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: hoff
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.

ACKNOWLEDGEMENTS
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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