Phenylalanine hydroxylase from the sponge Geodia cydonium: implication for allorecognition and evolution of aromatic amino acid HYDROXYLASESfn1

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Pergamon

Developmental and Comparative Immunology\ Vol[ 11\ No[ 4:5\ pp[ 358Ð367\ 0887 Þ 0887 Elsevier Science Ltd[ All rights reserved Printed in Great Britain 9034Ð294X:87 ,08[99¦9[99

PII] S9034Ð294X"87#99923Ð1

PHENYLALANINE HYDROXYLASE FROM THE SPONGE Geodia cydonium: IMPLICATION FOR ALLORECOGNITION AND EVOLUTION OF AROMATIC AMINO ACID HYDROXYLASES* Matthias Wiens,† Claudia Koziol,† Renato Batel‡ and Werner E. G. Mu¨ller†§ †Institut fu¨r Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universita¨t, Duesbergweg 6, 55099 Mainz, Germany ‡Center for Marine Research, ‘‘Ruder Boskovic’’ Institute, 52210 Rovinj, Croatia "Received February 0887^ Accepted May 0887#

Abstract*The prophenoloxidase activating system is a defense system\ frequently reported both in protostomes and in deuterostomes[ The _nal product of the phenoloxidase activity is mel! anin which is ubiquitously present throughout the metazoan kingdom[ The melanin synthesis path! way starts with the amino acid ðaaŁ phenylalanine which is converted to tyrosine by the phenyl! alanine hydroxylase ðPAHŁ[ We show that after allo!transplantation in the marine sponge Geodia cydonium PAH is upregulated in the grafts[ Enzyme determination studies revealed that PAH activity increases by three!fold two d after transplantation and reaches its maximum after 2 d "by 2[6!fold#[ This _nding was supported by determining the steady!state level of the mRNA for PAH[ Furthermore the cDNA\ encoding this enzyme was isolated from G[ cydonium[ Its deduced aa sequence encodes a protein of 40 kDa[ Alignment studies indicate that the sponge PAH shares the consensus pattern as well as one characteristic pterin!binding site with the biop! terin!dependent aromatic amino acid hydroxyl! ases[ Phylogenetic analysis of sponge PAH shows

 The sequence reported here is deposited in the EMBL:GenBank data base "Accession no[ Y05242#[ & Address correspondence to W[ E[ G[ Muller\ Institut fur Physiologische Chemie\ Abteilung Angewandte Molekularbiologie\ Universitat\ Duesbergweg 5\ 44988 Mainz\ Germany[ Tel[] ¦5020!284809^ Fax[] ¦5020!284132^ E!mail] WMUELLERÝmail[UNI!Mainz[DE[

that all metazoan PAH fall in one group with the sponge PAH as the oldest member[ The related classes of enzymes\ the tyrosine hydroxylases and the tryptophan hydroxylases are statistically sig! ni_cantly separated from PAH^ the tyrosine hydroxylase diverged as the _rst class from the common ancestor\ a process which was calculated to have occurred ¼499 million years ago[ It is concluded that in the sponge model system G[ cydonium allogeneic rejection involves an upre! gulation of PAH\ an enzyme initiating the path! way to melanin synthesis[ Þ 0887 Elsevier Science Ltd[ All rights reserved Keywords*Geodia cydonium^ Sponges^ Phenylalanine hydroxylase^ Evolution^ Allo! recognition^ Aromatic amino acid hydroxylases[

Introduction It is established that sponges ðPoriferaŁ rep! resent the phylogenetically oldest and sim! plest extant multicellular animals "0#[ Recent molecular biological data obtained from genes encoding proteins have con! vincingly contributed to resolve the phylo! genetic status of Porifera within the Metazoa[ Comparative sequence analyses strongly support monophyly of all Met! azoa "reviewed in] "1Ð3##[ Sponges have always been considered to lack basic elements characteristic for higher Meta! zoa\ mainly the abilities to form tissues

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and to di}erentiate speci_c sensory cells* nerve cells*"4#[ This assumption could be revised\ by the demonstration that sponges contain\ among others\ the genes coding for constitutive proteins of the basal lamina\ integrin "5# and _bronectin "6#[ In addition\ data are now available that the marine sponge Geodia cydonium contains elements of sensory organs\ known from higher metazoan phyla\ e[g[ the eye lens protein bg!crystallin "7#\ G!protein"s# related to transducin ""8#^ and unpublished results#\ and the metabotropic glutamate receptor "09#[ Sponges are known to possess remark! able regeneration capabilities "00\ 01#[ In addition\ they have developed mechanisms to distinguish between self and non!self "see as examples] "02\ 03##[ Smith and Hil! demann "04# have grouped sponge allo! immune responses into two major rejection processes^ "i# some species may form bar! riers to separate self from non!self tissue^ e[g[ the marine sponge Axinella verrucosa "05# or the freshwater sponge Ephydatia muelleri "06#\ while "ii# others may react by cytotoxic factors which destroy the trans! plant^ e[g[ the marine sponges Callyspon`ia diffusa "07# or Geodia cydonium "08#[ One potential molecule involved in sponge allo! recognition and host defense is the receptor tyrosine kinase with its immunoglobulin! like domains ðIg!likeŁ\ as analyzed in the sponge G[ cydonium^ both the Ig!like coding domains "19# and\ more surpris! ingly\ the introns between the two Ig units display an amazingly high nucleotide ðntŁ polymorphism "10#[ A defense system\ apparently common to both protostomes and deuterostomes\ is the prophenoloxidase activating system "for reviews] "11\ 12##[ The _nal product of the phenoloxidase activity is melanin which is ubiquitously present throughout the metazoan kingdom[ The enzyme is acti! vated by serine protease"s# from its inactive form\ the prophenoloxidase"s# "13#[ Mel! anin and:or its reactive intermediates have been shown to be fungistatic and bac! teriostatic\ and to display also antiviral

