Variability and evolution of bovine [beta]-defensin genes

Abstract

Defensins comprise an important family of antimicrobial peptides. Among vertebrates numerous defensin genes have been detected, but their evolutionary background is still discussed. We investigated the molecular evolution of bovine defensins via screening of different bovine species including the extinct ancestor of domestic cattle (Bos primigenius) for beta-defensin encoding genes. We detected a large variability of new defensin encoding sequences similar to previously published bovine neutrophil beta-defensin (bnbd), neutrophil beta-defensin 12 (nbd12), enteric beta-defensin (ebd), lingual antimicrobial peptide (lap), and tracheal antimicrobial peptide (tap). Our data suggest that variants of the same so-called subfamily (tap, lap, ebd, and nbd12) each share a common ancestry independent of their species origin, implicating several duplication events of tap, lap, ebd, and nbd12 before the different bovine lineages diverged. Variants of bovine neutrophil beta-defensins bnbd5 and bnbd9 were detected exclusively in domestic cattle and aurochs. Values of synonymous and nonsynonymous substitutions demonstrated lap, bnbd5, bnbd9 and nbd12 evolving under positive selection, whereas amino-acid altering substitutions among variants of ebd and tap are purified. Comparison of the amino-acid sequences with available structures of human and murine defensins suggested conservation of the typical secondary elements of defensins in the absence of high sequence similarity.

Genes and Immunity (2005) 6, 115122

& 2005 Nature Publishing Group All rights reserved 1466-4879/05 $30.00www.nature.com/geneFULL PAPERVariability and evolution of bovine b-defensin genesK Luenser1 and A Ludwig11Department of Evolutionary Genetics, Leibniz-Institute for Zoo and Wildlife Research, Berlin, GermanyDefensins comprise an important family of antimicrobial peptides. Among vertebrates numerous defensin genes have been

detected, but their evolutionary background is still discussed. We investigated the molecular evolution of bovine defensins via

screening of different bovine species including the extinct ancestor of domestic cattle (Bos primigenius) for b-defensin encoding

genes. We detected a large variability of new defensin encoding sequences similar to previously published bovine neutrophil

b-defensin (bnbd), neutrophil b-defensin 12 (nbd12), enteric b-defensin (ebd), lingual antimicrobial peptide (lap), and tracheal

antimicrobial peptide (tap). Our data suggest that variants of the same so-called subfamily (tap, lap, ebd, and nbd12) each

share a common ancestry independent of their species origin, implicating several duplication events of tap, lap, ebd, and nbd12

before the different bovine lineages diverged. Variants of bovine neutrophil b-defensins bnbd5 and bnbd9 were detected

exclusively in domestic cattle and aurochs. Values of synonymous and nonsynonymous substitutions demonstrated lap,

bnbd5, bnbd9 and nbd12 evolving under positive selection, whereas amino-acid altering substitutions among variants of ebd

and tap are purified. Comparison of the amino-acid sequences with available structures of human and murine defensins

suggested conservation of the typical secondary elements of defensins in the absence of high sequence similarity.Genes and Immunity (2005) 6, 115122. doi:10.1038/sj.gene.6364153

Published online 23 December 2004Keywords: b-defensin; innate immunity; gene duplication; positive selection; orthologous genes; molecular evolutionIntroductionDefensins are members of a large variety of antibacterial

peptides produced by vertebrate and invertebrate animals that function in innate immune defence.14Beside their antimicrobial activity, defensins have

chemoattractant activity for dendritic and T cells.5 Thus,

defensins provide the first barrier of host defence

combating broad spectrum of microorganisms, as well

as linkage of the innate and adaptive immune response.5

They are small (26 kDa), cationic, and active against

many Gram-negative and Gram-positive bacteria, fungi

and enveloped viruses.24 Six cysteines found to be

strongly conserved are involved in construction of three

disulphide bounds, stabilizing the tertiary structure of

the defensins.3 Based on their size and the spatial

position of the six cysteine residues, animal defensins

are classified as a-, b-, y-defensins of vertebrates

(mammalia and birds) and insect defensins. To date,

vertebrate defensins were identified in numerous species: mice,68 birdswhere they are called gallinacins

9 rats,10 cattle,11,12 goats,13 sheeps,14 humans,8,15,16 and

non-human primates1720 (reviewed in Martin et al,4

Hughes,21 Schroder,22 and Lehrer and Ganz23). Interestingly, domestic cattle (Bos taurus) was shown to share a

broad spectrum of different b-defensins, for example, tap

(tracheal antimicrobial peptide), lap (lingual antimicrobial peptide), bnbd (bovine neutrophil peptide), nbd12

(neutrophil beta defensin 12), and ebd (enteric beta

defensin).11,12Regarding the sites of expression of the three classes of

defensins, there appear to be substantial differences.

