3 seems to transiently bindONPG-6P, resulting in an increased absorption as an artifact,which needs to be taken into account.

ACKNOWLEDGEMENTS

The research in the laboratories of SC and AT was in partsupported by Grants from the FVG Region, the ItalianMinistry of the Universities and Scientific Research (MIURPRIN 2003 and Centre of Excellence for Biocristallography -CofinLab) and by the EU PANAD project QLK2-CT-2000-00411; MB and MVVF are recipients of a long termfellowship from University of Trieste.

ABBREVIATIONS

AMP = Antimicrobial peptide

CFU = Colony-forming units

dS = Number of synonymous nucleotide substitutionsper synonymous site

dN = Number of non-synonymous nucleotidesubstitutions per non-synonymous site

FISH = Fluorescence in situ hybridization

hBD = Human β-defensin

hcBD = Hylobates concolor β-defensin

HDP = Host defense peptide

mfaBD = Macaca fascicularis β-defensin

SNP = Single nucleotide polymorphism

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Primate β-defensins - Structure, Function and Evolution

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