Báo cáo khoa học: Isolation of a putative peroxidase, a target for factors controlling foot-formation in the coelenterate hydra docx

Chica Schaller Zentrum fu¨r Molekulare Neurobiologie, University of Hamburg, Germany In hydra, differentiated ectodermal cells of the foot region contain a peroxidase activity that can be

Isolation of a putative peroxidase, a target for factors controlling foot-formation in the coelenterate hydra

Sabine A H Hoffmeister-Ullerich, Doris Herrmann, Ju¨rgen Kielholz, Michaela Schweizer

and H Chica Schaller

Zentrum fu¨r Molekulare Neurobiologie, University of Hamburg, Germany

In hydra, differentiated ectodermal cells of the foot region

contain a peroxidase activity that can be used as a marker for

foot-specific differentiation processes Because the

expres-sion of the gene coding for the peroxidase must be tightly

regulated during foot-specific differentiation,

characteriza-tion of the protein and cloning of the corresponding gene

should provide valuable tools for getting deeper insights into

the regulation of foot-specific differentiation In this paper

we characterize the foot-specific peroxidase by biochemical,

histochemical, and molecular biological methods We show

that it is localized in granules, and that it consists of a single

component, the molecular mass of which is in the range of 43–45 kDa Purification of the protein and subsequent clo-ning of its complementary DNA yielded two closely related clones, ppod1 and ppod2 Transcripts of ppod2 are abundant

in the whole animal with the exception of the hypostome, the tentacles, and the foot; the expression of ppod1 matches exactly the localization of the foot-specific peroxidase Keywords: hydra; foot-specific peroxidase; differentiation processes; developmental regulation of gene expression

Hydrozoans such as the freshwater polyp Hydra vulgaris are

considered to be one of the most ancient multicellular

animal groups The radially symmetric animals have only

one prominent axis: the apical pole gives rise to

differenti-ated head structures with hypostome and tentacles, the basal

pole at the opposite end comprises the foot, with basal disc

and stalk region The head and the foot contain mainly

terminally differentiated cells, whereas epithelial and

inter-stitial cells in the body column are continuously proliferating

[1,2] Because of their striking ability to regenerate missing

parts even as adult animals, these polyps can be regarded as

permanent embryos, in which patterning and differentiation

processes have to be tightly regulated to maintain the body

structure Removal of head or foot induces the stem cells of

the remaining gastric column to differentiate into

hyposto-mal and tentacle cells of the head or into peduncle and foot

mucous cells of the foot In this process the original polarity

is maintained [3] The decision to undergo head- or

foot-specific differentiation is strictly regulated

Morphogeneti-cally active substances have been reported to be involved in

the control of growth and differentiation processes in hydra

[4–11] Numerous studies of patterning processes during

head regeneration have led to the characterization of

markers for tentacle and hypostome tissue [12–21] These

investigations show that the process of head regeneration

can be subdivided into two or

competence, as had been proposed before

markers specific for the hypostome can be detected very early in the regenerating tip, after which expression of specific markers is initiated Finally the tentacle-specific markers disappear from the regenerating tip and additional hypostome-specific markers start to be expressed Processes of patterning during foot regeneration are less well described Molecular markers of the foot region are the homeobox gene CnNK-2, which is expressed in the endo-derm, mainly in the peduncle region [23], the paired-like homeobox gene manacle, which is expressed at the differ-entiating edge of the basal disc, and the receptor protein tyrosine kinase gene shin guard being expressed in the ectoderm of the peduncle region [24] The ectoderm of the basal disc is built up by specifically differentiated epithelial cells, the foot mucous cells, which are characterized by the occurrence of granules or so called droplets Some of them contain acidic mucopolysaccharide material, and their size varies from 0.9 to 1.5 lm [25] Moreover, foot mucous cells have been shown to harbor a peroxidase activity that is an excellent marker for these cells [26] After excision of the foot the peroxidase starts to be expressed in the foot-regenerating tissue at about 12–15 h after cutting [26] The reappearance of the peroxidase correlates with the differen-tiation of epithelial stem cells to foot mucous cells; this was used to quantify the amount of foot mucous cell differen-tiation and therefore of foot regeneration [26] Accordingly, the effect of foot factors on foot-regeneration can be quantified by measuring the peroxidase activity in foot-factor treated and untreated foot-regenerating animals at a given time point after foot excision [7,26] Because the onset

of differentiation into foot mucous cells can be stimulated or inhibited by foot factors, they directly or indirectly control the expression of the peroxidase

In this paper we describe the localization, characteriza-tion, and isolation of the foot-specific peroxidase from Hydra vulgaris

Correspondence to S A H Hoffmeister-Ullerich, Zentrum fu¨r

Molekulare Neurobiologie, University of Hamburg,

Martinistraße 52, Hamburg, Germany.

