Evidence of Positively Selected Sites in Mammalian a-Defensins

E idence o Posi i ely Selec ed Si es in Mammalian a-De ensins Da id J. Lynn,*And ew T. Lloyd,*àMa io A. Fa es,§and Cliona O’Fa elly*k *Educa ion and Resea ch Cen e, S . Vincen ’s Uni e si y Hospi al, Dublin, I eland; Depa men o Medicine, Uni e si y College Dublin, Dublin, I eland; àDepa men o Gene ics, T ini y College Dublin, Dublin, I eland; §Biology Depa men , Na ional Uni e si y o I eland, Maynoo h, I eland; and kConway Ins i u e, Uni e si y College Dublin, Dublin, I eland a-De ensins a e a amily o mammalian an imic obial pep ides ha exhibi a iable ac i i y agains a panel o mic obes, including bac e ia, ungi, and en eloped i uses. We ha e employed a maximum-likelihood app oach o de ec e idence o posi i e selec ion (adap i e e olu ion) in he e olu ion o hese impo an molecules o he inna e immune esponse. We ha e iden i ied 14 amino acid si es ha a e p edic ed o be subjec o posi i e selec ion. Fu he mo e, we show ha all hese si es a e loca ed in he ma u e an imic obial pep ide and no in he p ep opep ide egion o he molecule, implying ha hey a e o unc ional impo ance. These esul s sugges ha mammalian a-de ensins ha e been unde selec i e p essu e o e ol e in esponse o po en ially in ec ious challenges by as -e ol ing mic obes. In oduc ion De ensins a e ca ionic an imic obial pep ides ha show a b oad spec um o an imic obial ac i i y agains g am-nega i e and g am-posi i e bac e ia, ungi, and en eloped i uses. They a e gene ally hough o ac by dis up ing he memb ane in eg i y o mic obes (Kagan e al. 1990; Sa chell e al. 2003). In mammals, a-de ensins and b-de ensins a e wo s uc u ally dis inc g oups, which di e in size and in hei spacing o a six-cys eine mo i . a- De ensins we e i s disco e ed as a amily o pep ides in abbi mac ophages (Sels ed, Szkla ek, and Leh e 1984) and we e subsequen ly iden i ied in he neu ophils o humans, macaques, a s, abbi s, guinea pigs, and hams e s and in human and mouse small in es ine Pane h cells (Raj and Den ino 2002). In humans, six a-de ensins ha e been iden i ied and s udied o da e. Human a-de ensins 1 o 4 a e localized in azu ophilic g anules o neu ophils, which has led o hem being e e ed o as human neu ophil pep ides (HNP1 o HPN4). The HNPs a e he mos abundan p o ein in neu ophils and con ibu e o he oxygen-dependen killing o phagocy osed mic oo ganisms (Ganz e al. 1985; Singh e al. 1988; Ba eman e al. 1991). Recen ly, HNP1 o HPN3 ha e been shown o be he majo componen s o a soluble ac o sec e ed om CD8 T-lymphocy es ha supp esses HIV-1 eplica ion (Zhang e al. 2002). Human a-de ensins (HAD) 5 and 6 a e p ima ily loca ed in he sec e o y g anules o Pane h cells wi hin he c yp s o Liebe ku¨hn in he small in es ine (Jones and Be ins 1992, 1993; Po e e al. 1997; Salzman e al. 2003b) whe e hey ha e been implica ed in mucosal hos de ense. Fu he e idence o he impo an ole hese pep ides play in mucosal hos de ense has been shown in a ecen s udy in which ansgenic mice exp essing HAD5 we e comple ely esis an o Salmonella yphimu ium (Salzman e al. 2003b). HAD5 has also been de ec ed in emale e- p oduc i e ac (S ina ich e al. 1997; Quayle e al. 1998) and in b onchial and nasal epi helia (F ye e al. 2000). Pep ides homologous o HAD5 and HAD6 ha e been ound in mouse Pane h cells whe e hey a e e med c yp dins (Ouelle e e al. 1992, 1994). a-De ensins a e encoded as p opep ides ha equi e p o eoly ic p ocessing o become ac i a ed. I has been sugges ed ha he p o egion is cy op o ec i