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

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 1 three different phases of tissue competence, as had been proposed before 2 [22]. Some markers specific for the hypostome can be detected very early in the regenerating tip, after which expression of tentacle-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) Eur. J. Biochem. 269, 4597–4606 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03159.x MATERIALS AND METHODS Animals and preparation of extracts from total hydra and from excised foot pieces H. vulgaris were cultured in a medium consisting of 1 m M CaCl 2 ,0.1m M KCl, 0.1 m M MgCl 2 ,and0.5m M NaH 2 PO 4 , pH 7.6. The temperature of the medium was kept at 19 ± 2 °C. 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. vulgaris were homogenized with a Teflon homogenizor in a buffer consisting of 20 m M citrate, 280 m M sucrose, 5m M EDTA, 3 m M EGTA, 0.3 m M phenylmethanesulfo- nyl fluoride (Serva), and 0.5 lgÆmL )1 leupeptin (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 4 °C (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) H 2 O 2 in 100 m M citrate, pH 5.0. The reaction was stopped after 30 min with 20 lLof100m M NaN 3 per mL of sample and the absorbance 3 was measured at 420 nm. As an insoluble substrate for the peroxidase, 0.06% (w/v) diaminobenzidine (Sigma) was used and 0.03% (v/v) H 2 O 2 in 100 m M citrate, pH 5.0. The reaction was stopped by several washes in H 2 O. Chromatographic procedures For anion-exchange chromatography an extract of 650 foot pieces in 500 lLofa20m M Tris/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 m M NaCl with a flow rate of 0.5 mLÆmin )1 . For cation-exchange chromatography 4300 foot pieces were sonicated in 20 m M citrate, pH 7.0, 100 m M NaCl. After centrifugation at 100 000 g for 15 min the pH was adjusted to 4.5 with 1 M citric acid. The column was equilibrated with 20 m M citrate, pH 4.5, 200 m M 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 m M NaCl. The foot-specific peroxidase eluted at 320–360 m M NaCl. To assay hydrophobic interactions, an extract of 4500 foot pieces in 1 mL of 50 m M citrate, pH 5.0, 1 M 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 m M citrate, pH 5.0, 20% glycerol. For chromato- graphy on hydroxyapatite columns an extract of 800 foot pieces in 200 lLofa20m M Tris/HCl, pH 6.9, 0.01 m M CaCl 2 buffer 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 m M 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 m M at 110 (90–130) m M 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 lLof20m M Tris/HCl, pH 7.0, 100 m M NaCl was applied to the column which was equilibrated with 20 m M Tris/HCl, pH 7.0, 300 m M NaCl. This buffer was also used for the elution of the column. The flow rate was 1 mLÆmin )1 and the volume of the collected fractions was 100 lL. The quotient of the elution volume, V e ,tothevoidvolume,V o , 4 was 1.38 for the peroxidase containing fractions, which corresponds to a molecular mass of 43–45 kDa. For all chromatographic procedures described elution was monitored at A 280 and fractions were assayed for peroxidase activity. Electron microscopy Animals were fixed in a mixture of 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2 for 1 h. They were washed several times in phosphate buffered saline (NaCl/P i ) 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) 5 for 10 min each. Following five washes in NaCl/P i (6 min each) the animals were finally reacted with 0.06% (w/v) diaminobenzidine and 0.03% (v/v) H 2 O 2 in NaCl/P i . Subsequently the animals were postfixed with 2% glutaraldehyde in NaCl/P i for 30 min. After several washes in NaCl/P i they were transferred into osmium tetroxide (2% in 0.1 M phosphate buffer) for 1 h, washed again, dehydrated, embedded in Araldit and cured for 48 h at 60 °C. 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/P i , 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) 6 -PAGE and SDS/PAGE 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 4598 S. A. H. Hoffmeister-Ullerich et al. (Eur. J. Biochem. 269) Ó FEBS 2002 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 m M Tris/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 lLby ultrafiltration-centrifugation (Centricon, Beckman, M r 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 SuperScript TM Preamplification System for First Strand cDNA Synthesis (GibcoBRL) and mRNA from H. vulgaris as 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 65 °C annealing temperature, going down to 55 °C, and performing 25 more cycles at 55 °C, followed by a reamplification of an aliquot with 30 cycles and an annealing temperature of 55 °C. 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 · 10 6 independent 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 NotIorXhoI, 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 42 °C over night. Filters were washed with 2 · NaCl/Cit, 0.1% SDS at 50–65 °Cand 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- 32 P]-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 m M citrate, 5 m M EDTA, 3 m M EGTA, 0.3 m M phenylmethanesulfonylfluoride, and 0.5 lgÆmL )1 leupeptin (Boehringer Mannheim), pH 4.5. The homogenate was centrifuged for 15 min at 13 000 g at 4 °C, 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 m M 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. RESULTS 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 H 2 O 2 , 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 Ó FEBS 2002 Foot-specific peroxidase from hydra (Eur. J. Biochem. 269) 4599 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 m M 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 m M NaCl (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)Overviewofa 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 lmin(B),and 1.5 lmin(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 lLof100m M NaN 3 ,andthe 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. 4600 S. A. H. Hoffmeister-Ullerich et al. (Eur. J. Biochem. 269) Ó FEBS 2002 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 situ staining 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 7 -gel 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 H 2 O 2 . 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 chromatography. 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. Ó FEBS 2002 Foot-specific peroxidase from hydra (Eur. J. Biochem. 269) 4601 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 C 18 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 the SWISS PROT database 8 . 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 NADIW as 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 GNKPLTAN(R) Fragment 2 V Y T V A I K Fragment 3 D/S N A D I W E 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. 4602 S. A. H. Hoffmeister-Ullerich et al. (Eur. J. Biochem. 269) Ó FEBS 2002 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,blottedandprobedwiththeantisera.Thestained 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 situ hybridization. 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- 32 P]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. Ó FEBS 2002 Foot-specific peroxidase from hydra (Eur. J. Biochem. 269) 4603 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. DISCUSSION 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 lmintheinset. 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. 4604 S. A. H. Hoffmeister-Ullerich et al. (Eur. J. Biochem. 269) Ó FEBS 2002 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-2 in 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. 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Isolation of a putative peroxidase, a target for factors controlling foot-formation in the coelenterate hydra Sabine A. H. Hoffmeister-Ullerich,. 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