Mammalian a-defensins were detected in neutrophils

and alveolar macrophages,24 as well as in Paneth cells of

humans,25,26 rats,27 and mice.28 Vertebrate b-defensins are

predominantely produced by epithelial cells, lining

various organs, for example, the human skin and

bronchial tree, the tongue, and genitourinary tract.11,2931The b-defensins are encoded by genes consisting of

two exons. The primary translation product is an inactive

precursor (prepropeptide) constructed of an N-terminal

signal sequence, a short propiece and a C-terminal

mature peptide, which is cleaved from the propiece.32

The first exon encodes the signal sequence, the second

exon encodes the pro- and the mature peptide.32The aim of the present study was to investigate the

sequence divergence and evolutionary relations of

bovine b-defensins. We examined the variety of b-defensins screening genomic DNA of different bovine

species including the aurochs, Bos primigenius, which

became extinct in the Middle Ages, and is regarded as

the ancestor of domestic cattle.33Comparison of orthologous defensin genes was used

to infer the molecular evolution of bovine b-defensins.

Comparison of deduced amino-acid sequences of the

presented bovine defensins with structures of human

and murine defensins allows speculation about the

functional properties of distinct regions within the

mature defensin peptide.Correspondence: Dr K Luenser, Institut fur Zoo- und Wildtierforschung,Postfach 601103, 10252 Berlin, Germany. E-mail: [email protected] 2 September 2004; revised 6 October 2004; accepted 7

October 2004; published online 23 December 2004Bovine b-defensinsK Luenser and A LudwigTable 1 Nucleotide-alignment of bovine defensins116Vertical numbers indicate nucleotide position.Genes and ImmunityBovine b-defensinsK Luenser and A Ludwig117ResultsGenomic DNA of domestic cattle and its extinct ancestor,

the aurochs, as well as DNA of gaur, yak, banteng and

the African buffalo was screened for b-defensin coding

sequences, using a PCR approach. Previous studies have

shown the sequence region corresponding to the active,

mature peptide to be highly variable and subjected to

positive selection, whereas the regions corresponding to

the signal and propeptide are more conserved.21,3437

Thus, concerning our interest in the evolutionary history

and the mode of selection, we regarded the second exon

to be more informative than the first exon, and

concentrated our study on the second exon coding for

the propeptide (propiece and mature peptide). The

alignment of nucleotide sequences of bovine defensins

is shown in Table 1.In total, we found 26 b-defensin coding sequences.

Five of these were identical to previously published ones:

tap (AF014106), lap (446811481), bnbd4 (AF008307), ebd

(AF000362) and bnbd9 (AF016394). In addition, we

detected 21 sequences similar to tap (AF014106), lap

(446811481), bnbd4 (AF008307), ebd (AF000362) bnbd9

(AF016394) bnbd5 (AJ278799), and nbd12 (AF105371).

Their distribution among the species investigated is

given in Table 2. Owing to the detection of sequences

identified as variants of previously published bovine

defensins, we numbered all newly detected variants.Three defensins were found to be present in different

species without any substitutions: (i) tap originally

described as tracheal antimicrobial peptide from the

tracheal mucosa of domestic cattle11 was also detected in

this study in banteng and gaur; (ii) ebd_1 was found in

domestic cattle, gaur and banteng; (iii) finally, the same

sequence, termed nbd12_2, was amplified from domestic

cattle and the African buffalo.Figure 1 shows the neighbour-joining (NJ) tree based