e because addi ion o i o in i o assays inhibi ac i i y (Ouelle e e al. 1999; Wu e al. 2003). Pane h cell ypsin has been iden i ied as he p ocessing enzyme o HAD5, bu he p ocessing enzyme o HAD6 has ye o be iden i ied (Ghosh e al. 2002). In mice, ac i a ion o c yp dins is media ed by ma ix me allop o einase-7 (MMP-7) (Wilson e al. 1999; Ayabe e al. 2002). The signal pep ide and p o egion in a-de ensins exhibi mo e amino acid conse a ion han does he ma u e an imic obial pep ide egion. Indeed only he six cys eine esidues and a glycine esidue a posi ion 18 o he ma u e pep ide a e conse ed be ween all a-de ensins. These esidues a e impo an o he s uc u e o he molecule (Hill e al. 1991), whe eas he o he esidues a e ee o a y. Mos molecula a ia ion ei he wi hin species o be ween species is caused by he andom ixa ion o mu a ions ha a e neu al. Mu a ions ha a e dele e ious o an o ganism a e emo ed by pu i ying selec ion. Occasionally, mu a ions con e selec i e ad an age o he o ganisms ha ing hem, and, he e o e, hese mu a ions a e ixed in he popula ion by posi i e selec ion (adap i e e olu ion) a a highe a e han expec ed unde neu al e olu ion. One o he mos s ingen me hods o de ec ing adap i e e olu ion is o compa e he a e o nonsynonymous subs i u ions (d N ) wi h he a e o synonymous subs i u ion (d S ). The a io be ween hese a es (x¼d N /d S ) is hen a eliable measu e o he selec i e p essu e ac ing in a p o ein-coding gene. I amino acid changes a e neu al, hey will be ixed a he same a e as synonymous mu a ions (x¼1). I mu a ions (d N ) a e dele e ious, d N /d S ¼0, and he mu a ions will be emo ed by selec ion. I nonsynonymous mu a ions a e sligh ly dele e ious xis less han 1, and he coe icien o selec ion ac ing agains he mu a ion will depend on he popula ion size. Finally, i he amino acid changes a e selec i ely ad an ageous, hey will be ixed a a highe a e (x.1). A numbe o me hods ha e been de eloped o calcula e d N and d S among lineages (see o e iew, Yang [2002]). Maximum-likelihood (ML) me hods a e mos s a is ically sa is ac o y because hey employ an explici model o e olu ion, aking in o accoun he e ec s o unequal ansi ion and ans e sion a es, unequal base Key wo ds: Adap i e e olu ion, an imic obial pep ide, a-de ensin. E-mail add ess: [email p o ec ed]. Mol. Biol. E ol. 21(5):819–827. 2004 DOI:10.1093/molbe /msh084 Ad ance Access publica ion Feb ua y 12, 2004 Molecula Biology and E olu ion ol. 21 no. 5 ÓSocie y o Molecula Biology and E olu ion 2004; all igh s ese ed. and codon equencies, and a iable x alues among lineages in a phylogeny (Yang 1998; Yang and Nielsen 1998). As mos amino acid si es a e expec ed o be conse ed o main ain key s uc u al and unc ional cha ac e is ics o a p o ein, and only a small numbe o si es in a p o ein a e likely o be a ec ed by adap i e e olu ion, me hods ha a e age subs i u ion a es o e all si es in a sequence ha e li le powe in de ec ing cases o posi i e selec ion. Apa om models ha de ec posi i e selec ion among lineages, models ha can de ec amino acids si es unde adap i e e olu ion ha e also been de eloped (Nielsen and Yang 1998; Yang e al. 2000). Because a-de ensins a e a c i ical componen o he inna e immune esponse in an ‘‘a ms ace’’ agains as - e ol ing mic obes, i is possible ha hese molecules a e subjec o adap i e e olu ion. I has been p e iously demons a ed ha xis g ea e han 1 in he an imic obial pep ide egion o a-de ensins, indica i e o posi i e selec ion (Hughes and Yeage 1997). The Hughes and Yeage s udy was, howe e , unable