A(1fi3)-b-
D
-glucan recognition protein from the sponge
Suberites domuncula
Mediated activation of fibrinogen-like protein and epidermal growth factor gene
expression
Sanja Perovic
´
-Ottstadt
1
, Teresa Adell
1
, Peter Proksch
2
, Matthias Wiens
1
, Michael Korzhev
1
, Vera Gamulin
3
,
Isabel M. Mu¨ ller
1
and Werner E. G. Mu¨ ller
1
1
Institut fu
¨
r Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universita
¨
t, Mainz, Germany;

epidermal growth factor precursor. Expression of the
(1fi3)-b-
D
-glucan-binding protein and the fibrinogen-like
protein occurred in cells on the sponge surface, in the pin-
acoderm. By Western blotting, the product of the fibrin-
ogen-like protein gene was identified, the recombinant
protein isolated, and antibodies raised to this protein. Their
application revealed that a 5 kDa factor is produced, which
is apparently processed from the 77 kDa epidermal growth
factor precursor. Finally, we provided evidence that a
tyrosine kinase pathway is initiated in response to exposure
to
D
-glucan; its phosphorylation activity could be blocked
by aeroplysinin. In turn, the increased expression of the
downstream genes was suppressed. We conclude that
sponges possess a molecular mechanism for recognizing
fungi via the
D
-glucan carbohydrates on their surfaces.
Keywords:
D
-glucan binding protein; epidermal growth
factor; fungi; sponges; symbiosis.
Sponges (phylum Porifera) are, among all metazoan taxa,
those animals which contain the widest range of specific and
very effective bioactive compounds [1,2]. It has been
assumed that most of these secondary metabolites are
produced by symbiotic microorganisms which are harbored

¨
t,
Duesbergweg 6, 55099 Mainz, Germany.
Fax: + 49 6131 39 25243, Tel.: + 49 6131 39 25910,
E-mail:
Abbreviations: EGF, epidermal growth factor; LPS, lipopolysaccha-
ride; MAPK, mitogen-actived protein kinase; PoAb, polyclonal
antibody.
Note: This article is dedicated to Professor Zeeck (University of
Go
¨
ttingen) on the occasion of his 65th birthday.
Note: The cDNA sequences from Suberites domuncula have been
deposited in EMBL/GenBank as follows: the (1fi3)-b-
D
-glucan-
binding protein (GLUBPp_SUBDO) under the accession number
AJ606470, the fibrinogen-like molecule (FIBl_SUBDO) under the
accession number AJ606471, and the epidermal growth factor
precursor (EGFl-PREC_SUBDO) under the accession number
AJ606469.
(Received 23 January 2004, revised 9 March 2004,
accepted 22 March 2004)
Eur. J. Biochem. 271, 1924–1937 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04102.x
which have been isolated from cell walls of plants, but
also from bacteria and fungi [8]. Prominent purified glu-
can molecules of this group are (a) curdlan, a linear
polysaccharide from Alcaligenes faecalis [9,10] and (b) lami-
narin, a poly (1fi3)-b-
D

had to be identified. Such a molecule has previously been
isolated and cloned from a number of protostomians – from
crustaceans [13,17], earthworm [12] and insects [18], as well
as from sea urchins [19]. After successfully cloning the
(1fi3)-b-
D
-glucan-binding protein from S. domuncula,we
continued our search for other potential binding proteins
that might be involved in recognizing the
D
-glucan. Prom-
ising candidates were molecules that display lectin proper-
ties, e.g. the horseshoe crab acetyl group-recognizing lectin
[20] or lectin molecules with fibrinogen domains, e.g. the
ficolins [21]. This rationale led to the isolation of a
fibrinogen-like molecule from S. domuncula.
It is known that cells from deuterostomians react to
fungal cell wall polysaccharides by producing cytokines [22].
The epidermal growth factor (EGF) domain occurs very
frequently in cytokines; for sponges this domain has already
been described [23]. Therefore, degenerate primers were
designed to identify genes which comprise this domain and
that are expressed by the stimulation of sponges with
D
-glucans. This approach resulted in the identification of a
cDNA whose deduced polypeptide, termed EGF precursor,
comprises three EGF domains.
Our data provide, for the first time, an insight into the
response of sponges to stimulation with (1fi3)-b-
D