Fax: + 49 040 42803 510120246, Tel.: + 49 040 42803 5076,

E-mail: hoffmeis@zmnh.uni-hamburg.de

Abbreviations: ABTS, 2,2¢-azino-bis-(3-ethylbenzthiazoline-6-sulfonic

acid) ammonium salt; LDS, lithium dodecyl sulfate; V e , elution

volume; V o , void volume.

(Received 26 June 2002, accepted 1 August 2002)

M A T E R I A L S A N D M E T H O D S

Animals and preparation of extracts from total hydra

and from excised foot pieces

H vulgariswere cultured in a medium consisting of 1 mM

CaCl2, 0.1 mM KCl, 0.1 mM MgCl2, and 0.5 mM

NaH2PO4, pH 7.6 The temperature of the medium was

kept at 19 ± 2C The animals were fed daily between 9

and 10 am with nauplii of Artemia salina and washed 6 h

later For the preparation of total extracts, 2 g of lyophilized

H vulgariswere homogenized with a Teflon homogenizor

in a buffer consisting of 20 mM citrate, 280 mM sucrose,

5 mMEDTA, 3 mMEGTA, 0.3 mM

phenylmethanesulfo-nyl fluoride (Serva), and 0.5 lgÆmL)1leupeptin (Boehringer

Mannheim), pH 7.0 After centrifugation at 45 000 g for

30 min the supernatant was collected and used for further

analysis For extractions of foot pieces, feet were cut shortly

above the end of the peduncles, collected batchwise, and

frozen before use For the extraction the feet were sonified

for 3· 7 s on ice (Branson Sonifier 250) in a buffer

appropriate for the consecutive chromatographic method

The homogenate was centrifuged for 15 min at 100 000 g at

4C (Beckman TL-100) Mono Q, Mono S, S-Sepharose

fast flow, Superose 12 HR 10/30, and phenyl-Sepharose 6

fast flow were from Pharmacia, the TSK BIO-SIL SEC

125-column from Bio-Rad Protein concentrations were

determined by the method of Bradford (Bio-Rad protein

assay) using bovine serum albumin as standard

Determination of the peroxidase activity

The peroxidase activity was measured in a solution

containing 0.1% (w/v)

2,2¢-azino-bis-(3-ethylbenzthiazo-line-6-sulfonic acid) ammonium salt (ABTS, Sigma) and

0.0003% (v/v) H2O2 in 100 mM citrate, pH 5.0 The

reaction was stopped after 30 min with 20 lL of 100 mM

NaN3per mL of sample and the absorbance

at 420 nm As an insoluble substrate for the peroxidase,

0.06% (w/v) diaminobenzidine (Sigma) was used and

0.03% (v/v) H2O2in 100 mMcitrate, pH 5.0 The reaction

was stopped by several washes in H2O

Chromatographic procedures

For anion-exchange chromatography an extract of 650 foot

pieces in 500 lL of a 20 mMTris/HCl, pH 7.4 solution was

applied to a Mono Q column, which was equilibrated with

the same buffer After washing of the column with two

column volumes of the Tris/HCl solution, the salt

concen-tration of the chromatography buffer was increased in a

linear gradient from 0 to 500 mMNaCl with a flow rate of

0.5 mLÆmin)1 For cation-exchange chromatography 4300

foot pieces were sonicated in 20 mM citrate, pH 7.0,

100 mM NaCl After centrifugation at 100 000 g for

15 min the pH was adjusted to 4.5 with 1M citric acid

The column was equilibrated with 20 mM citrate, pH 4.5,

200 mM NaCl The sample (1 mL) was applied to the

column with a flow rate of 1 mLÆmin)1 The peroxidase was

eluted with a linear gradient from 200 to 600 mMNaCl The

foot-specific peroxidase eluted at 320–360 mM NaCl To

assay hydrophobic interactions, an extract of 4500 foot

pieces in 1 mL of 50 m citrate, pH 5.0, 1 phosphate

(with sodium as counter ion) was applied to a phenyl-Sepharose 6 fast flow (highly substituted) column with a flow rate of 0.5 mLÆmin)1 At an elution volume of 10 mL after start of the chromatography the buffer was exchanged with 25 mMcitrate, pH 5.0, 20% glycerol For chromato-graphy on hydroxyapatite columns an extract of 800 foot pieces in 200 lL of a 20 mM Tris/HCl, pH 6.9, 0.01 mM