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activity "reviewed in] "11##^ it has been sug! gested that melanization occurs also during allorecognition tissue responses "14#[ Mel! anin is a polymer formed via the phe! noloxidase"s# from tyrosine\ an amino acid ðaaŁ which is converted from phenylalanine by the phenylalanine hydroxylase ðPAHŁ "EC 0[03[05[0# "see] "15##[ The PAH is the rate!limiting enzyme in the pathway to cat! abolize phenylalanine "15#[ This enzyme belongs together with the tyrosine hydroxylase ðTHŁ "EC 0[03[05[1# and the tryptophan hydroxylase ðTRHŁ "EC 0[03[05[3# to the family of pterin!depen! dent aromatic L!amino acid hydroxylases "16#[ In sponges melanization has been observed in a number of species\ e[g[ Hal! ichondria\ Hymeniacidon\ Microciona and Veron`ia "17#[ Until now\ no speci_c cells producing melanin\ such as melanocytes in some invertebrates\ have been identi_ed in sponges "18#[ Some evidence has been pre! sented indicating that melanin is syn! thesized in those cells which are destined to die "29#[ In the present study we report on the cloning of the cDNA encoding the PAH from the sponge G[ cydonium and we show that this enzyme is induced in those tissues which undergo death in response to allorecognition[

Materials and Methods Materials Restriction endonucleases and other enzymes for recombinant DNA techniques and vectors were from Stratagene "Heidel! berg^ Germany#\ QIAGEN "Hilden^ Ger! many# and USB "Cleveland\ OH#[ TRIzol Reagent was from GibcoBRL "Grand Island\ NY#\ CDP ðdisodium 1!chloro!4! "3!methoxyspiro"0\1!dioxetane!2\1?!"4?! chloro#!tricyclo"2[2[0[0 2\6 #decan#!3!yl# phenyl phosphateŁ\ DIG ðdigoxigeninŁ\ RNA labeling kit and DIG!00!UTP were obtained from Boehringer "Mannheim^ Germany#[

Phenylalanine hydroxylase from the sponge Geodia cydonium

Spon`e Live specimens of Geodia cydonium "Porifera\ Demospongiae\ Geodiidae# were collected near Rovinj "Croatia#[ After termination of the rejection experi! ments the tissue samples were immediately frozen in liquid nitrogen[

Graftin` Procedure The technique for grafting applied\ the {{insertion technique||\ was described earl! ier "19\ 20#[ Tissue pieces were removed from one specimen with a cork drill "diam! eter of 0 cm^ approximate length of 3 cm# and inserted into holes in the recipients\ which had a slightly narrower diameter "9[8 cm#[ The animals were kept in 1\999 l tanks at 05>C under constant seawater cur! rent and moderate aeration[ After 0 to 09 days the grafts were removed and analyzed for PAH activity as well as for the amount of transcript coding for this enzyme[

Extracts Extracts were prepared from grafts by grinding frozen tissue samples in three times their volume of phosphate!bu}ered saline "pH 5[7#\ supplemented with 0 mM EDTA and 0 mM phenylmethylsulfonyl ~uoride[ After centrifugation the super! natant was collected and protein content was determined "21#[ RNA was extracted from liquid!nitro! gen pulverized sponge tissue with TRIzol Reagent "GibcoBRL# as recommended by the manufacturer[

Isolation of G[ cydonium GCPAH cDNA The complete sponge cDNA named GCPAH was cloned by polymerase chain reaction ðPCRŁ from a G[ cydonium cDNA library in lambda ZAP ExpressTM "22#[

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The degenerate reverse primer PAH*R\ 4? ! TARTACTGDGTGSAGTGRAA ! 2? "RA:G^ DA:G:T^ SG:C# in con! junction with the ZAPII 4?!end vector! speci_c primer T2 as well as the forward primer PAH*F\ 4?!TGTACTGGATT CAGACTGAGA!2? together with the ZAPII 2?*end vector*speci_c primer T6\ were used[ The PCR reaction with the pair PAH*R!T2 was carried out at an initial denaturation at 84>C for 2 min\ then 24 ampli_cation cycles of 84>C for 29 sec\ 47>C*34 sec\ 63>C*0[4 min\ and a _nal extension step at 63>C for 09 min[ The reac! tion mixture of 49 ml included 19 pmoles of the degenerate primer and 09 pmoles of the primer T2\ 199 mM of each nucleotide\ 0 ml of the cDNA library "approximately 096 pfu#\ bu}er and 1[4 units of Taq DNA polymerase "Boehringer Mannheim#[ A fragment of 729 bp was obtained[ The PCR reaction with the pair PAH*F "speci_c primer within the sponge 729 bp frag! ment#!T6 was performed accordingly with an annealing temperature of 59>C[ The clone was termed GCPAH and was sequenced using an automatic DNA sequ! enator ðLi!Cor 3199Ł[