)
and were then processed as described previously [28]. Where
indicated, the tissue was additionally treated with 1 lgÆmL
)1
of aeroplysinin. Samples were homogenized in lysis buffer
[1 · Tris-buffered saline (TBS), pH 7.5, 1 m
M
EDTA, 1%
Nonidet-P40, 10 m
M
NaF, protease inhibitor cocktail (one
tablet per 10 mL) and 1 m
M
sodium orthovanadate],
centrifuged and the supernatants analysed by Western blot.
To determine the phosphorylation of tyrosine, the tissue
samples were treated for 6 h with polysaccharide.
Total tissue extracts (20 lg per lane) were subjected to
electrophoresis in 8% polyacrylamide gels containing 0.1%
SDS, as described by Laemmli [29]. Western blotting
experiments were performed as described previously [30].
The membranes were incubated with mouse mAb-anti-
phosphotyrosine (mAb-aTyr) (1 : 2000 dilution). After
washing, the blots were incubated with peroxidase-coupled
goat antimouse IgG (1 : 2000 dilution). Detection of
the immunocomplex was carried out using the BM
Chemoluminescence Blotting Substrate kit from Roche
(Mannheim, Germany).
Ligand-binding blot
The assay was performed as described previously [12].

Ó FEBS 2004 Activation of sponge cells by (1fi3)-b-
D
-glucan (Eur. J. Biochem. 271) 1925
S. domuncula cDNA library [24] by PCR. The primers
were designed against the highly conserved region within
the (1fi3)-b-
D
-glucan-binding proteins; in the b-1,3-glucan-
binding protein from the black tiger shrimp, Penaeus mon-
odon (accession number AF368168-1) the stretch reads
MLWPAIWM (amino acids 160–167). The degenerate
primer, 5¢-TGGCTITGGCCIGCIATA/C/GTGGATG-3¢,
was used in the PCR reaction, together with the vector
primer. The PCR was carried out as follows: initial
denaturation at 95 °C for 4 min, followed by 30 amplifi-
cation cycles at 94 °Cfor30s,62°C for 45 s and 70 °C
for 1.5 min, and a final extension at 70 °C for 10 min. The
reaction mixture was as described previously [31]. The
fragmentsobtainedwereusedtoisolatethecDNAfrom
the library [32] and identified one clone with a 1327
nucleotide insert [excluding the poly(A) tail]. The clone
was termed SDGLUBP; it was sequenced using an
automatic DNA sequencer (Li-Cor4200; MWG Biotech,
Ebersberg, Germany).
cDNA corresponding to the fibrinogen-like protein
Following the strategy described in the Introduction, a
conserved fibrinogen domain was selected for the design
of degenerate primers. Aligning different fibrinogen-
domain containing proteins, fibrinogens, fibroleukins/
techylectins, angiopoietins, ficolins and tenascins, the

CLUSTAL W
, Version 1.6 [35]. Phylogenetic
trees were constructed on the basis of amino acid sequence
alignments by neighbour-joining, as implemented in the
NEIGHBOR
program from the
PHYLIP
package [36]. The
distance matrices were calculated using the
DAYHOFF PAM
matrix model, as described previously [37]. The degree of
support for internal branches was further assessed by
bootstrapping [36]. The graphic presentations were pre-
pared using
GENEDOC
[38].
Recombinant EGF precursor and production of antibodies
The sponge SDEGFI-PREC sequence was isolated by
PCR using the forward primer, f1 [5¢-
CCATGGAGA
AGATTCTAGCAACAGTCAATTCAAATGAC-3¢ (the
NcoI restriction site is underlined), nucleotides 1060–1098],
and the reverse primer, r1 [5¢-
GCGGCCGCTG
TATCTGAAGTTGGGGAATTACTGTGTTTGTTGTT-3¢
(the NotI restriction site is underlined); nucleotides 2206–
2241]. The full-length PCR product (1143 bp) was
expressed in E. coli. The cDNA was cloned into the
bacterial glutathione-S-transferase/oligohistidine/S expres-
sion vector, pET41a (Novagen, Madison WI, USA) via