CaCl2buffer was applied to the column with a flow rate of

200 lLÆmin)1 The column was equilibrated with the same buffer After an elution volume of 8 mL, the phosphate concentration was raised continuously from 0 to 350 mM

phosphate in a volume of 18.4 mL (stippled line), in Fig 3D The foot-specific peroxidase was eluted with a linear phosphate gradient from 0 to 350 mMat 110 (90–130)

mM phosphate For the determination of the molecular mass of the foot-specific peroxidase an TSK BIO-SIL SEC 125-column was used The column was calibrated with eight different molecules of known molecular mass (inset of Fig 4) An extract of 200 foot pieces in 50 lL of 20 mM

Tris/HCl, pH 7.0, 100 mMNaCl was applied to the column which was equilibrated with 20 mM Tris/HCl, pH 7.0,

300 mMNaCl This buffer was also used for the elution of the column The flow rate was 1 mLÆmin)1and the volume

of the collected fractions was 100 lL The quotient of the elution volume, Ve, to the void volume, Vo,

peroxidase containing fractions, which corresponds to a molecular mass of 43–45 kDa For all chromatographic procedures described elution was monitored at A280 and fractions were assayed for peroxidase activity

Electron microscopy Animals were fixed in a mixture of 4% paraformaldehyde and 1% glutaraldehyde in 0.1Mphosphate buffer, pH 7.2 for 1 h They were washed several times in phosphate buffered saline (NaCl/Pi) and incubated in 1% sodium borohydride for 30 min Thereafter they were processed in

a series of solutions of ethanol/water (10, 20, 40, 20, 10% ethanol, v/v)

NaCl/Pi(6 min each) the animals were finally reacted with 0.06% (w/v) diaminobenzidine and 0.03% (v/v) H2O2 in NaCl/Pi Subsequently the animals were postfixed with 2% glutaraldehyde in NaCl/Pifor 30 min After several washes

in NaCl/Pi they were transferred into osmium tetroxide (2% in 0.1M phosphate buffer) for 1 h, washed again, dehydrated, embedded in Araldit and cured for 48 h at

60C Ultrathin sections from diaminobenzidine positive regions and control animals, respectively, were prepared and analyzed with an electronmicroscope Zeiss 902 For cryosectioning specimens were fixed for 3 h in 4% paraformaldehyde and, after several washes in NaCl/Pi, they were embedded in Tissue Tek II (Miles Laboratories), and frozen on solid carbondioxide Cryostat sections (7 lm) were mounted on gelatin-coated slides and then subjected to the diaminobenzidine-procedure as described above

Electrophoresis Lithium dodecyl sulfate (LDS)

were performed as described in and Proteins in gels were stained either with Coomassie Brilliant Blue R or with silver stain Preparative electrophoresis was carried out in a

preparative cell Model 491 (Bio-Rad) according to the

instructions of the manufacturer Electrophoresis was

performed at 40 mA under cooling for about 8–10 h The

proteins were eluted with elution buffer (150 mMTris/HCl,

pH 7.5) Fractions of 2 mL were collected and analyzed by

ABTS-peroxidase reactions and by SDS/PAGE The

pooled fractions were concentrated to about 100 lL by

ultrafiltration-centrifugation (Centricon, Beckman, Mr

cut-off 30 000)

Cloning and sequence analysis of the peroxidase

mRNA was isolated from H vulgaris with a Quick Prep

Micro mRNA Purification Kit (Pharmacia)