Sequence Comparisons The sequence was analyzed using com! puter programs BLAST "23# and FASTA "24#[ Multiple alignment was performed with CLUSTAL W Ver[ 0[5 "25#\ and the graphic presentation was prepared with GeneDoc "26#[

Northern Blot RNA was extracted from liquid!nitro! gen pulverized sponge tissue with TRIzol Reagent "GibcoBRL# as recommended by the manufacturer[ An amount of 4 mg of total RNA was electrophoresed through a formaldehyde:agarose gel and blotted onto a Hybond N¦ membrane following the manufacturer|s instructions "Amersham#

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"27#[ Hybridization was performed with a ¼699 bp PCR!product of GCPAH as a probe[ As a control\ the cDNA encoding bg!crystallin from G[ cydonium\ GCCRBG ""7#^ accession number Y97660#\ was used[ The probes were labeled with DIG!00! dUTP by the DIG DNA labeling kit "Boehringer\ Mannheim#[ For quantitation of the signals of North! ern blots the chemiluminescence procedure was applied "28#^ CDP was used as substr! ate[ The screen was scanned with the GS! 414 Molecular Imager "Bio!Rad#[

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mouse!rat!human aa sequence was used[ In the second round the speci_c forward primer PAH*F located within the frag! ment obtained from the _rst reaction was used[ Four identical nt sequences com! prising 0\378 bp "excluding the poly"A# tail# have been analyzed^ they were termed GCPAH "accession number Y05242#[ The open reading frame of GCPAH begins with the ATG!start codon Met ðnt 17Ł and extends for 0\249 bp^ Fig[ 0"A#[ Northern blot analysis revealed a single band of approximately 0[4 kb "Fig[ 1#\ sug! gesting that the complete cDNA was cloned[

Enzyme Activity Determination PAH activity in G[ cydonium extracts was determined spectrophotometrically as described "39# and is based upon the increase in absorbance at 164 nm[ The reaction mixture contained in a _nal reac! tion volume of 0 ml 099 mM K!phosphate bu}er "pH 5[7#\ 9[1 mM lysolecithin\ 9[1 mM L!phenylalanine\ 099 mM 5! methyltetrahydropterin\ 09 mM dithio! threitol and 19 mg catalase[ The reaction was initiated by addition of enzyme extract and performed for 29 min at 26>C[ The D absorbance "at 164 nm# per mol of tyrosine formed is 0559 cm−0 "39#[ The activity is given in unit"s# de_ned as the conversion of one nmole of phenylalanine into tyrosine formed during the incubation period and is correlated to one mg of soluble protein[

Results Identi_cation of G[ cydonium PAH cDNA The cDNA encoding the putative sponge PAH has been cloned to com! pletion by PCR technique as described under {{Materials and Methods|| from the marine sponge G[ cydonium[ In the _rst step a degenerate reverse primer PAH* R\ directed against the conserved region within the Drosophila melano`aster!

Deduced aa Sequence of Spon`e PAH The deduced aa sequence of GCPAH\ termed PAHÐGEOCY\ has a putative size "Mr# of 40\193 and an isoelectric point "pI# of 3[87 30^ 0"a#[ The estimated half!life of the putative sponge PAH is 2[4 hr\ indi! cating an unstable protein ""30#^ program PHYSCHEM#[ Homology searches "BLAST\ BLITZ and FASTA# with PAHÐGEOCY revealed highest similarity with metazoan PAHs "BLAST score] 678Ð628# from rat "30) aa identity and 50) similarity#\ Branchio! stoma ~oridae "45)\ 60)# and Drosophila melano`aster "41)\ 69)#[ Only slightly lower were the similarities to the trypto! phan hydroxylases ðTRH^ score 569Ð540Ł from human "35)\ 56)# and Caenorhab! ditis ele`ans "21)\ 49)# and to tyrosine hydroxylases ðTH^ score 516Ð498Ł with the examples of rat "30)\ 50)#\ eel "39)\ 46)#\ C[ ele`ans "21)\ 43)# or the trema! tode Schistosoma mansoni "23)\ 43)#[ Alignment studies revealed that the sponge PAH shares the consensus pattern for biopterin!dependent aromatic amino acid hydroxylases\ P!D!x1!H!ðDEŁ!L! LIVMF!G!H!ðLIVMŁ "the aa present in the sponge sequence are in bold^ the stretch spans from aa179 to aa189# "16\ 31#[ Based on the analyses of Hufton et al[ "15# the PAH sequence can be divided into