Zealand), as previously described [39]. Animal experiments
were registered and performed according to German law.
After three booster immunizations, the serum was collected;
the PoAbs were termed PoAb-EGF protein. In control
experiments, 100 lL of the PoAb-EGF protein was adsor-
bedto20 lg of r-EGF_SUBDO (30 min; 4 °C) prior to use.
Western blotting of EGF
For the identification of EGF in extracts from sponge tissue,
extracts were prepared, as described above, and subjected to
electrophoresis through 15% polyacrylamide gels contain-
ing 0.1% SDS, as described previously [29]. The membranes
were incubated with rabbit PoAb-EGF precursor (1 : 500
dilution); the immune complexes were visualized by incu-
bation with alkaline phosphatase-conjugated antirabbit
IgG, followed by staining with 4-chloro-1-naphthol. To
quantify a given signal on the blots, scanning with the
GS-525 Molecular Imager (Bio-Rad) was performed. The
relative value, with respect to the signal seen in the
nontreated extract, is given for the signal seen in extract
from curdlan-treated tissue.
1926 S. Perovic
´
-Ottstadt et al. (Eur. J. Biochem. 271) Ó FEBS 2004
RNA preparation and Northern blot analysis
RNA was extracted from liquid-nitrogen pulverized tissue
using TRIzol reagent (GibcoBRL, Grand Island, NY,
USA), as described previously [40]. Then, 5 lgoftotal
RNA was electrophoresed and blotted onto Hybond-N
+
nylon membrane (Amersham, Little Chalfont, Bucks, UK).

controls in the experiments.
Results
Effect of incubation with curdlan on the phosphorylation
of tyrosine in sponge tissue
It is known that (1fi3)-b-
D
-glucans are activators of gene
expression in mammalian cells [22]. Therefore, we investi-
gated whether sponges react to curdlan with an increased
phosphorylation of tyrosine. Tissue samples were incubated
in the presence or absence of 10 lgÆmL
)1
curdlan. Extracts
were prepared and the proteins were size separated by
SDS/PAGE. After transfer, the blot was incubated with
mAb-aTyr and then with a labeled secondary antibody. The
results show that in the absence of curdlan no bands were
detected on the blots (Fig. 1B; lane a); however, in the
extracts from curdlan-treated tissue a strongly staining band
of 32 kDa was observed (lane b). When the tissue was
treated with curdlan and the tyrosine kinase-inhibitor,
aeroplysinin (1 lgÆmL
)1
), no 32 kDa band was detected
(lane c). In parallel, the gels were stained with Coomassie
Brilliant Blue (Fig. 1A) and no change in the banding
pattern and their intensities occurred.
Detection of glucan-binding activity in extracts
from
S. domuncula

b-
D
-glucan-binding protein
Sequence. The insert with SDGLUBP comprises one
ORF, which ranges from nucleotides 46–48 to nucleo-
tides 1252–1254(stop); the cDNA is of full length, as
shown by Northern blot analysis (1.4 kb; see below). The
deduced protein shows high sequence similarity to the
Fig. 1. Phosphorylation of a 32 kDa protein after incubation of sponge
tissue with curdlan. Tissue samples were incubated for 6 h with or
without 10 lgÆmL
)1
curdlan. Protein extracts were then prepared and
size-separated by PAGE (8% gel). (A) The gel was stained with
Coomassie Brilliant Blue. (B) Proteins were blot transferred and
reacted with mouse antiphosphotyrosine mAb and then with labeled
goat anti-mouse IgG. Detection of the immunocomplex was carried
out as described in the Materials and methods. Protein extract from
tissue incubated in the absence (lane a, – cur), or in the presence (lane
b, + cur) of curdlan. In one series of experiments, the tissue was
additionally treated with 1 lgÆmL
)1
aeroplysinin (lane c, + aero). M,
protein size markers.
Ó FEBS 2004 Activation of sponge cells by (1fi3)-b-
D
-glucan (Eur. J. Biochem. 271) 1927
(1fi3)-b-
D
-glucan-binding proteins and was therefore