o p edic which pa icula amino acid si es we e unde posi i e selec ion, as app op ia e models we e unde eloped a ha ime. In his i s comp ehensi e s udy o all a ailable mammalian a-de ensins, we implemen ML models o es o posi i e selec ion among e olu iona y lineages and among amino acid si es. Ma e ials and Me hods Coding sequences and co esponding p o ein se- quences o 34 a-de ensins om se en di e en species, Homo sapiens (human), Macaca mula a ( hesus monkey), Mus musculus (mouse), Ra us no egicus ( a ), O yc o- lagus cuniculus ( abbi ), Ca ia po cellus (domes ic guinea pig), and Ca ia cu le i (wild guinea pig) we e downloaded om GenBank ( able 1). These sequences ep esen all he ull-leng h a-de ensins cu en ly a ailable in he da abase. Molecules anno a ed as a-de ensin– ela ed we e no included in his s udy, as hey con ain a di e en cys eine mo i in he ma u e pep ide and do no align well in his egion wi h o he a-de ensins. Inclusion o hese molecules could bias he esul s by o e es ima ing d N . The p o ein sequences we e aligned using he T-Co ee p og am ( ig. 1 in Supplemen a y Ma e ial online) (No edame, Higgins, and He inga 2000). A Neighbo -Joining (NJ) ee was in e ed om he p o ein alignmen using MEGA e sion 2.1, wi h he gamma dis ibu ion model implemen ed o accoun o he e ogenei y among si es (Kuma e al. 2001). The shape pa ame e o he gamma dis ibu ion (a) was es ima ed using he BASEML p og am (implemen ing he REV model) om he PAML package e sion 3.12 (Yang 1997). One- housand boo s ap eplica es we e ca ied ou o es he signi icance o each node in he ee. B anches leading o nodes wi h a boo s ap alue o less han 500 we e collapsed. The opology o he ee was used as he inpu ee o CODEML and CODEMLSITES p og ams, also om he PAML package e sion 3.12 (Yang 1997). To cons uc an alignmen o he coding sequences, he p o ein alignmen was used as a empla e, and a copygaps Pe l sc ip was used o align he DNA, main aining he gaps ha we e p esen in he p o ein alignmen . Any columns in he DNA alignmen ha had mo e han h ee gaps we e emo ed. Va iable Selec i e P essu es Among Lineages Models o a iable x a ios among lineages we e i ed by ML o he alignmen o he 34 a-de ensin sequences. The one- a io model assumes an equal x a io o all b anches in he phylogeny. The ee- a ios model assumes an independen x a io o each b anch. The wo models can be compa ed by a likelihood a io es (LRT). Twice he log-likelihood di e ence be ween he wo models is compa ed wi h a 2 dis ibu ion wi h N-1 deg ees o eedom whe e N is he numbe o b anches. Pos e io Bayesian p obabili ies we e es ima ed o codon subs i u ions in each b anch o he phylogene ic ee. Va iable Selec i e P essu es Among Amino Acid Si es Models o a iable x a ios among si es we e used o es o he p esence o si es unde di e si ying selec ion (x.1) and o iden i y hem. Fi e models o he x dis ibu ion implemen ed in he CODEMLSITES p og am o he PAML package we e es ed. Model M1 (neu al) assumes wo classes o si es in he p o ein: he conse ed Table 1 Accession Numbe s o he 34 a-De ensin Genes Used in he Analyses Gene Species Accession Numbe HNP1 Homo sapiens NM_004084 HNP3 Homo sapiens NM_005217 HNP4 Homo sapiens NM_001925 HAD5 Homo sapiens NM_021010 HAD6 Homo sapiens NM_001926 MNP1 Macaca mula a AF184159 MNP1A Macaca mula a AF184160 MNP2 Macaca mula a AF184161 Mde 1 Macaca mula a AF188268 Mde 3 Macaca mula a AF188269 Mde 4 Macaca mula a AF188271 Mde 6 Macaca mula a AF188272 Mde 8 Macaca mula a AF188270 CRP1 Mus musculus NM_010031 CRP2 Mus musculus