Phenylalanine hydroxylase from the sponge Geodia cydonium

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Figure 1. Sponge phenylalanine hydroxylase. A. Nucleotide (GCPAH) and deduced aa sequences (PAH–GEOCY) of G. cydonium phenylalanine hydroxylase-like protein. The three parts of the PAH sequence, the regulatory domain, the catalytic domain and the intersubunit binding region (subunit binding); the potential two binding sites A and B of the enzyme to the coenzyme tetrahydrobiopterin (Pterin-A and Pterin-B); and the two repeated motifs (RM-1 and RM-2) are marked. B. Rooted phylogenetic tree using sponge phenylalanine hydroxylase [PAH] (PAH–GEOCY) with related metazoan sequences from this class; the following species have been used. Rat (PAH–RAT; accession no. P04176), Branchiostoma floridae PAH (PAH–BRANF; AJ001677) and Drosophila melanogaster (PAH–DROME; g2117656). In addition, one sequence from the class of tryptophan hydroxylases [TRH] the one from rat (TRH–RAT; P09810) and one from the tyrosine hydroxylases [TH], rat (TH– RAT; P04177), has been included. A distantly related sequence, the bacterial PAH from Chromobacterium violaceum (PAH–CHROV; g78222), was used as an outgroup. The numbers at the nodes refer to the level of confidence as determined by bootstrap analysis (1000 bootstrap replicates). Scale bar indicates an evolutionary distance of 0.1 aa substitutions per position in the sequence.

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Figure 2. Northern blot analysis to determine the size and the amount of the transcripts of the mRNA encoding the sponge PAH. RNA from sponge tissue samples from allogeneic transplants was extracted, size-separated and after blot transfer hybridized with the GCPAH probe (upper blot) or with GCCRBG (lower blot). 5 mg of total RNA, each, were analyzed. Samples were taken immediately after transplantation (0 days; lane a), or 1 (lane b), 2 (lane c) or 3 days (lane d) later.

a regulatory domain "in the G[ cydonium sequence] aa0 to aa026#\ the catalytic domain "aa027 to aa397# and the intersubunit binding region "aa398 to aa349#^ Fig[ 0"A#[ Experi! mental evidence suggested that the co! enzyme tetrahydrobiopterin binds at two sites to the enzyme "15#[ The _rst pterin! binding site A has been proposed to be located adjacent "direction to the N!ter! minus# to the region H!x2!E!x1!H which is involved in the potential binding of non! haem iron "in G[ cydonium at aa199 to aa195#[ The second site!pterin!binding site B!is present in PAHÐGEOCY "aa153 to aa178#[ In addition\ the two repeat motifs A!G!L! L!S!S!x2!L "RM0 aa134 to aa143 and RM1 aa232 to aa241#\ found in PAH sequences of other invertebrates "32#\ are present in the sponge sequence[

Sequence Comparison and Phylo`enetic Analysis of Spon`e PAH Earlier studies revealed that the sub! classes of PAHs\ THs and TRHs form the family of aromatic L!amino acid hydroxyl!

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ases and are phylogenetically closely related[ Therefore\ sequences from those classes of enzymes from Metazoa as well as from Prokaryotae were chosen for com! parisons[ Sequences from Protozoa have not yet been described[ Sequences from the class of "i# PAHs\ from rat\ B[ ~oridae and from D[ melano`aster\ one member of "ii# TRHs from rat\ and one of "iii# THs from rat\ were included in the alignment[ The tree ðFig[ 0"B#Ł was constructed using the distantly related bacterial PAH from Chro! mobacterium violaceum as an outgroup[ The rooted tree shows ðFig[ 0"B#Ł that all three classes of enzymes fall into distinctly separated branches^ the THs diverged _rst from the common ancestor of the aromatic L!amino acid hydroxylases while the TRH and the PAH appeared later in evolution[ Based on bootstrap analysis this branching pattern is robust[

Evolutionary Rate It is well known that the evolutionary rates\ expressed as kaa!values\ vary between di}erent proteins "summarized in] "33##[ As shown by Kimura "34#\ kaa!values range from 7[2×09−8 ð_brinopeptidesŁ to 9[90×09−8 ðhistone H3Ł\ with an average kaa!value of 0×09−8\ meaning 0 aa sub! stitution per site in 098 years[ Previously it was calculated that the galectin protein from the sponge G[ cydon! ium "22# has a kaa!value of 0[6×09−8 "35# while the receptor tyrosine kinase "36# has a kaa!value of 0[13×09−8[ The heat shock protein Hsp69 from the same sponge has a comparably low evolutionary rate with a kaa!value of 9[014×09−8 "the calculations have been performed according to Kimura "34##[ The sponge sequence for PAH has a kaa!value of 0[9×09−8 demonstrating an average evolutionary rate[ Taking a stan! dard value for the average rate of 9[86×09−8 substitutions per site and year "35# the divergence of sponge PAH from the common ancestral gene can be

Phenylalanine hydroxylase from the sponge Geodia cydonium

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Figure 3. Transplantation of allogeneic tissue from the sponge G. cydonium applying the ‘‘insertion technique’’. The grafts (arrows) were analyzed by sectioning immediately after grafting (A), or 2 d (B) and 5 d later (C). Magnification ×2.