one side and the molecule from crabs on the other
(Fig. 3B).
cDNA encoding the fibrinogen-like protein
Sequence. One species of insert was identified – the ORF,
which spanned nucleotides 31–33 to nucleotides
877–879(stop). The full size cDNA (SDFIBI; 1.1 kb by
Northern blot analysis; see below) encoded the predicted
protein, termed FIBI_SUBDO, comprising 282 amino acid
residues (giving a calculated M
r
of 31 997). Domain searches
revealed that within the polypeptide, one fibrinogen domain
for b-andc-chains (PFAM: PF00147) exists between amino
acids 81–270. One conserved disulphide bond exists
connecting Cys225 to Cys239 and one eukaryotic secretory
signal sequence can be predicted [44] (Fig. 4A). The highest
similarity exists with vertebrate fibrinogens; therefore the
sequence was named fibrinogen-like protein.
Fibrinogens are the principal proteins of the vertebrate
clotting system and form hexamers, composed of the three
different chains: a, b and c [45]. As outlined by Spraggon
et al.[46],theb-andc-chains are homologous throughout
the complete sequence, while the a-chain comprises the
highest similarity only in the first 200 residues. Alignment
studies with the sponge and three mammalian fibrinogens
showed that the sponge fibrinogen-like protein, even though
the full-length sequence is available, shares similarity only
within the middle segment of the a-, b-andc-chains. Hence,
no further classification of the sponge protein to any of the
three vertebrate chains can be made. In the sponge

library. The 2446 nucleotide contains an ORF, from
nucleotides 100–102 to nucleotides 2242–2244(stop); the
Fig. 2. Detection of a glucan-binding protein in extracts from Suber-
ites domuncula. A protein sample was prepared from tissue that had
been incubated for 1 day with 10 lgÆmL
)1
curdlan, as described in the
Materials and methods. Extract from curdlan-treated tissue was size
separated by SDS-PAGE (lane a). After separation, the protein ex-
tracts were transferred to poly(vinylidene difluoride)-Immobilon and
incubated with biotin-labeled curdlan (cur, 5 lgÆmL
)1
)(laneb).
Alternatively, the blots were first preincubated with 10 lgÆmL
)1
lam-
inarin (lam, lane c), or 2 lgÆmL
)1
lipopolysaccharide (LPS, lane d), for
5 h, and then washed and probed with biotin-curdlan (cur), as des-
cribed in the Materials and methods.
1928 S. Perovic
´
-Ottstadt et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Fig. 3. The Suberites domuncula potential beta-1,3-glucan-binding protein (GLUBPp_SUBDO). (A) The deduced sponge sequence (GLU-
BPp_SUBDO) is aligned with the most related sequence, the b-1,3-glucan-binding protein from the black tiger shrimp Penaeus monodon
(GLUBP_PENMO, AF368168-1). Identical amino acids are shown in white on black. The positions of the two potential transmembrane regions
(TM) and the Ôglycosyl hydrolases-16Õ domain (glyco-hydr) are indicated. The segment towards which the degenerate primers were designed is
underlined by dashes. (B) Phylogenetic analysis of these two sequences with the GLUBP from the blue shrimp, Litopenaeus stylirostris
(GLUBP_LITSTY, AF473579-1), the putative Gram-negative bacteria-binding proteins from the Diptera Anopheles gambiae (ENSAN1_ANGA,