U02996 CRP3 Mus musculus NM_007850 CRP4 Mus musculus NM_010039 CRP5 Mus musculus NM_007851 CRP6 Mus musculus NM_007852 NP1 Ra us no egicus U16686 NP2 Ra us no egicus NM_173329 NP3 Ra us no egicus U50353 NP3B Ra us no egicus U50354 ED Ra us no egicus AF115768 NP4 Ra us no egicus NM_173299 NP3A O yc olagus cuniculus M64599 NP4 O yc olagus cuniculus M64601 NP5 O yc olagus cuniculus M64602 MCP1 O yc olagus cuniculus M28883 MCP2 O yc olagus cuniculus M28072 DEF1A Ca ia po cellus D14119 DEF1B Ca ia po cellus D14118 DEF2 Ca ia po cellus X63676 DEF1A Ca ia cu le i X57705 NOTE.—Whe e Re Seq (h p://www.ncbi.nlm.nih.go /Re Seq/) accession num- be s ha e been assigned, hese ha e been used, o he wise, GenBank accession numbe s a e gi en. 820 Lynn e al. si es (x¼0) and he neu al si es (x¼1). Model M2 (selec ion) adds a hi d class o si e, wi h xas a ee pa ame e , allowing o si es wi h xg ea e han 1. Unde he disc e e model M2, he p opo ion o si es unde pu i ying selec ion (p 0 ) and p opo ion o si es unde neu ali y (p 1 ) a e es ima ed om he da a. Model M3 (disc e e) uses a gene al disc e e dis ibu ion wi h h ee classes o si e, wi h he p opo ions (p 0 ,p 1 , and p 2 ) and he x a ios (x 0 ,x 1 , and x 2 ). Model M7 (be a) uses a be a dis ibu ion, which, depending on pa ame e s pand q, can ake di e en shapes in he in e al (0, 1). Model M8 (be a and x) adds an ex a class o si es o he be a (M7) model, wi h he p opo ion and he x a io es ima ed om he da a, hus allowing o si es wi h xg ea e han 1. F om hese models, h ee LRTs compa e M0 (one a io) wi h M3 (disc e e), M1 (neu al) wi h M2 (selec ion), and M7 (be a) wi h M8 (be a and x), espec i ely. Models M2, M3, and M8 a e es s o posi i e selec ion among si es. Pos e io Bayesian p obabili ies o si e classes we e calcula ed o each amino acid si e. I he x a ios o some si e classes a e g ea e han 1, si es wi h high pos e io p obabili ies o hose classes a e likely o be unde posi i e selec ion. Sliding-Window Analysis o De ec Selec i e Cons ain s by Maximum Pa simony Se e al s udies ha e shown ha maximum-likelihood me hods a e sensi i e o he iola ion o assump ions made in models o de ec adap i e e olu ion and ha alse posi i e esul s could be ob ained unde ce ain condi ions (Suzuki and Nei 2002). We, he e o e, applied a maximum- pa simony me hod o es o adap i e e olu ion in ou sequences. We applied he Kimu a-based model o Li (1993) using a sliding-window p ocedu e (Fa es e al. 2002). B ie ly, he me hod in e s a s a is ically op imum codon-window size and slides i along he alignmen . We hen es in each sliding s ep he signi icance o he nonsynonymous nucleo ide subs i u ions (d S ), synonymous subs i u ions (d N ), and he nonsynonymous- o-synonymous a e a io (x). The ma hema ical app oach used is based on he maximum-pa simony me hod o Suzuki and Gojobo i (1999). The main di e ence howe e esides in he ac ha he window size is selec ed unde a s a is ical p ocedu e (Fa es e al. 2002). Ano he ad an age o using his me hod is ha he numbe o synonymous subs i u ions is es ed o signi icance, and, hence, sa u a ed synonymous si es, i any, can be highligh ed and emo ed om he analysis. Resul s An NJ phylogene ic ee was econs uc ed om he amino acid alignmen using MEGA e sion 2.1 ( ig. 1). The gamma dis ibu ion model was implemen ed o accoun o he e ogenei y among si es (Kuma e al. 2001). The gamma shape pa ame e was es ima ed using he BASEML p og am o be a¼1.39. This ee opology was used in he subsequen analyses o de ec adap i e e olu ion. To ensu e