Oligonucleo-tide primers were synthesized according to the sequences of

the tryptic peptides For fragment 1, LVTAEEAGNKPL

TAN, and fragment 3, NADIWER, the following sense and

antisense primers were designed: GAG/A GAG/A GCG/T/

C GGG/T/C AAT/C AAG/A CC for fragment 1 and

AAT/C GCG/T/C ATA/T/C TGG GAG CG for fragment

3, GG T/CTT A/GTT C/G/TCC C/G/TGC C/TTC

C/TTC for fragment 1 and CCA G/TAT GTC G/T/CGC

GTT GTC for fragment 3 With these primers in different

combination polymerase chain reactions were performed

with the SuperScriptTM Preamplification System for First

Strand cDNA Synthesis (GibcoBRL) and mRNA from

H vulgarisas template The reactions were carried out on a

TRIO-Thermoblock (Biometra) applying different

proto-cols for different given combinations of primers For the

isolated peroxidase-clones, ppod1 and ppod2, the conditions

used were 10 cycles of a touch-down protocol, starting with

65C annealing temperature, going down to 55 C, and

performing 25 more cycles at 55C, followed by a

reamplification of an aliquot with 30 cycles and an

annealing temperature of 55C From the sequence of this

PCR-fragment new primers were designed and used for the

generation of the 3¢ and 5¢ ends by performing PCRs either

with the 3¢ RACE system (GibcoBRL) or with DNA of a

(ZAP cDNA library of H vulgaris as template For library

construction the mRNA was reverse-transcribed into

cDNA and ligated into the Uni-ZAP XR vector using the

ZAP cDNA synthesis kit (Stratagene) The vector was

packaged with the Gigapack II packaging extract

(Strata-gene) The library contained 0.8· 106independent plaques

and was amplified once As template for PCR the cDNA

was excised and cloned into XL1-blue cells The plasmid

DNA was linearized with NotI or XhoI, respectively, prior

to PCR The ppod1 and ppod2 cDNA sequences are stored

in GenBank, accession numbers AY034096 and AY034095,

respectively

DNA sequencing was performed on both strands using

the dideoxy chain termination method and a automated

sequencer Sequence data were analyzed using the GCG

package of programs (Genetics Computer Group, Inc.,

Wisconsin, USA) and the PSORT program (prediction of

protein localization sites,

www.expasy.ch/sprot/sprot-top.html)

In situ hybridization

Nonradioactive in situ hybridization was carried out as

described in using as templates the 3¢-terminal first 395 and

483 nucleotides for ppod1 and for ppod2, respectively The

probes were derived from the NcoI linearized pGem-T easy plasmid with SP6 polymerase for the antisense probe and from the same SpeI linearized plasmid with T7 polymerase for the sense probe

Northern blot analysis Preparation and blotting of poly(A)+RNA from cut and pooled tissue pieces of H vulgaris were carried out as described Hybridization was performed with 50% forma-mide, 5· NaCl/Cit, 0.1% SDS, 5· Denhardt’s,

100 lgÆmL)1 tRNA at 42C over night Filters were washed with 2· NaCl/Cit, 0.1% SDS at 50–65 C and autoradiographed by means of a phosphoimager (Fuji Bas 2000) or Kodak Biomax film Probes for ppod2 and ppod1 were the same fragments as for the in situ hybridization, labeled with [a-32P]-dCTP by random priming (Amersham) Western blot analysis

For the preparation of extracts, 30 mg of lyophilized

H vulgaris(500–600 animals) or frozen foot pieces (about 1000) were sonified for 3· 7 s on ice (Branson Sonifier 250)

in a buffer consisting of 20 mMcitrate, 5 mMEDTA, 3 mM

EGTA, 0.3 mM phenylmethanesulfonylfluoride, and 0.5 lgÆmL)1 leupeptin (Boehringer Mannheim), pH 4.5 The homogenate was centrifuged for 15 min at 13 000 g at

4C, and the supernatant was subjected to cation-exchange chromatography on Sartobind-S membranes The peroxi-dase was recovered by elution with a buffer consisting of

100 mM citrate, pH 7.0 containing a protease inhibitor cocktail Finally, the active fractions were pooled, concen-trated by ultrafiltration-centrifugation, and then applied to SDS/PAGE Protein samples were separated on reducing 12% SDS-polyacrylamide gels and transferred to Immobi-lon-P membranes The peroxidase was detected on the blots with polyclonal antisera directed against amino acids 20–28

of PPOD1, generated in mice (Eurogentec), used at a dilution of 1 : 250, and visualized with an alkaline phos-phatase conjugated secondary antibody (Sigma) at a dilution of 1 : 7500

R E S U L T S

Subcellular localization of the foot-specific peroxidase

in foot mucous cells Previous work had implied that the foot-specific peroxidase occurs in or is closely associated with granules For a more detailed analysis of the subcellular localization, the peroxi-dase was detected in situ by the addition of diaminobenzi-dine and H2O2, and the tissue was prepared for electron microscopy Figure 1A shows a semithin section demon-strating the darkly stained foot mucous cells in the ectoderm

of the foot Stained diaminobenzidine containing granules are concentrated in the apical part of foot mucous cells (Fig 1B) The amount of stained granules per cell varies depending on the position of the foot mucous cell with respect to the body axis of the animal Freshly matured foot mucous cells, in the transition zone between gastric region and foot contain fewer granules than mature foot mucous cells, which lie closer to the foot Foot mucous cells very close to the centre of the basal disc, the aboral porus, are