77 901 (putative EGF precursor, EGFI-PREC_SUBDO).
By comparison with the Isrec-Server [49] domain database,
three EGF domains were identified in EGFI-PREC_SUB-
DO; they span the regions amino acids 331–368 (EGF1),
amino acids 364–410 (EGF2) and amino acids 407–455
(EGF3). Furthermore, three low-density lipoprotein
1930 S. Perovic
´
-Ottstadt et al. (Eur. J. Biochem. 271) Ó FEBS 2004
receptor repeats and one serine-rich segment were predicted
(Fig. 5A). One strong transmembrane region is present
between amino acids 624 and 663. The EGF domains are
characterized by three typical intramolecular disulfide
bonds [50], which are found in the sponge domains 2 and
3 with high similarity (Fig. 5A). The EGF domains from
Fig. 5. The putative epidermal growth factor (EGF) precursor, EGFl-PREC_SUBDO, from Suberites domuncula. (A) The 714 amino acid poly-
peptide comprises three potential low-density lipoprotein receptor repeats (LDL) and also three EGF-like domains (EGF). Within the EGF
domains 1 and 2, the three characteristic intramolecular disulfide bonds (C ¼¼ C) are marked. In addition, a Ser-rich segment (<Ser>) and a
transmembrane region () exists. The essential amino acid residues involved in binding to a receptor (#) and necessary for the biological function
of the factor (§) are marked in domain 2. (B) Unrooted tree constructed from the three sponge EGF domains (EGF_SUBDO) and the next similarity
domains present in the pro-EGF precursor from human (EGF_HOMO, P01133), the bovine fibrillin 1 precursor (MP340) (FBN1_BOVIN,
P98133; amino acids 2205–2229), the transforming growth factor-a precursor (TGF-a)fromOvis aries (sheep) (TGFA_SHEEP, P98135; amino
acids 7–49), fibrillin 1 from the Cnidaria Podocoryne carnea (FBN1_PODCA, AAA91336; amino acids 443–487), rat cubilin (CUBIL_RAT,
NP_445784; amino acids 2819–2864), frog (Xenopus laevis) neurogenic locus notch protein homolog precursor (NOTC_XENLA, P21783; amino
acids 1735–1780), and the hypothetical protein ZC116.3 from Caenorhabditis elegans (ZC116_CAEEL, CAA98952; amino acids 3000–3054).
Ó FEBS 2004 Activation of sponge cells by (1fi3)-b-
D
-glucan (Eur. J. Biochem. 271) 1931
S. domuncula can be grouped to the calcium-binding EGF-
like domains (NCBI:cd00054.2, EGF_CA); calcium is

curdlan, a strong upregulation of the expression
is seen, which increases during the following 2 days (Fig. 6).
Parallel experiments were performed to determine the
expression of the SDFIBI gene (fibrinogen-like protein).
Again, in the absence of curdlan, no transcripts can be
detected by this technique, while, in the presence of the
polysaccharide, a strong increase in the expression of
SDFIBI occurs. Likewise, a strong expression pattern was
determined for the SDEGFI-PREC gene (Fig. 6). The level
of expression of the housekeeping gene, tubulin, was not
altered by the presence of curdlan.
In view of the above finding, that curdlan causes
phosphorylation of Tyr residue(s) in polypeptides of
S. domuncula, which is prevented by aeroplysinin, it seemed
necessary to determine whether this protein tyrosine kinase
inhibitor also has an effect on the pronounced increase of
expression of the three genes under study. Therefore, the
tissue was incubated with 10 lgÆmL
)1
curdlan, together
with 1 lgÆmL
)1
aeroplysinin. In this co-incubation experi-
ment it was evident that the inhibitor, aeroplysinin,
completely prevented any upregulation of the expres-
sion of SDGLUBP [(1fi3)-b-
D
-glucan-binding protein],
SDFIBI (fibrinogen) or SDEGFI-PREC (EGF precursor)
(Fig. 6).