ha he opology o he ee was no dependen on he si es unde posi i e selec ion o he in a ian si es, he phylogene ic analysis was epea ed wi hou hese si es. The opology o his ee was essen ially he same as he ee in igu e 1, wi h some o he esolu ion los (see Supplemen- a y Ma e ial online). The CODEMLSITES analysis was epea ed using his opology as inpu and esul ed in he de ec ion o he same si es as being subjec o posi i e selec ion (da a no shown). Mul igene amilies whose membe s ha e he same unc ion may e ol e in a conce ed ashion ha homog- enizes he sequences o he membe genes by in e locus ecombina ion o gene con e sion, such ha sequences wi hin a species a e mo e simila o each o he han hose be ween species (Liao 1999). The physical clus e ing ypical o a-de ensin amilies wi hin species may imply conce ed e olu ion. The e a e, howe e , wo excep ions o phylogene ic clus e ing in ou ee ( ig. 1), which may mean ha he bi h-and-dea h model o e olu ion is mo e app op ia e (Nei, Gu, and Si niko a 1997). Nei he he p ima es no he oden s show species-speci ic clades o a- de ensins. In ac , in he case o he mouse and a , he FIG. 1.—Neighbo -Joined ee o mammalian a-de ensins. Con- s uc ed using MEGA e sion 2.1 (gamma dis ibu ion model, 1,000 boo s ap eplica es). B anches wi h less han 50% boo s ap suppo ha e been collapsed. Mm ¼Mus musculus (mouse), Rn ¼Ra us no egicus ( a ), Hs ¼Homo sapiens (human), Mma ¼Macaca mula a ( hesus monkey), Oc ¼O yc olagus cuniculus ( abbi ), Cp ¼Ca ia po cellus (domes ic guinea pig), and Cc ¼Ca ia cu le i (guinea pig). Posi i ely Selec ed Si es in Mammalian a-De ensins 821 phylogene ic clus e ing appea s o be caused by a unc- ional ela ionship be ween he di e en a-de ensins. Mice do no ha e neu ophil de ensins (Eisenhaue and Leh e 1992) and mos o he a a-de ensins a e exp essed in neu ophils; howe e , he one a a-de ensin exp essed in he in es ine (ED_Rn) in a simila manne o he mouse c yp dins, clus e s wi h he mouse a-de ensins. Fu he - mo e, he le el o sequence di e gence, e en a he p o ein le el ( ig. 1 in supplemen a y ma e ial a ailable online), is inconsis en wi h he homogeniza ion o sequences expec ed unde he conce ed e olu ion model. In he bi h-and-dea h model o e olu ion, duplica e genes a e p oduced in a gene amily. Some o he genes unc ionally di e ge, o he s may be los om he genome, and s ill o he s become pseudogenes (Liao 1999). Consis en wi h his model, we ha e de ec ed mul iple a-de ensin pseudo- genes in he human and mouse genomes (unpublished da a). Examining Sa u a ion o Synonymous Si es Sa u a ion o synonymous si es is an impo an issue in he de ec ion o posi i e selec ion by he c i e ion ha d N /d S is g ea e han 1, as sa u a ion will lead o he unde es ima ion o d S and an in la ion o he d N /d S a io. To ensu e ha sa u a ion was no an issue in ou da a se , pai wise d N and d S alues we e calcula ed using he ML me hod implemen ed by CODEML (Goldman and Yang 1994). A co ela ion analysis be ween d N and d S was pe o med using SPSS e sion 11.0 ( ig. 2). I synony- mous si es a e sa u a ed, we migh expec a quad a ic model wi h an inc easing slope o be be e i o he da a han a linea model. I synonymous si es a e no sa u a ed, a linea model should i he da a be e han a quad a ic model. A quad a ic model wi h a dec easing slope implies ha when a e age alues o nonsynonymous nucleo ide subs i u ions a e examined, mos o hem show s ong pu i ying selec ion and no sa u a ion o synonymous si es exis s in ou da a ha could in la e d N /d S alues. Va