considered to be aged cells, and they contain less stained

granules than the mature ones Higher power micrographs

of the foot mucous cells show that the stained granules are

0.5–1.5 lm in diameter (Fig 1C) The peroxidase is

asso-ciated with the granular matrix, and not all granules are

stained The intensity of the labeling varies between different

granules, implying that the content of peroxidase is variable

For comparison, tissue of the same region not subjected to

the diaminobenzidine reaction is shown in Fig 1D

Properties of the foot-specific peroxidase

The localization of the peroxidase in granules implies that

this enzyme might be active under acidic pH conditions

Determination of its pH optimum showed that the maximal

enzymatic activity is observed at pH 4.5 (Fig 2) The

enzymatic activity is inhibited by azide and is totally blocked

by heating For a biochemical characterization of the

foot-specific peroxidase foot extracts were subjected to different

chromatographic procedures (Fig 3) The foot-specific

peroxidase activity was eluted from an anion exchanger at

a salt concentration of less than 100 mM with a yield of

about 10% (Fig 3A) For a comparison the interaction of

the foot-specific peroxidase with a cation exchanger,

Mono S was tested The foot-specific peroxidase eluted at

320–360 mMNaCl (Fig 3B), the yield of activity was 66%

To assay hydrophobic interactions the peroxidase was

applied to a phenyl-Sepharose 6 fast flow (highly

substi-tuted) column As can be seen in Fig 3C, the peroxidase

bound to the column and was eluted by decreasing the ionic

strength Hydroxyapatite often resolves multiple

compo-nents that behave homogeneously in other

chromatogra-phic and electrophoretic techniques Therefore, we tested

whether the foot-specific peroxidase could be bound to hydroxyapatite and whether it could be eluted as a single peak comprising activity No further proteins, measured as

Fig 1 Subcellular localization of the foot-specific peroxidase (A) Overview of a longitudinal section of H vulgaris with the tentacles (t) at the distal, and the foot (f) at the proximal end of the animal The arrow points

at peroxidase containing cells lying in the ectoderm of the foot The diaminobenzidine-stained granules are localized mainly in the apical part of the foot mucous cells as indicated by the arrow in (B) (C,D) Higher power electron micrographs, which show (C) the diaminobenzidine-stained granules in the foot mucous cells (fm), and granules without diaminobenzidine-staining as a control (D) Scale bars are 1 mm in (A), 20 lm in (B), and 1.5 lm in (C) and (D).

Fig 2.

9 Determination of the pH-optimum of the foot-specific peroxi-dase Equal amounts of an extract of foot pieces were reacted with

1 mL of a solution containing 100 m M citrate, 0.1% ABTS, and 0.0003% H 2 O 2, which was adjusted to the different pH values by titration with NaOH After an incubation time of 30 min, the reaction was stopped by the addition of 10 lL of 100 m M NaN 3 , and the absorbance

10 was measured at 420 nm Maximal activity was found at

pH 4.5 Shown are the mean values and their standard deviations of three independent experiments.

absorption at 280 nm, and no activity eluted with higher salt

concentrations (Fig 3D) For the determination of the

molecular mass of the foot-specific peroxidase an extract of

200 foot pieces was applied to an analytical size-exclusion

TSK-column The linear range of separation for this column

lies between 0.5 and 100 kDa As can be seen in Fig 4, the

molecular mass of the foot-specific peroxidase was 43–

45 kDa Taken together, these results show that the

foot-specific peroxidase is optimally active under acidic pH

conditions as can be achieved intracellularly in granules,

that it displays an overall positive rather than a negative

charge, is able to interact with hydrophobic surroundings,

and that it is most likely enzymatically active as a single

component of 43–45 kDa

Characterization of hydra’s peroxidase activities by gel

electrophoresis

In situstaining of whole mounts of hydra had shown that a

main peroxidase activity is present in the foot, but that there

exists at least one more peroxidase activity that is distributed

over the rest of the animal For a comparison of these

different peroxidase activities we applied extracts from

whole animals and from foot pieces to LDS

electro-phoresis, which had been shown to be compatible with the detection of peroxidase activities For the visualization of the peroxidase activities the gel was reacted with diam-inobenzidine and H2O2 As can be seen in Fig 5A, a major and a minor activity exist in the animal Only the major activity (band I) is present in the feet of the animals Therefore, band I was regarded as the foot-specific peroxi-dase activity of hydra For a further characterization an extract of about 30 000 foot pieces was first purified on cation-exchange chromatography, then applied to LDS-PAGE Several stained bands I were excised from LDS gels, pooled, and applied to SDS/PAGE under standard dena-turing conditions After silver staining of the gel, the only detectable band migrated slightly below the 45 kDa marker protein, ovalbumin (Fig 5B) These results confirm the result obtained by size exclusion chromatography and show that the foot-specific peroxidase can be separated by gel electrophoresis from another peroxidase activity which resides predominantly in the footless part of hydra Purification of the band I peroxidase

For the purification of the foot-specific peroxidase cytosolic fractions of several enzymatically active preparations were

Fig 3 Chromatography of the foot-specific peroxidase (A) Mono Q anion-exchange chromatography (B) Mono S cation-exchange chromatog-raphy The stippled line shows the NaCl concentration (C) Phenyl-Sepharose chromatogchromatog-raphy The stippled line shows the change to the buffer with low ionic strength (D) Chromatography of the foot-specific peroxidase on a hydroxyapatite column After an elution volume of 8 mL, the phosphate concentration was raised continuously from 0 to 350 m M phosphate in a volume of 18.4 mL (stippled line) Elution was monitored at