calculated as ¼499 million years ago ðMYAŁ[

Induction of PAH in Allo`rafts from G[ cydonium Tissue level[ After transplantation of allogeneic tissue from G[ cydonium by the {{insertion technique||\ the graft undergoes necrotic degeneration "Fig[ 2#[ Already after 1 d the colour of the graft turns from yellow ðFig[ 2"A#Ł to brownish ðFig[ 2"B#Ł[ After a transplantation period of 4 d the graft tissue is black ðFig[ 2"C#Ł[ Enzyme level[ Tissue samples from the graft were analyzed for PAH activity[ The initial PAH activity\ determined immedi! ately after transplantation is 1[5 units:mg of protein "Fig[ 3#[ The activity of PAH increases after already 1 d to 7[1 units:mg and reaches a maximum with 8[6 units:mg after 2 d[ Then the enzyme activity decreases which is attributed to the nec! rotic process occurring in the grafts[ Transcript level[ The upregulation of the PAH could also be demonstrated on the level of PAH transcripts[ mRNA isolated

from the grafts contained after 1 d a 6!fold higher level if compared with the controls[ After 2 d the level of mRNA for PAH decreases and was determined to be 4[1! fold higher than at time zero "Fig[ 1#[ No di}erence in the degree of expression was seen in non!grafted tissue and the expres! sion in the grafts at time zero "data not shown#[ As a control\ the cDNA encoding bg!crystallin from G[ cydonium\ GCCRBG "7#\ was used to determine the expression in the grafts[ In contrast to the observed increase in the expression of sponge PAH

Figure 4. Increase of PAH activity in allografts from G. cydonium. Tissue was removed immediately after grafting (time zero) or 1 to 10 d after transplantation. The activity of PAH is given in units/mg of protein. The S.D. is less than 10%; n = 10.

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transcripts\ no induction was seen for bg! crystallin "Fig[ 1#[

Discussion Only after application of molecular bio! logical techniques and identi_cation of genes coding for structural! and regulatory proteins\ and not relying only on sequence data from rDNA "37#\ it became evident that sponges are provided with the basic elements which are characteristic for Met! azoa "reviewed in] "1Ð3##[ In the past few years also elements of the immune system of vertebrates have been discovered in sponges^ the scavenger receptor cysteine! rich molecules\ widely found on the cell surface of mammalian macrophages or lymphocytes "38#\ short consensus repeat modules\ characteristic for complement receptor in mammals "2#\ or a Rhesus!fac! tor!like protein were isolated from the mar! ine sponge G[ cydonium "49#[ While the functions of these molecules are currently under investigation\ _rst studies are avail! able on the role of the Ig!like domains which are linked to a receptor tyrosine kin! ase "10\ 36\ 40#[ Allo! and autograft experi! ments have been performed which indicated distinct polymorphic alleles[ In the course of these experiments it was found that the rejected grafts underwent necrotic degeneration accompanied by melanization "08\ 19#[ The process of melanization has been well studied in some invertebrate phyla and is considered as an immediate system involved in recognition and defense^ the major biological systems which have been studied in detail are crustaceans "reviewed in] "11\ 12## and insects "41\ 42#[ The formation of melanin involves a series of enzymatic steps\ among which phenol! oxidase"s# play"s# a major role[ The mel! anin pathway starts with the essential amino acid phenylalanine which has to be converted to tyrosine via the PAH[ In the present study we show that sponges possess this enzyme and express it with increasing

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activity in grafts during the rejection process[ The activity of PAH in allografts rises by 3[4!fold "with respect to the con! trol# at day three after transplantation^ also the expression of the PAH gene increases considerably after transplantation[ The potential role of the G[ cydonium PAH as a regulatory protein is also supported by the calculation that the estimated half!life is only 2[4 hr[ From this it has to be deduced that this type of immune reaction is not a passive one but involves induction of several genes[ Recently it was found that during the apoptotic process in sponges\ which is induced by de_ned extracellular stimuli\ the expression of genes charac! teristic for apoptosis in vertebrates\ e[g[ the MA!2 gene "43#\ is upregulated[ This result may also support the assumption that allo! geneic incompatibility reaction in sponges is under genetic control[ The PAHs form together with the THs and the TRHs the family of pterin!depen! dent aromatic L!amino acid hydroxylases "16#[ It was suggested earlier that these enzymes originate from a common met! azoan ancestor from which the THs bran! ched o} _rst\ approximately 649 MYR ago\ while the TRHs and the PAHs evolved later ¼599 MYA ago "16#\ very likely by gene duplication "15#[ Having cloned and sequenced the PAH from the lowest Met! azoa this suggestion could be revised^ the calculations were examined statistically by bootstrap analysis[ The results show that the THs are the phylogenetically oldest members of this family from which the two other groups derived later[ This process\ according to the calculation given here\ occurred ¼499 MYA ago[ Taken together\ the _ndings presented here show that in the sponge model system G[ cydonium allogeneic rejection involves an upregulation of the PAH\ the _rst enzyme in the pathway starting from the essential amino acid phenylalanine and leading to melanin formation[ Phylo! genetic analysis of the deduced aa sequence of the PAH reveals that this class of enzymes is evolutionarily younger than the