iable Selec i e P essu es Among Lineages To es o a iable x a ios among lineages, he one- a io model (Goldman and Yang 1994), which assumes he same x a io o all lineages, was compa ed wi h he ee- a io model (Yang 1998), which assumes an independen x a io o each b anch, using he LRT. The log-likelihood alue o he one- a io model is ‘ 0 ¼23888.76, while he alue o he ee- a io model is ‘ 1 ¼23862.20. Compa ison o 2‘¼2(‘ 1 2‘ 0 )¼53.12 (d ¼60) e eals ha he ee- a io model is no signi ican ly be e han he one- a io model (P.0.5). The ee- a io model does, howe e , p edic a iable x alues among lineages, some o which a e g ea e han 1 ( ig. 3). Because he LRT did no e eal a signi ican di e ence, we canno conclude ha he e is e idence o posi i e selec ion among he a- de ensin lineages. The ela i ely low x alues in some o he mouse lineages may be a esul o he ac ha all he mouse a-de ensins a e exp essed only in he in es ine and no in he neu ophils (Eisenhaue and Leh e 1992), and as a g oup hey may no be subjec o he same selec i e p essu es as he o he species ha ha e bo h neu ophil and in es ine a-de ensins. Va iable Selec i e P essu es Among Amino Acid Si es To es o posi i e selec ion a indi idual amino acid si es, LRTs we e ca ied ou be ween model M0 (one a io) and M3 (disc e e), M1 (neu al) and M2 (selec ion), and M7 (be a) and M8 (be a and x) ( able 2). All 3 models (M2, M3 and M8) ha allow o selec ion ( able 3) a e signi ican ly a o ed o e he o he models (P,0.001) in all cases ( able 3). The si es p edic ed o be unde posi i e selec ion wi h pos e io p obabili ies g ea e han 0.95 a e in ag eemen be ween models M2, M3 and M8, excep o FIG. 2.—Co ela ion be ween d N and d S alues as es ima ed by maximum likelihood. 822 Lynn e al. si es 25 and 62. Nei he o hese si es is p edic ed by M8, and si e 25 is only p edic ed by M3. a-De ensins a e encoded as p ep opep ides ha a e p o eoly ically clea ed o elease he C- e minal an i- mic obial pep ide. All o he si es p edic ed o be unde posi i e selec ion unde model M8, he mos s ingen model, a e loca ed in he ma u e an imic obial pep ide and no in he p ep opep ide egion ( ig. 4a). Compa ison o he dis ibu ion o posi i ely selec ed si es o a Poisson dis ibu ion e ealed ha he clus e ing o hese si es in he an imic obial pep ide was s a is ically signi ican (P, 0.001). Adap i e e olu ion o he ma u e pep ide is likely o ha e d i en he conside able amino acid a ia ion in his egion compa ed wi h he p ep opep ide egion. The si es p edic ed o be subjec o posi i e selec ion ha e been displayed on he h ee-dimensional s uc u e o HNP3 ( ig. 4b). Al hough si es unde posi i e selec ion occu h oughou he molecule, i is no able ha almos all o he si es in he coil egion ( esidues 7 o 14) a e subjec o adap i e e olu ion. The only si e no p edic ed is Glu14, which is known o o m a sal b idge wi h A g6 and as such is ela i ely in a iable (Hill e al. 1991). I is likely ha his egion has an impo an pa o play in he unc ion and ac i i y o he an imic obial pep ide. Many o he si es de ec ed o be unde posi i e selec ion using ML-based models we e also de ec ed using he sliding-window–based me hod. Some o he s, howe e (amino acid si es 62, 63, 68, 69, 78, and 82), did no show signi ican di e ences be ween d S and d N . Among he posi i e-selec ed amino acid si es, he a e age x alue was 2.972, being signi ican ly highe han 1 and highe han he expec a ion unde neu ali y, e en a e co ec ing o mul iple es s (mul iple sliding-window es s) using Bon e oni co ec ion (Z ¼11.47; P,0.001). We ha e no de ec ed sa u a ion o synonymous si es and hence x alues a e no in la ed by his bias. We ha e o s ess, howe e , ha maximum-pa simony me hods a e e y conse ed and may be subjec o he p oblem o possible con e gences. Despi e his ac , ou esul s using maxi- mum pa simony a e qui e coinciden wi h hose using ML me hods and do no a ec he inal conclusions o his s udy. FIG. 3.