A 280 and fractions were assayed for peroxidase activity Grey bars indicate the active fractions.

pooled, subjected batchwise to cation-exchange

chromato-graphy by Mono-S and concentrated on Mono-S mini filter

cartridges (Sartorius) After elution the pooled fractions

were processed by preparative gel electrophoresis The

eluted fractions were analyzed for the size of the proteins

they contained and for their peroxidase activity The

appropriate fractions were pooled, concentrated by ultrafil-tration-centrifugation, and then applied to SDS/PAGE The band that corresponded to a size of 43 kDa was excised from the gel After extraction from the gel and evaporation

of the solvent this material was incubated with trypsin to generate peptides for sequencing The peptides were separ-ated by reverse-phase C18 chromatography and then sequenced with an automated sequencer The amino acid sequences of four peptides (Table 1), derived from the puri-fied protein, were not present in theSWISS PROTdatabase

Cloning of the peroxidase The information obtained from the amino acid sequences of the tryptic peptides provided the basis for a cloning strategy using PCR In the first step, single-strand complementary DNA was generated by reverse transcription from the messenger RNA isolated from hydra feet Different pools of oligonucleotides were designed as primers Those encoding EEAGNK as sense (upstream) primer and NADIWas antisense (downstream) primer, for two of the obtained tryptic fragments, yielded a product of 475 base pairs, encoding a putative protein of 154 amino acids This included the six amino acids of the peptide used to design the sense primer and additional five amino acids of the same tryptic fragment, the five amino acids of the fragment for the antisense primer and seven amino acids derived from fragment four, SYLIANR, which was not used as a primer (underlined in Fig 6B) From the nucleotide sequence, two new forward and two new reverse primers were generated for

Fig 5 Detection of the peroxidase in polyacrylamide gels (A) Extracts

of 500 foot pieces and extracts of 1000 whole animals were applied to a

12% polyacrylamide gel which was stained with 0.06% (w/v)

diam-inobenzidine and 0.03% H 2 O 2 in 100 m M citrate, pH 5.0 The

incu-bation was stopped after 35 min by four or five washes of the gel in

H 2 O The arrows indicate the stained bands in the gel I is the major

peroxidase activity of hydra residing in the foot of the animal, II

indicates another peroxidase activity present in the rest of the animal.

(B) An extract of  30 000 foot pieces was first purified on

cation-exchange chromatography, then applied to LDS-PAGE The LDS gel

consisted of 8% polyacrylamide The active bands were excised from

the gel, pooled and applied to a 12% SDS/PAGE The proteins were

visualized by silver stain On the left panel the molecular mass markers

are shown, on the right panel the purified peroxidase protein.

Table 1 Amino acid sequences of the tryptic fragments derived from the purified peroxidase protein.

Fragment 1 L V T A E E A G N K P L T A N (R) Fragment 2 V Y T V A I K

Fragment 3 D/S N A D I WE R (R) Fragment 4 S Y L I A N R

Fig 4 Determination of the molecular mass of the foot-specific peroxidase A TSK BIO-SIL SEC 125-column was calibrated with eight different molecules of known molecular mass

as shown in the inset An extract of 200 foot pieces was applied to the column V e /V o was 1.38 for the peroxidase containing fractions which corresponds to a molecular mass of 43–45 kDa Elution was monitored at A 280

and fractions were assayed for peroxidase activity Grey bars indicate the active fractions.

the cloning of the 5¢ and 3¢ end of the clone, respectively.

Analysis of the nucleotide sequences of the newly obtained

PCR-fragments yielded two different, highly homologous

clones, which we designated ppod1 and ppod2 (pp standing

for putative peroxidase) The lengths of ppod2 and ppod1 are

1092 and 1099 base pairs, respectively Northern blot analysis

revealed that the size of the messages for both clones is

 1.2 kb implying that full-length cDNAs had been obtained

(Fig 7) The two cDNAs show 80% sequence homology at

the nucleotide level They comprise an open reading frame of

888 nucleotides for ppod2 and 873 nucleotides for ppod1,

coding for 295 and 290 amino acids, respectively Moreover,

the Northern blot analysis gave a first hint that ppod1

encodes the foot-specific peroxidase (Fig 7)