treatment with curdlan
As a first step, antibodies were required that could identify
the (mature) EGF product and the approximate level in
tissue by Western blotting. The recombinant protein was
prepared in E. coli using the cDNA (clone SDEGFI-PREC)
spanning the three EGF domains (corresponding to amino
acids 321–713). After induction with isopropyl thio-b-
D
-
galactoside, the bacterial extract was isolated and purified
by affinity chromatography (Fig. 8A; lanes a and b). The
68 kDa recombinant fusion protein (r-EGF_SUBDO) was
used to raise PoAbs, as described in the Materials and
methods. After cleavage with enterokinase, the sponge
Fig. 6. Steady-state expression of the SDGLUBP gene [(1fi3)-b-
D
-
glucan-binding protein], the SDFIBI gene (fibrinogen-like protein) and
the SDEGFI-PREC gene (epidermal growth factor EGF precursor) in
tissue from Suberites domuncula after exposure to 10 lgÆmL
)1
curdlan.
The housekeeping gene, b-tubulin, SDTUB (accession number
AJ550806), of S. domuncula was used as an internal standard. In one
series of experiments the tissue samples were co-incubated with
1 lgÆmL
)1
of aeroplysinin (aero). RNA extraction was performed 0, 1
or 3 days after incubation with curdlan. Equal amounts were loaded
onto the gel. The RNA was size separated, blot transferred and then

recognition system which is able to discriminate between self
and symbiont. On the next level of specificity, the sponges
must be provided with pattern recognition molecules serving
as biosensors for the detection of invading pathogens
(parasitic), of commensalic (of benefit only for one partner)
or of mutualistic organisms (benefit for both partners). It
has been shown, in sponges, that some microbial secondary
metabolites, e.g. okadaic acid [4], are beneficial for the host.
However, the origin of most secondary metabolites identi-
fied in sponges is not clear. The understanding of the
pathways which result in the synthesis of these compounds
in sponges is crucial for their sustainable production/
exploitation for human benefit [53,54].
It is now established that sponges can recognize bacteria
and react to them with an increased phosphorylation of key
kinases of the MAPK pathway [7,28]. Furthermore, the first
genes encoding antibacterial proteins, such as perforin [55]
or the lectin tachylectin [6], have been identified in the
Fig. 8. Level of epidermal growth factor (EGF) in tissue after treatment
with curdlan. (A) Antibodies against the EGF precursor were prepared
from the recombinant protein expressed in Escherichia coli (A, a;
stained with Coomassie Brilliant Blue). The purified recombinant
41 kDa polypeptide (A, b; stained with Coomassie Blue) was used for
immunization. (B) The polyclonal antibody (PoAb)-EGF precursors
were used to identify the protein in extracts from sponge tissue. The
extracts were size separated and the gels stained with Coomassie
Brilliant Blue (B, a). Then, the proteins were blot transferred and
reacted with PoAb-EGF precursor. The level of cross-reacting proteins
was assessed by molecular imaging, as described in the Materials and
methods (B, b and c). In one series (B, d) the PoAb-EGF precursor was

compound of choice. Fungi are known to bind, via this
carbohydrate, to the surface of insects [58], crustaceans [59],
and also to human cells [22]. The (1fi3)-b-
D
-glucan-binding
protein was first identified in crustacean blood [60] and then
cloned from the earthworm Eisenia foetida [12]. In plants,
the (1fi3)-b-
D
-glucan binds to a b-glucan elicitor-binding
protein of different structure [61].
Following a previously described approach [12], the
(1fi3)-b-
D
-glucan-binding protein was identified biochem-
ically. The binding of the linear carbohydrate, curdlan, was
abolished by the branched molecule, laminarin, indicating
that in S. domuncula a binding protein with specificity to
(1fi3)-b-
D
-glucan exists. Subsequent cloning studies re-
vealed a cDNA coding for a putative protein which shares
the characteristic feature of other metazoan (1fi3)-b-
D
-
glucan-binding proteins. The expression of the gene was
induced after exposure to (1fi3)-b-
D
-glucan. The sponge
polypeptide shares a high similarity to the related insect/