—Phylogeny o mammalian a-de ensins. The abb e ia ions used a e he same as in igu e 1. B anch leng hs we e es ima ed by maximum likelihood unde he ee- a io model, which assumes an independen x alue o each b anch. B anches wi h no x alues shown had alues ¼‘.x alues g ea e han 1 a e shown in bold. The opology and numbe o b anches is he same as in igu e 1 (some b anches a e e y sho and a e no isible on his ee). Posi i ely Selec ed Si es in Mammalian a-De ensins 823 FIG. 4.—Si es p edic ed o be unde posi i e selec ion in mammalian a-de ensins. (a) Si es p edic ed o be unde posi i e selec ion a e highligh ed in he mul iple sequence alignmen and (b) he dime s uc u e o he ma u e HNP3 pep ide (PDB en y ¼1DFN). Si es shown in ed a e hose si es p edic ed o be unde posi i e selec ion (model 8). Pos e io p obabili ies o hese si es a e all g ea e han 0.95. Si es shown in blue a e he si es ha a e 100% conse ed ac oss all OTUs. The ma u e an imic obial pep ide o HNP3 is highligh ed. The ma u e pep ides o o he a-de ensins may di e in size sligh ly a ei he e minus. The numbe ing o esidues in he ma u e pep ide o HNP3 in (a) co esponds o he numbe ing assigned o he HNP3 s uc u e in he P o ein Da a Bank (PDB) da abase (h p://www. csb.o g/pdb/) (b). No e ha esidue 2 in (a) and (b) co esponds o esidue 63 in able 3, and so on. The s uc u e o HNP3 was displayed using RasMol e sion 2.7.2.1(h p://www.open asmol.o g/so wa e/ asmol/). 824 Lynn e al. Discussion In his s udy, we ha e de ec ed posi i e selec ion a se e al amino acid si es loca ed in he ac i e an imic obial pep ide egion o a-de ensins. I is likely ha as mammals e ol ed o occupy new niches, hey we e aced wi h a new ange o mic obial pa hogens. E olu ion o an imic obial pep ides wi h new sensi i i ies capable o a ge ing no el in ec ious agen s would con e a selec i e ad an age. Ou esul s a e consis en wi h adap i e e olu ion o he a- de ensins in esponse o such a challenge and he e olu ion o speci ici y o di e en mic obes. The e is expe imen al e idence ha a-de ensin pep ides a e di e se in hei po ency agains di e en pa hogens. The human neu ophil de ensins HNP1, HNP2, and HNP3 a e no ac i e agains he o al g am-nega i e bac e ium Ac inobacillus ac ino- myce emcomi ans (Miyasaki e al. 1990a), whe eas abbi NP-1 shows s ong ac i i y (Miyasaki e al. 1990b). T ophozoi es o Gia dia lamblia a e e y sensi i e o mouse c yp dins 2 and 3 bu no o c yp dins 1 and 6 (Aley e al. 1994). No ably, he gia dicidal ac i i y has been a ibu ed o he p esence o a posi i ely cha ged a ginine esidue a posi ion 15 in he ac i e pep ide (Ouelle e and Be ins 2001). In ou s udy, his si e is p edic ed o be unde adap i e e olu ion. Ra and abbi de ensins ha e also been shown o ha e a iable ac i i ies agains a panel o g am-posi i e and g am-nega i e bac e ia (Kohashi e al. 1992). Small changes in he p ima y s uc u e o hese molecules can ha e eno mous e ec on hei po ency. HNP1 and HNP3 di e only by one esidue a he N- e minus o he ma u e pep ide, and, ye , HNP1 exhibi s po en ac i i y agains Candida albicans, whe eas HNP3 has li le e ec . (Leh e e al. 1988; Raj, An ony aj, and Ka