Analysis of the structure of ppod2 and ppod1

Sequence analysis of both cDNAs revealed 75% identity at

the protein level Both predicted proteins have a modular

structure of 34 amino acids in common The six modules

that can be found in PPOD1 and PPOD2, respectively, are

schematically shown in Fig 6B Two similar modules

(43.7% identity in a stretch of 72 amino acids) can be

found in the C-terminal region (amino acids 399–471) of

chitinase C However, the conserved amino acids between this chitin binding region and the modules of PPOD1 and PPOD2 are not considered to be essential for chitin binding The deduced protein sequences of ppod1 and ppod2 contain several putative phosphorylation sites and, in the case of ppod2,also a putative glycosylation site These findings may explain why the native peroxidase migrates with an apparent molecular mass of 43–45 000, whereas the deduced molecular masses of ppod1 and ppod2 are 32 020 and 32 927, respectively Antisera were generated in mice against a peptide comprising amino acids 20–28 of PPOD1 Extracts of feet and whole animals were applied to SDS/ PAGE, blotted and probed with the antisera The stained band migrated with an apparent mass of about 45 000, thus confirming the identity of the cloned peroxidase (Fig 8) Localization of ppod2 and ppod1 in hydra tissue The ppod1 and ppod2 expression patterns were analyzed by

in situhybridization These experiments showed that ppod2

is expressed along the gastric column of the animal (Fig 9B), whereas expression of ppod1 is restricted to the foot of the animal (Fig 9A) Both signals are localized in the outer cell layer, in ectodermal epithelial cells, which

Fig 6 Protein sequences of PPOD1 and

PPOD2 (A) Sequence comparison between

the deduced amino acids of the two obtained

clones The sequences of the originally

obtained tryptic fragments are underlined in

bold (B) Schematic drawing of PPOD1 and

PPOD2 showing the arrangement of the

modules (M1–M6) Also indicated are the

putative phosphorylation sites (P), the

puta-tive glycosylation site (G), the signal peptide

(SP) and the hydrophobic region at the

carboxyterminal end of PPOD1 and PPOD2

(hashed region).

Fig 7 Northern-blot analysis of ppod1 and

ppod2 Northern blot analysis reveals ppod1

expression in feet of hydra H vulgaris were

cut into feet (F), gastric regions (B), and heads

(H), and about 2 lg of poly(A) + RNA from

each fraction were subjected to Northern blot

analysis using [a-32P]dATP-labeled ppod1 and

ppod2-specific probes, respectively

Methy-lene-blue staining of the same filter revealed

the amounts of RNA loaded per lane The

sizes of an RNA marker are indicated.

corresponds to the localization of the peroxidase activity as

shown before Therefore, the ppod1 clone is regarded as the

cDNA for the foot-specific peroxidase For a comparison of

ppod1 and the described peroxidase, foot-regenerating

animals of H vulgaris were subjected to in situ hybridization

After cutting off the feet of the animals the ppod1 signal

vanished and started to reappear at 10–13 h after foot

removal (Fig 10A–C), which is about 2–5 h earlier than the

measurable start of the reappearance of the protein At 10

and 13 h after cutting the expression of ppod1 is confined to

the regenerating area (Fig 10B,C), later the area of ppod1

expression extends more into the head direction

(Fig 10D,E), which is similar to what was found for the

expression of pedibin during foot regeneration After

completion of foot regeneration, 30 h after cutting, the

level of ppod1 expression is still elevated in comparison to

the mature adult foot region (Figs 10F and 9A) In buds,

which are close to maturity and departure from the parental

animal, the timing of the appearance and the localization of

the mRNA was also in accordance with the peroxidase

protein

D I S C U S S I O N

The finding that a peroxidase activity occurs in foot mucous cells of the basal disc in hydra has provided a valuable tool for the study of foot-specific differentiation processes By use of a precipitable substrate, like diaminobenzidine, foot mucous cells can be reliably identified in histological preparations [26,40–43] Alternatively, by application of a soluble substrate like ABTS, the presence of foot mucous

Fig 8 Western blot analysis of extracts enriched in peroxidase from

H vulgaris Antibodies directed against amino acids 20–28 of PPOD1

were subjected to the blot carrying peroxidase enriched extracts

sep-arated on a 12% reducing polyacrylamide gel A band in the range of

45 kDa is detected.

Fig 9 Expression pattern of ppod1 and ppod2 in tissue of H vulgaris Expression of peroxidase transcripts was detected in whole mount prepa-rations with digoxigenin labeled riboprobes (A) ppod1 expression exclusively in the foot of the animal Inset: higher magnification of a foot region showing intense staining of the ectoderm (B) ppod2 expression along the gastric region of the animal excluding the foot region ec, ectoderm; f, foot; h, head Bar corresponds to 1 mm in (A) (B) and to 140 lm in the inset.