Phenylalanine hydroxylase from the sponge Geodia cydonium

two other aromatic L!amino acid hydroxyl! ases\ TH and TRH[ Future experiments will be performed to elucidate the second pathway originating from phenylalanine: tyrosine which leads to the synthesis of cat! echolamines[ From studies in vertebrates it is known that catecholamines control the activity of all three classes of aromatic L! amino acid hydroxylases "15# and hence

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might be involved in the tuned rejection process as well[

Acknowled`ements*Supported by the Deut! sche Forschungsgemeinschaft ðMu 237:02!0Ł\ the Minerva!Foundation and the International Human Frontier Science Program ðRG!222: 85!MŁ[

References 0[ Mehl\ D[\ Muller\ I[\ Muller\ W[ E[ G[\ Molecular biological and palaeontological evidence that Eumetazoa\ including Porifera "sponges#\ are of monophyletic origin[ In Spon`e Sciences\ eds[ Y[ Watanabe\ N[ Fusetani[ Tokyo] Springer!Verlag\ 0887\ pp[022!045[ 1[ Muller\ W[ E[ G[ Molecular phylogeny of met! azoa ðanimalsŁ] monophyletic origin[ Natur! wissenschaften\ 0884\ 71\ 210Ð218[ 2[ Muller\ W[ E[ G[ Origin of metazoan adhesion molecules and adhesion receptors as deduced from their cDNA analyses from the marine sponge Geodia cydonium[ Cell Tissue Res[\ 0886\ 178\ 272Ð284[ 3[ Muller\ W[ E[ G[ Molecular phylogeny of Eume! tazoa] experimental evidence for monophyly of animals based on genes in sponges ðPoriferaŁ[ Pro`r[ Molec[ Subcell[ Biol[\ 0887\ 08\ 78Ð021[ 4[ Nielsen\ C[\ Animal evolution[ Oxford] Univ[ Oxford Press\ 0884[ 5[ Pancer\ Z[\ Kruse\ M[\ Muller\ I[ and Muller\ W[ E[ G[ On the origin of adhesion receptors of metazoa] cloning of the integrin a subunit cDNA from the sponge Geodia cydonium[ Molec[ Biol[ Evol[\ 0886\ 03\ 280Ð287[ 6[ Pahler\ S[\ Blumbach\ B[\ Muller\ I[ and Muller\ W[ E[ G[\ A putative multiadhesive basal lamina protein from the marine sponge Geodia cydonium] Cloning of the cDNA encoding a _bronectin!\ an SRCR! as well as a complement control protein module[ J[ Exp[ Zool[\ in press[ 0887[ 7[ Krasko\ A[\ Muller\ I[ M[ and Muller\ W[ E[ G[ Evolutionary relationships of the metazoan bg! crytallins\ including the one from the marine sponge Geodia cydonium[ Proc[ R[ Soc[ Lond[\ 0886\ B153\ 0966Ð0873[ 8[ Seack\ J[\ Kruse\ M[ and Muller\ W[ E[ G[ Evol! utionary analysis of G!proteins in early met! azoans] Cloning of a! and b!subunits from the sponge Geodia cydonium[ Biochim[ Biophys[ Acta\ 0886\ 0390\ 82Ð092[ 09[ Muller\ W[ E[ G[\ Perovic\ S[\ Krasko\ A[ and Meesters\ E[ Nachweis neuronaler Elemente in Zellen des Meeresschwammes Geodia cydonium[ BIOspektrum\ 0886\ 4\ 70Ð72[ 00[ Wilson\ H[ V[ On some phenomena of coales! cence and regeneration in sponges[ J[ Exptl[ Zool[\ 0896\ 4\ 134Ð147[ 01[ Humphreys\ T[ Chemical dissociation and in vitro