fibrinogen-like domain. Molecules with this domain exist
from the crown taxa to echinoderms [65] or ascidians [21] in
the deuterostomian branch, and to the horseshoe crabs [20]
in the protostomian line; this domain is thought to play a
role in carbohydrate binding [66]. PCR-based identification
and subsequent cloning of a full-length cDNA coding for a
fibrinogen-like protein was successful. The putative protein
comprises only one domain, which, however, has high
similarity to the fibrinogen domain; unexpectedly, no
further domain, e.g. a collagen domain as in the ficolins
[21], or lectin as in the horseshoe crab [20], or a coiled-coil
domain in angiopoietin [67], could be detected in the sponge
protein.
The sponge fibrinogen-like protein identified shares the
highest sequence similarity with the deuterostomian fibrin-
ogens, with the sea lamprey (Petromyzon marinus) sequence
as the most closely related. The lamprey fibrinogens [68]
and, to a smaller extent, also the echinoderm related
molecule [65], are known to be the ancestors for the three
homologous polypeptide chains (a
2
b
2
c
2
) in the vertebrate
blood clotting system. The a-, b-andc-subunits of the
fibrinogens are evolutionarily closely related; they differ
especially in the N- and/or C-terminal regions [45,46]. Only
one characteristic disulfide bridge exists in the predicted

allograft inflammatory factor-1, the glutathione peroxidase,
the endothelial monocyte-activating polypeptide or the pre-
B-cell colony-enhancing factor [74], which, in higher meta-
zoan taxa, are transported in vascular systems.
Next, the level of expression of the fibrinogen-like protein
was analysed and its spatial distribution determined. It was
demonstrated that the expression is strongly upregulated
after exposure to curdlan. Even after only 1 day of
incubation, upregulation is seen. To strengthen the conclu-
sions, in situ hybridization studies were performed, which
again show that the expression of this gene is primarily seen
in the cells lining the canals – the pinacoderm.
From these two series of experiments it can be concluded
that curdlan initiates an activation circuit, from the surface
1934 S. Perovic
´
-Ottstadt et al. (Eur. J. Biochem. 271) Ó FEBS 2004
of the sponge cell to the gene expression level and back to a
further molecule potentially involved in the recognition of
the carbohydrate, the fibrinogen-like protein. In line with
previous data in human blood [66], it could be concluded
that the sponge ancestral protein, with its fibrinogen
domain, is also involved in binding to the carbohydrate.
The (1fi3)-b-
D
-glucans are known to activate a cell
metabolism/defense system, both in protostomians and in
deuterostomians. In the horseshoe crab, this carbohydrate
activates the prophenoloxidase activating system [75]. In
vertebrates, (1fi3)-b-

performed. They revealed that the expression level strongly
increased during the course of exposure to the carbohy-
drate.
The human prepro-EGF contains, like the sponge
polypeptide, a transmembrane region, which anchors the
molecule into the membrane [78]. During activation, the 53
amino acid EGF becomes released. In order to establish
whether the sponge putative EGF precursor molecule also
undergoes processing during incubation with curdlan, an
antibody was raised against the recombinant sponge
protein. This antibody was used to determine the size of
the mature peptide in the sponge tissue, before and after
exposure to curdlan. The data revealed that, almost
exclusively, a protein was identified with a size of 5 kDa.
The signal of this peptide was higher in Western blots of
extracts from curdlan-exposed sponge tissue. Based on this
finding, it is concluded that in the sponge an EGF molecule
of similar size to that found in mammals exists. Further-
more, the synthesis of this processed protein is upregulated
in the presence of curdlan.
As first proof that the (1fi3)-b-
D
-glucan-mediated chan-
ges in the expression level of the genes studied here might be
controlled by the same (or a coupled) pathway, inhibition
studies were performed. First, it was shown that at least one
protein undergoes phosphorylation at tyrosine residue(s) in
response to curdlan. Therefore, it was then studied whether
inhibition of tyrosine kinase-mediated phosphorylation
resulted also in a reduction of expression of the three genes

Acknowledgements
This work was supported by grants from the Deutsche Forschungsg-
emeinschaft (Mu
¨
/14–3), the Bundesministerium fu
¨
r Bildung und
Forschung Germany (project: Center of Excellence BIOTECmarin)
and the International Human Frontier Science Program (RG-333/
96-M).
References
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¨
ller, W.E.G. (1993) Secondary
Metabolites from Marine Sponges. Akademie gemeinnu
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