Fig 10 Kinetics of the reappearance of ppod1 in foot regenerating tissue

of H vulgaris Whole mount in situ hybridization of regenerates shows that the ppod1 mRNA starts to reappear between 10 and 13 h at the cut surface, if the cut was carried out just above the stalk region (A) Immediately after cutting off the foot there is no expression of ppod1 in the tissue detectable (B) After 10 h, ppod1 positive cells become visible and expression increases steadily in the regenerating tissue after 13, and

18 h, (C) and (D), respectively After 24 h, the ppod1 expressing area is not further expanding (E), and after completion of foot regeneration at

30 h there is a very high level of expression in the mature basal disc with the adjacent cells still expressing ppod1 (F) Bar corresponds to 1 mm.

cells can be easily quantified [6,7,44] Peroxidases are widely

distributed in the plant as well as in the animal kingdom

serving different metabolic tasks One of their most

important functions is probably the protection of cells from

oxidative stress, provoked by the presence of peroxides, but

they can also play an important role in processes like growth

and differentiation, inflammation, phagocytosis, and

apop-tosis [36,45–48] In hydra the basal disc is the most proximal

region of the polyp, and it is the area of the animal that

attaches to any type of substrate It is also one of the

extremities at which cells die and are sloughed off Hence,

the foot-specific peroxidase may be involved in defence

mechanisms of this exposed body region and/or may be

involved in differentiation or aging processes of these cells

The activity of the foot-specific peroxidase appears to be

best stabilized at pH values in the range of pH 4–5, which

under physiological conditions in the cells of the animal is

probably achieved by the compartmentalization in granules

The occurrence of secretory, so called mucous granules,

which are reactive to diaminobenzidine in foot mucous cells

had been shown previously, and it was assumed that the

diaminobenzidine stain was due to the action of a secretory

peroxidase [49] Here we show that the foot-specific

peroxi-dase from hydra can be eluted as a single enzymatically

active component after binding to hydroxyapatite

More-over, the foot-specific peroxidase was found to display

hydrophobic interactions We purified this foot-specific

per-oxidase by means of cation-exchange chromatography,

pre-parative gel electrophoresis, and subsequent SDS/PAGE

Two cDNAs, ppod1 and ppod2, encoding highly

homo-logous proteins were isolated based on tryptic fragments of

the purified protein Both proteins contain the tryptic

fragments obtained from the isolated protein, which

con-firms that the corresponding cDNAs encode the purified

protein Northern blot analysis revealed that the cDNAs

most likely represent full-length transcripts Comparison of

the expression patterns of the ppod1 and ppod2 mRNA

strongly implies that ppod1 is encoding the foot-specific

peroxidase, because the expression of this clone is restricted

to the ectoderm of the foot of hydra In addition, we could

show that the timing of the reappearance of ppod1

transcripts in foot-regenerating tissue slightly precedes the

reappearance of the enzymatically active protein The fact

that the deduced amino acid sequence of ppod1 comprises a

signal peptide implies that the protein can be secreted, as

had been proposed before [49] The analysis of the

expres-sion pattern of the ppod2 transcripts demonstrates, that they

are abundant in the whole animal with the exception of the

hypostome, the tentacles, and the foot This second cDNA

might correspond to another peroxidase activity that can be

detected in hydra The modular composition of the proteins

may be taken as a hint for the early origin of a modular

composition of enzymes during evolution

The foot mucous cells are derived from epithelio

muscu-lar cells of the gastric column, which are gradually forced

proximally to the basal disc During this process the cells are

transformed into foot mucous cells Therefore, under steady

state conditions this is one of the regions of the animal

where differentiation processes have to be initiated

perma-nently The transformation from epithelio muscular cells to

foot mucous cells can be visualized by means of the

expression of the foot-specific peroxidase as described

previously [34] Thus, the foot-specific peroxidase is a target

of factors, which control foot-specific differentiation pro-cesses This becomes also evident during foot regeneration

In this situation, epithelial stem cells of the regenerating tip start to express ppod1 10–13 h after the initiation of regeneration, which is 2–5 h before the enzymatic activity can be measured [26] From the presently available data for patterning during foot regeneration, the following picture arises Between 5 and 7 h after cutting the expression of pedibin, a foot formation stimulating factor, is up-regulated [39] Next, the expression of the transcription factors CnNK-2in the endoderm [23] and manacle in the ectoderm [24] is initiated in the regenerating tip, followed by the expression of the marker for differentiated foot mucous cells, ppod1 Later, when the regeneration of the basal disc is complete, shin guard, another putative target gene for factors controlling foot formation, is expressed in the peduncle region [24] Hence, analysis of the regulation of ppod1 expression should shed some more light on the mechanisms of pattern formation in the foot of hydra and will be the subject of further investigations

A C K N O W L E D G E M E N T S

We thank Saskia Siegel for excellent technical assistance, Dr Fritz Buck for producing and sequencing of the tryptic fragments, Marion Da¨umigen for DNA sequencing, Dr Timo Wittenberger for helping

to analyze the modular structure of the clones, Dr Irm Hermans-Borgmeyer for critical reading of the manuscript, and Oliver Sperl and Simon Hempel for help with the figures This work was supported part

of the time by the DFG (Ho 1296/1–2).

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