on amino-acid sequences of bovine b-defensins. To

facilitate understanding, we will introduce the term

subfamily as the name of the different types of bovine

defensins. All newly found sequences clustered within

known subfamilies (eg tap, lap, nbd12 and bnbd4, bnbd9,

bnbd5). In addition, variants of the bovine neutrophil

b-defensins bnbd9 and bnbd5 cluster together in a speciesspecific manner with bnbd5 to bnbd5_2 and bnbd9 to

bnbd9_5 detected exclusively in domestic cattle. The

defensin sequence amplified from aurochs (bnbd5_3)

clusters within the bnbd5 subfamily, which seems to be

restricted to domestic cattle among recent bovine species

investigated in this study. Sequences belonging to the

defensin subfamilies tap, lap, ebd, bnbd4, and nbd12 cluster

over several species. These results may allow speculation

that members of these defensin subfamilies are homologous to each other and share common ancestors. Inbnbd4 Bos taurusbnbd4_1 Bos javanicuslap 44681481 (Bos taurus) lap Bos taurus

lap_1 Bos frontalis

lap_2, lap_6 Bos grunniens

lap_3 Bos frontalis

lap_4, lap_5 Syncerus caffer

lap_7 Bos javanicusbnbd9 AF016394

(Bos taurus)bnbd9 Bos taurusbnbd9_15 Bos taurusTable 2 Distribution of bovine b-defensin genesPublished similar sequence This study Speciesbnbd4 AF008307

(Bos taurus)bnbd5 AJ278799

(Bos taurus)bnbd5_1, bnbd5_2 Bos taurus

bnbd5_3 Bos primigeniusebd AF000362 (Bos taurus) ebd Bos taurus

ebd_1 Bos taurus, Bos

frontalis, Bos

javanicustap AF014106 (Bos taurus) tap Bos javanicus, Bos

taurus, Bos frontalis

tap_1 Bos taurustap_2 Bos grunniensnbd12 AF105371

(Bos taurus)nbd12_1 Bos frontalis

nbd12_2 Bos taurus, Syncerus

cafferFigure 1 NJ-tree based on proportion difference at 45 amino-acid

sites. Abbrevations indicate the species as follows: BtBos taurus;

BjBos javanicus; BgBos grunniens; BfBos frontalis; BpBos

primigenius; ScSyncerus caffer. Previously published defensin

genes are underlined.Genes and ImmunityBovine b-defensinsK Luenser and A Ludwigcontrast, the fact that bnbd5 and bnbd9 coding genes could

only be amplified from domestic cattle and their ancestor

points toward independent evolution after divergence ofB. primigeniuslineage from the other Bovinae.

The bovine b-defensins are characterized by a high

degree of variability, as indicated by the number of

variable positions (Table 3). In order to analyse the mode

of evolution, we estimated the values of nonsynonymous

substitutions per nonsynonymous site (dN) and synonymous substitutions per synonymous site (dS) for all

within-species comparisons of defensin subfamilies

(Table 4, Figure 2). We found that the mean dN value

was higher than the mean dS value in comparisons of

lap4 vs lap5, nbd12 vs nbd12_2, bnbd5 and bnbd9 coding

genes indicating that positive selection has acted on these

gene subfamilies. In contrast, comparison of tap vs tap_1

and ebd vs ebd_1 indicates that these genes are negatively

selected.The alignment of deduced amino-acid sequences is

reported in Figure 3. The following positions were fixed

across all amino-acid sequences: (i) with one exception

(bnbd5_2) the six-cysteine array forming three disulphide

bonds stabilizing the tertiary structure is strongly118Table 4 Mean numbers of synonymous (dS) and nonsynonymous

(dN) nucleotide substitutions per 100 sites (7s.e.) in within gene

cluster comparisons of bovine b-defensinsdS per 100 sites dN per 100 sitestap AF014106 vs tap_1 0.8670.000 0.009470.000

lap_4 vs lap_5 0.00070.000 0.018770.000

ebd AF016539 vs ebd_1 0.25070.000 0.059770.000

nbd12 AF105371 vs nbd12_2 0.00070.000 0.009570.000

bnbd5 AJ277899 vs others bnbd5 0.00070.000 0.032570.012

bnbd9 AF016394 vs others bnbd9 0.00070.000 0.022770.012Sequence subfamilies followed classification of sequences in Figure1. No comparisons between different species are included.Figure 2 Plots of numbers of nonsynonymous (dN) substitutions