02[

03[

04[

05[ 06[

07[

08[

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reconstruction of sponge cell adhesion[ Devel[ Biol[\ 0852\ 7\ 16Ð36[ Evans\ C[ W[\ Kerr\ J[\ Curtis\ A[ S[ G[\ Graft rejection and immune memory in marine sponges[ In Phylo`eny of Immunolo`ical Memory\ ed[ M[ J[ Mannig[ Amsterdam] Elsevier\ 0879\ pp[ 16Ð23[ Hildemann\ W[ H[ and Linthicum\ D[ S[ Trans! plantation immunity in the Palauan sponge Xestospon`ia exi`ula[ Transplantation\ 0870\ 21\ 66Ð79[ Smith\ L[ C[ and Hildemann\ W[ H[ Allogeneic cell interactions during graft rejection in Cally! spon`ia diffusa "Porifera^ Demospongia#^ a study with monoclonal antibodies[ Proc[ R[ Soc[ Lond[\ 0875\ B115\ 266Ð354[ Buscema\ M[ and Van de Vyver\ G[ Variability of allograft rejection processes in Axinella verrucosa[ Devel[ Comp[ Immunol[\ 0872\ 6\ 502Ð505[ Mukai\ H[ Allogeneic recognition and sex di}er! entiation in chimeras of the freshwater sponge Ephydatia muelleri[ J[ Exp[ Zool[\ 0881\ 153\ 187Ð 200[ Hildemann\ W[ H[\ Johnston\ I[ S[ and Jokiel\ P[ L[ Immunocompetence in the lowest metazoan phylum] Transplantation immunity in sponges[ Science\ 0868\ 193\ 319Ð311[ Pfeifer\ K[\ Schroder\ H[ C[\ Rinkevich\ B[\ Uhlenbruck\ G[\ Hanisch\ F[!G[\ Kurelec\ B[\ Scholz\ P[ and Muller\ W[ E[ G[ Immunological and biological identi_cation of tumor necrosis factor in sponges] Role of this factor in the for! mation of necrosis in xenografts[ Cytokine\ 0881\ 3\ 050Ð058[ Pancer\ Z[\ Kruse\ M[\ Schacke\ H[\ Sche}er\ U[\ Ste}en\ R[\ Kovacs\ P[ and Muller\ W[ E[ G[ Polymorphism in the immunoglobulin!like domains of the receptor tyrosine kinase from the sponge Geodia cydonium[ Cell Adhesion Commun[\ 0885\ 3\ 216Ð228[ Pancer\ Z[\ Skorokhod\ A[\ Blumbach\ B[ and Muller\ W[ E[ G[ Multiple Ig!like featuring genes divergent within and among individuals of the marine sponge Geodia cydonium[ Gene\ 0887\ 196\ 116Ð122[ Johansson\ M[ W[ and Soderhall\ K[ The pro! phenoloxidase activating system and associated proteins in invertebrates[ Pro`r[ Molec[ Subcell[ Biol[\ 0885\ 04\ 35Ð55[ Smith\ V[ J[ The prophenoloxidase activating sys!

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tem] a common defense pathway for deut! erostomes and protostomes[ Adv[ Comp[ Environ[ Physiol[\ 0873\ 12\ 64Ð003[ Aspan\ A[ and Soderhall\ K[ Puri_cation of pro! phenoloxidase from cray_sh blood cells and its activation by an endogenous serine proteinase[ Insect Biochem[\ 0880\ 10\ 252Ð262[ Sabbadin\ A[ Formal genetics of ascidians[ Am[ Zool[\ 0871\ 11\ 654Ð662[ Hufton\ S[ E[\ Jennings\ G[ I[ and Cotton\ R[ G[ H[ Structure and function of the aromatic amino acid hydroxylases[ Biochem[ J[\ 0884\ 200\ 242Ð 255[ Grenett\ H[ E[\ Ledley\ F[ D[\ Reed\ L[ L[ and Woo\ S[ L[ C[ Full!length cDNA for rabbit tryp! tophan hydroxylase] functional domains and evolution of aromatic amino acid hydroxylases[ Proc[ Natl[ Acad[ Sci[ U[S[A[\ 0876\ 7\ 4429Ð4423[ Kennedy\ G[ Y[\ Pigments of marine invert! ebrates[ In Advances in Marine Biolo`y\ Vol[ 05\ eds[ F[ S[ Russell\ M[ Young[ Academic Press\ New York\ 0886\ pp[203Ð270[ Simpson\ T[ L[\ The cell biology of sponges[ Spr! inger!Verlag\ New York\ 0885[ Pavans de Ceccatty\ M[ La melanisation chez quelques eponges calcaires et siliceuses] Ses rap! ports avec le systeme reticulo!histocytaires[ Arch[ Zool[ Exp[ Gen[\ 0847\ 85\ 0Ð40[ Muller\ W[ E[ G[\ Bernd\ A[\ Zahn\ R[ K[\ Kure! lec\ B[\ Dawes\ K[\ Muller\ I[ and Uhlenbruck\ G[ Xenograft rejection in marine sponges[ Europ[ J[ Biochem[\ 0870\ 005\ 462Ð468[ Lane\ E[ Spectrophotometric and turbidimetric methods for measuring proteins[ Methods Enzymol[\ 0846\ 2\ 336Ð343[ Pfeifer\ K[\ Haasemann\ M[\ Ugarkovic\ D[\ Bret! ting\ H[\ Fahrenholz\ F[ and Muller\ W[ E[ G[ S! type lectins occur also in invertebrates] unusual subunit composition and high conservation of the carbohydrate recognition domain in the lectin genes from the marine sponge Geodia cydonium[ Glycobiol[\ 0882\ 2\ 068Ð073[ BLAST ncbi[nlm[nih[gov] 79:cgi!bin:BLAST: nph!blast^ INTERNET^ 0886[ FASTAÝebi[ac[uk^ INTERNET^ 0886[ Thompson\ J[ D[\ Higgins\ D[ G[ and Gibson\ T[ J[ CLUSTAL W] improving the sensitivity of progressive multiple sequence alignment through sequence weighting\ positions!speci_c gap pen! alties and weight matrix choice[ Nucleic Acids Res[\ 0883\ 11\ 3562Ð3579[ Nicholas\ K[ B[\ Nicholas\ H[ B[ Jr[\ GeneDoc] a tool for editing and annotating multiple sequence alignments[ Version 0[0[993[ Distributed by the author^ cris[com:½ketchup:genedoc[shtml^ INTERNET^ 0886[ Koziol\ C[\ Wagner!Hulsmann\ C[\ Cetkovic\ H[\ Gamulin\ V[\ Kruse\ M[\ Pancer\ Z[\ Schacke\ H[ and Muller\ W[ E[ G[ Cloning of the heat! inducible biomarker\ the cDNA encoding the 69! kDa heat shock protein\ from the marine sponge Geodia cydonium] Response to natural stressors[ Mar[ Ecol[ Pro`[ Ser[\ 0885\ 025\ 042Ð050[ Stanley\ P[ E[ and Kricka\ L[ J[\ Bioluminescence and chemiluminescence] current status[ New York] John Wiley + Sons^ 0889[