unaka an 2000). This si e was also p edic ed o be subjec o posi i e selec ion in ou s udy. Simila ly, he ac i i y o mouse c yp din 4, he mos po en o he mouse a-de ensins agains a b oad spec um o mic obes (Ouelle e e al. 1994), is dependen on he p esence o one o wo esidues a he N- e minus o he ac i e pep ide. Remo al o hese esidues can o ally elimina e he an imic obial ac i i y o c yp din 4 (Ouelle e e al. 2000). I is p obable ha he e olu ion o di e en mechanisms o in e ac ion wi h mic obial memb anes has been impo an in he e olu ion o speci ici y o pa icula a-de ensins o pa icula mic obes. Human HNP2 o ms s able mul ime ic po es in model memb anes (Wimley, Sels ed, and Whi e 1994), whe eas abbi NP-1 does no , bu pe meabilizes memb anes by c ea ing la ge, sho - li ed de ec s (H is o a, Sels ed, and Whi e 1996). These al e na i e mechanisms a e likely o ha e a ying e ec s, depending on he cons i u ion o he memb ane, which is a iable among mic obes. Many mic obes ha e e ol ed mechanisms ha a emp o e ade and sub e he ac ions o an imic obial molecules (Ganz 2001). I is likely ha his ongoing ‘‘a ms ace’’ wi h mic obes has been a signi ican o ce d i ing he adap i e e olu ion o he a-de ensins. Fo example, he human pa hogens, Pseudomonas ae uginosa, En e ococ- cus aecalis, and S ep ococcus pyogenes elease de ma an sulpha e, a compound ha binds o and neu alizes HNP1 (Schmid chen, F ick, and Bjo ck 2001). Fu he mo e, he pa hogen Salmonella en e ica can elici a dec ease in he exp ession o mouse c yp dins (Salzman e al. 2003a). In his s udy we ha e p o ided e idence ha se e al amino acid si es in he ac i e pep ide o mammalian a- de ensins a e unde posi i e Da winian e olu ion. This wo k will assis in he design o in i o analyses o unc ionally and s uc u ally ele an si es. By syn he i- cally changing he esidues a si es p edic ed o be unde posi i e selec ion, which a e he si es mos likely o be o unc ional impo ance, i is possible o al e he ac i i y o hese molecules agains pa icula pa hogens and gain insigh in o hei mechanisms o ac i i y. Posi i e selec ion in he ma u e an imic obial egion o o he an imic obial pep ide amilies has also been demons a ed. E idence o posi i e selec ion has been de ec ed in p ima e b-de ensins (Bonio o e al. 2003; Semple, Rol e, and Do in 2003), mu ine b-de ensins (Mo ison e al. 2003), he D osophila and opin an ibac- Table 2 E idence o Adap i e E olu ion Among Si es in Mammalian a-De ensins Model P Pa ame e s ‘d N /d S Posi i ely Selec ed Si es M0: one a io 1 x¼0.9342 23888.76 ¼x M1: neu al 1 p 0 ¼0.108, x 0 ¼023837.45 0.8918 p 1 ¼0.892, x 1 ¼1 M2: selec ion 3 p 0 ¼0.107, x 0 ¼023751.88 1.6211 62,63,68,69,70,72,73,74,77,78,82,84,86,87,89 p 1 ¼0.670, x 1 ¼1 p 2 ¼0.223, x 2 ¼4.263 M3: disc e e 5 p 0 ¼0.188, x 0 ¼0.075 23740.19 1.264 25,62,63,68,69,70,72,73,74,77,78,82,84,86,87,89 p 1 ¼0.585, x 1 ¼0.821 p 2 ¼0.227, x 2 ¼3.386 M7: be a 2 p¼0.479, q¼0.273 23797.38 0.6374 M8: be a and x4p 0 ¼0.791, p¼0.452, q¼0.230 –3738.80 1.258 63,68,69,70,72,73,74,77,78,82,84,86,87,89 p 1 ¼0.209, x¼3.513 Table 3 Likelihood Ra io Tes (LRT) o De ec Adap i e E olu ion Models 2‘ 2 Value d P- alue M1 e sus M2 2(–3837.45 23751.88) 171.14 2 ,0.001 M0 e sus M3 2(–3888.76 23740.19) 297.14 4 ,0.001 M7 e sus M8 2(–3797.38 23738.80) 117.16 2 ,0.001 Posi i ely Selec ed Si es in Mammalian a-De ensins 825 e ial pep ide (Da e-I o e al. 2002), and in a numbe o amphibian an imic obial pep ides (Duda, Vanhoye, and Nicolas 2002). 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