per 100 sites vs synonymous (dS) substitutions per 100 sites in

within-gene cluster comparisons of bovine b-defensins. The line is a

451line (x y); plots above the line indicate dN exceeding dS.Table 3 Variable positions of nucleotide sequencesVertical numbers indicate nucleotide positions (see Table 1).Genes and ImmunityBovine b-defensinsK Luenser and A Ludwig119Figure 3 Multiple sequence alignments of bovine, human and murine b-defensins. Numbering of the sites starts at the beginning of the

mature peptide. Amino acids found to be strictly conserved among bovine b-defensins are marked by black boxes. Corresponding sites in

human and murin defensins, characterized by little variations, are marked by grey boxes. a-helical and b-sheet positions known from

structure determination of murine and human defensins are schematically indicated below the alignment.conserved; (ii) the N-terminus of the propiece always

shows the sequence Gly-Phe-Thr-Gln; (iii) the fourth

cysteine Cys27 is flanked by a conserved stretch of Gln23-Ile24-Gly25-Thr26; (iv) the third cysteine Cys17 is flanked

by Pro18; (v) the sixth cysteine Cys35 is followed by a

conserved Arg36; (vi) at position eight a conserved Asn8

is found.The highest variability was observed at the region

localized between the fourth and fifth cysteines (Cys27

and Cys34).DiscussionIn this study, we cloned the second exon of 26 genes

belonging to seven b-defensin gene subfamilies (bnbd4,

bnbd5, bnbd9, tap, lap, ebd, nbd12). The presented NJ-genetree suggests that the variants of the different defensin

subfamilies evolved by several gene duplication events

of ancestral forms of these defensin subfamilies. Values

of the synonymous and nonsynonymous substitutions

produced evidence of both positive and negative selection. Negative selection of accumulated nonsynonymous

substitutions was found for variants of tap and ebd. These

results conform to the predictions generated by the

neutral theory of molecular evolution.38 Contrarily,

bovine neutrophil b-defensins belonging to bnbd5 and

bnbd9 subfamilies, as well as lap_4 vs lap_5 and nbd12 vs

nbd12_2 were shown to evolve under positive selection.

The idea of gene duplication and the following positive

selection of accumulated amino-acid altering mutations

on the gene copy was already proposed by Ohno39 in

1970. Almost 20 years ago, Hill and Hastie40 demonstrated dN exceeding dS in certain regions of serine

protease inhibitors. Nowadays, the process of gene

duplication and positive selection is a widely accepted

model and is used to explain the evolution of new genes

with beneficial new functions.The evolution of defensins under positive or Darwinian selection was already described in detail for murine,Genes and ImmunityBovine b-defensinsK Luenser and A Ludwigprimate and mammalian defensins.21,3437 In addition, the

loci of the major histocompatibility complex (MHC)41,42

and immunoglobulin genes43,44 were described as examples for Darwinian selection to create diversity among

gene family members. Thus, genes involved directly or

indirectly in the immune defence system seem to be

favoured for an evolutionary mode of positive selection.

The most simple explanation for the diversifying positive

selection of the bovine defensins may be an evolutionary

pressure to acquire a broad range of antimicrobial

defence tools. This hypothesis is supported by findings

of Morrison et al,45,46 who reported functional differences

within members of the murine b-defensin gene family in

terms of their ability to kill, specifically, certain pathogens. In addition, diversifying positive selection may

reflect a coevolutionary process between invading

pathogens and the hosts defence system. In conclusion,

the provided evidence that several bovine b-defensins

seem to evolve under positive selection is in agreement

with the current opinion of evolutionary mode of

defensins. Surprisingly, we also found the atypical

substitution pattern of dN exceeding dS with dS 0

(Table 4, Figure 2), indicating that every substitution

affects the amino acid sequence. Although regarded as

statistically unlikely, these data are supported by

published data concerning the evolution of the b-defensin 2 gene in primates.18However, in a further approach, we were interested in

the phylogeny of the bovine b-defensins and calculated a

NJ-tree based on proportion difference at the amino acid

sites similar to the approach by Hughes,21 who investigated the phylogeny among mammalian a-defensins