M. Wiens et al. 39[ Miller\ M[ R[\ McClure\ D[ and Shiman\ R[ p! Chlorophenylalanine e}ect on phenylalanine hydroxylase in hepatoma cells in culture[ J[ Biol[ Chem[\ 0864\ 149\ 0021Ð0039[ 30[ PC:GENE Data Banks CD!ROM^ Release 03[9[ IntelliGenetics\ Inc[ Mountain View\ CA\ 0884[ 31[ Li\ W[ H[\ Tanimura\ M[ and Sharp\ P[ M[ An evaluation of the molecular clock hypothesis using mammalian DNA sequences[ J[ Mol[ Evol[\ 0876\ 14\ 229Ð231[ 32[ Onishi\ A[\ Liotta\ L[ J[ and Benkovic\ S[ J[ Clon! ing and expression of Chromobacterium viola! ceum phenylalanine hydroxylase in Escherichia coli and comparison of amino acid sequence with mammalian aromatic acid hydroxylases[ J[ Biol[ Chem[\ 0880\ 155\ 07348Ð07434[ 33[ Morales\ G[\ Requena\ J[ M[\ Jimenez!Ruiz\ A[\ Lopez\ M[ C[\ Ugarte\ M[ and Alonso\ C[ Sequence and expression of the Drosophila phe! nylalanine hydroxylase mRNA[ Gene\ 0889\ 82\ 102Ð108[ 34[ Kimura\ M[\ The Neutral Theory of Molecular Evolution[ Cambridge] Cambridge University Press\ 0872[ 35[ Hirabayashi\ J[ and Kasai\ K[ The family of met! azoan metal!independent b!galactoside!binding lectins] structure\ function and molecular evol! ution[ Glycobiol[\ 0882\ 2\ 186Ð293[ 36[ Schacke\ H[\ Schroder\ H[ C[\ Muller\ I[ M[\ Muller\ W[ E[ G[\ Gamulin\ V[ and Rinkevich\ B[ The immunoglobulin superfamily includes mem! bers from the lowest invertebrates to the highest vertebrates[ Immunol[ Today\ 0883\ 04\ 386Ð387[ 37[ Wagele\ J[ W[ and Wetzel\ R[ Nucleic acid sequence data are not per se reliable for inference of phylogenies[ J[ Natl[ History\ 0883\ 17\ 638Ð 650[ 38[ Pancer\ Z[\ Munkner\ J[\ Muller\ I[ and Muller\ W[ E[ G[ A novel member of an ancient super! family] sponge "Geodia cydonium\ Porifera# puta! tive protein that features scavenger receptor cysteine!rich repeats[ Gene\ 0886\ 082\ 100Ð107[ 49[ Seack\ J[\ Pancer\ Z[\ Muller\ I[ M[ and Muller\ W[ E[ G[ Molecular Cloning and primary struc! ture of an Rh "Rhesus# antigen!like protein from the marine sponge Geodia cydonium[ Immuno! `enetics\ 0886\ 35\ 382Ð387[ 40[ Schacke\ H[\ Rinkevich\ B[\ Gamulin\ V[\ Muller\ I[ M[ and Muller\ W[ E[ G[ Immunoglobulin!like domain is present in the extracellular part of the receptor tyrosine kinase from the marine sponge Geodia cydonium[ J[ Molec[ Reco`nition\ 0883\ 6\ 161Ð165[ 41[ Nappi\ A[ J[ and Vass\ E[ Melanogenesis and generation of cytotoxic molecules during insect cellular immune reactions[ Pi`ment Cell Res[\ 0882\ 5\ 006Ð015[ 42[ Sugumaran\ M[ and Kanost\ M[ R[\ Regulation of insect hemolymph phenoloxidases[ In Para! sites and Patho`ens of Insects\ eds[ N[ E[ Beckage\ S[ N[ Thompson\ B[ A[ Frederici[ Academic Press\ San Diego\ 0886\ pp[ 206Ð231[ 43[ Wagner\ C[\ Ste}en\ R[\ Koziol\ C[\ Batel\ R[\ Lacorn\ M[\ Steinhart\ H[\ Simat\ D[ and Muller\ W[ E[ G[ Apoptosis in marine sponges] a bio! marker for environmental stress "cadmium and bacteria#[ Marine Biol[\ 0887\ 020\ 300Ð310[