presenting a NJ-tree with species-specific defensin-gene

clusters. In agreement with the species-specific clustering, Hughes concluded that a-defensins had duplicated

repeatedly after divergence of the included mammalian

species.21 The results presented in our study deviate

from this idea regarding the species-specificity of

defensin genes. The tree reported in Figure 1 demonstrates that the bovine b-defensins form gene subfamilyspecific clusters (tap, lap, nbd12, bnbd4, bnbd5, bnbd9). But

in contrast to the results of Hughes21, only the bnbd5 and

bnbd9 coding genes are distributed in a species-specific

manner. Bovine b-defensin subfamilies, despite bnbd5

and bnbd9 coding sequences, cluster in an over different

species spanned mode. In addition, three sequences (tap,

ebd_1, nbd12_2) were detected in different species without any nucleotide substitution (Figure 1). Given the

clustering of bovine defensins, it could be concluded that

these over-species clustering defensin subfamilies (tap,

lap, ebd, nbd12, bnbd4) harbour orthologous genes,

implicating that members of the same gene subfamily

descended from a common ancestor. The distribution of

variants of bnbd5 and bnbd9 coding genes indicates

independent evolution of both subfamilies after divergence of B. primigenius. In agreement to the separate

domestic cattle-specific clustering of all bnbd5 variations,

the detected sequence from the aurochs clustered

together with these genes. The clusters of the NJ-tree

and the high sequence similarity implicate several

duplication events of ancestral forms of tap, lap, ebd

and nbd12 before the bovine species diverged, suggesting

homology of these genes. Nevertheless, the observed

similarities could be explained alternatively by an

independent or convergent evolution resulting in genes

with similar features. Positively selected genes are

characterized by an accelerated evolution making development of structural similar features or genes likely. To

shed light on this issue, investigation of the noncoding

intron sequences expected to evolve under a neutral

mode of evolution should be addressed.To assess the question of whether variations in the

amino-acid sites could result in functional implications,

we compared them to structural data available for

human b-defensin 1 and 2 (hBD1, hBD2),47,48 murine

defensins mBD-748 and mBD-8,48 as well as for bovine

defensin NBD12.49 These defensins show the conservation of a triple-stranded b-sheet, the general fold, and

with the exception of NBD12,49 a short amino terminal

a-helix.47,48 Figure 3 shows an alignment of bovine b-defensins corresponding to nucleotide sequences reported in Table 1 and includes human and murine

defensin sequences whose secondary and tertiary structures are known. a-helical positions (no. 58) and b-sheet

elements (no. 1014, 2126, 3237) are indicated schematically below the alignment (Figure 3). Amino acids

corresponding to these indicated sites seem to be

relatively conserved among all bovine defensins.

Furthermore, we found conservation of the six-cysteine

pattern as well as conservation of the small amino acid

glycin on position no. 10. Because this site is buried in the

interior of the molecule,48 it may be argued that this site

was favoured as evolutionary for steric reasons. Summarizing these observations, we propose that the bovine

defensins found in this study exhibit the same threedimensional structure of a short a-helix on top of three

antiparallel b-strands as described for murine and

human defensins.47,48Most amino acid changes were observed at the C-

terminus between residues 28 and 33 located between

the fourth and fifth cysteines (Cys27 and Cys34). The

significance of these variations remains to be determined. Nevertheless, we propose these variations to

affect the pathogen-specificity.In conclusion, in addition to previously described

bovine b-defensins, we report numerous newly detected

variants. These defensins are characterized by a high

degree of nucleotide substitutions resulting in a high

number of amino-acid altered positions. Estimation of

the ratio of synonymous and nonsynonymous substitutions provide strong evidence for an evolutionary mode

under positive selection, most likely in response to fast

evolving microbes. Surprisingly, despite the high number of variable positions, the bovine defensins share

regions with moderate conservation, suggesting a common structure for all defensins with the typical b-defensin feature of a triple-stranded b-sheet and an

N-terminal a-helix. The observed variations are likely to

affect the biological activity. To obtain more information

concerning the functional implication of single positions

within the mature peptide, their recombinant expression

and purification or their synthesis followed by functional

analysis may be useful.Materials and methodsSamplesThe following species of the genera Bos and Syncerus

belonging to the subfamily of Bovinae have been used120Genes and ImmunityBovine b-defensinsK Luenser and A Ludwigfor molecular investigation: domestic cattle (B. taurus),

gaur (B. frontalis), banteng (B. javanicus), yak (B.

grunniens), African buffalo (Syncerus caffer). In addition,

fossil bone sample of aurochs (B. primigenius), the extinct

ancestor of domestic cattle, was obtained. Fossil bone

material of aurochs were discovered in Svodin, Slovakia

and inventoried with the number SVBA 0625/56 by

Uerpmann, H-P, Tu bingen, Germany. Dating revealed an

age of 5000 years BP.DNA extractionTotal DNA of modern specimens was purified from

blood according to the standard protocols (QIAGEN Inc.,

The Netherlands). Ancient DNA of the aurochs sample

was kindly provided by Burger J and Bollongino R from

the Molecular Archeology Group, Institute of Anthropology, Johannes Gutenberg-University, Mainz, Germany. Ancient DNA work was carried out in Mainz in

two distinct laboratories located in different buildings to

separate pre-PCR procedures from post-PCR analysis to

avoid contaminations with modern DNA. Extraction of

ancient DNA was carried out following the protocol as

previously described by Hagelberg and Clegg,50 designated the Centricont (Millipore, USA) approach. All

samples of recent species were analysed at the Leibniz-

Institute for Zoo and Wildlife Research (IZW), Berlin.Primers and amplificationFor molecular studies of modern DNA, the primers P1 50-gagggtccaccagagcct-30 and P2 50-caacctcaatgaccagtgg-30

were designed. Annealing of the forward primer (P1)

occurred 203 bp upstream the second exon, the reverse

primer (P2) annealed 132 bp downstream the second

exon. For molecular studies of ancient aurochs DNA, the

forward primer (P3) was 50-RRTCCTCWAAGCTGCCGT

HKGAA-30 the reverse primer (P4) was 50 TGCAGTTT

CTGACTCCGCA-TTGGT-30. Annealing of P3 occurred

at the region coding for the beginning of the mature

peptide, annealing of P4 occurred 30 bp downstream

the second exon. Amplification was performed using

standard conditions with annealing at 581C.Cloning and sequencingPCR-products with 473 bp (amplified from modern

DNA), and 144 bp (amplified from ancient DNA) in

length, were cloned with TA-TOPO cloning kit (Invitrogen, The Netherlands). Competent cells of Echerichia coli

(DH5a) (Invitrogen, The Netherlands) were transformed

with the ligation products. The cloned PCR-products were

amplified and sequenced using the vector primers T7 and

M13 reverse (Invitrogen, The Netherlands). Each PCR-

product was sequenced completely from both strands.

Sequencing was carried out in an automated ABI 3100

DNA Sequencer (Applied Biosystem; USA). Second exon

coding sequences were aligned to published beta defensin

genes stored in GenBank (AJ278799, AF105370, AF105371,

AF01539, AJ009877) using MEGA 2.0.51The phylogenetic tree was constructed by NJ algorithms implemented by PAUP* 4.0b1052 and MEGA 2.051

based on the proportion of different amino acid sites. Its

reliability was assessed with 1000 bootstrap replications.

The significance of the branch lengths in the NJ-tree was

also examined by a standard error test using the

confidence probability (CP) program of MEGA.51Assignment of the bovine b-defensins obtained in this

study to the different defensin subfamilies occurred via a

nucleotide blast search in the NCBI gene bank.Translation of the nucleotide sequences into the

deduced amino-acid sequence was performed using the

expasy translator (http://us.expasy.org/tools/dna.html).

Calculation of the number of synonymous (dS) substitutions per synonymous site and nonsynonymous (dN)

substitutions per nonsynonymous site were done by the

method of Nei and Gojobori53 using the program

Dnasp400.54121AcknowledgementsWe thank C Pitra, J Fickel and H-M Seyfert for helpful

comments and discussions. In addition, we thank for the

cooperative support of the Molecular Archaeology

Group, J Burger and R Bollongino from the Institute of

Anthropology, Mainz, Germany. We recognize the

following veterinarians and biologists: M Reimann, A

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