Mu¨ller, Institut fu¨r Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universita¨t, Duesbergweg 6, 55099 Mainz, Germany Fax: +49 6131 3925243 Tel: +49 6131 3925910 E-mail
Trang 1Lubomirskia baicalensis
Role of okadaic acid produced by symbiotic dinoflagellates
Werner E G Mu¨ller1,2, Sergey I Belikov2, Oxana V Kaluzhnaya1,2, Sanja Perovic´-Ottstadt1,
Ernesto Fattorusso3, Hiroshi Ushijima4, Anatoli Krasko1and Heinz C Schro¨der1
1 Institut fu¨r Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universita¨t Mainz, Germany
2 Limnological Institute of the Siberian Branch of Russian Academy of Sciences, Irkutsk, Russia
3 Dipartimento di Chimica delle Sostanze Naturali, Universita` di Napoli ‘Federico II’, Italy
4 Department of Developmental Medical Sciences, Institute of International Health, The University of Tokyo, Japan
The taxon sponges (phylum Porifera) has been
surpris-ingly successful during evolutionary development This
metazoan phylum is the only one to have survived the
severe Varanger–Marinoan ice age (605–585 million years ago) of the Neo-Proterozoic eon (1000–520 million years ago), during which the earth was almost
Keywords
dinoflagellates; heat shock protein;
Lubomirskia baicalensis; okadaic acid;
protein phosphatase
Correspondence
W E G Mu¨ller, Institut fu¨r Physiologische
Chemie, Abteilung Angewandte
Molekularbiologie, Universita¨t,
Duesbergweg 6, 55099 Mainz, Germany
Fax: +49 6131 3925243
Tel: +49 6131 3925910
E-mail: wmueller@uni-mainz.de
Website: http://www.biotecmarin.de/
Database
The sequences from Lubomirskia
baicalen-sis reported here have been deposited in
the GenBank database under the accession
numbers AM392283 (protein phosphatase
LUBAIHSP70PP1) and AM392284 (heat
shock protein-70 LUBAIHSP70)
Note
This article is dedicated to Professor Michele
Sara`, Professor of Zoology at the University
of Genova, for his outstanding contributions
to marine biology, 1926–2006
(Received 16 August 2006, revised 21
October 2006, accepted 27 October 2006)
doi:10.1111/j.1742-4658.2006.05559.x
The endemic freshwater sponge Lubomirskia baicalensis lives in Lake Bai-kal in winter (samples from March have been studied) under complete ice cover at near 0C, and in summer in open water at 17 C (September) In March, specimens show high metabolic activity as reflected by the produc-tion of gametes L baicalensis lives in symbiosis with green dinoflagellates, which are related to Gymnodinium sanguineum Here we show that these dinoflagellates produce the toxin okadaic acid (OA), which is present as a free molecule as well as in a protein-bound state In metazoans OA inhibits both protein phosphatase-2A and protein phosphatase-1 (PP1) Only cDNA corresponding to PP1 could be identified in L baicalensis and sub-sequently isolated from a L baicalensis cDNA library The deduced poly-peptide has a molecular mass of 36 802 Da and shares the characteristic domains known from other protein phosphatases As determined by west-ern blot analysis, the relative amount of PP1 is almost the same in March (under ice) and September (summer) PP1 is not inhibited by low OA con-centrations (100 nm); concon-centrations above 300 nm are required for inhibi-tion A sponge cell culture system (primmorphs) was used to show that at low temperatures (4C) expression of hsp70 is strongly induced and hsp70 synthesis is augmented after incubation with 100 nm OA to levels measured
at 17C In the enriched extract, PP1 activity at 4 C is close to that meas-ured at 17C Immunoabsorption experiments revealed that hsp70 contri-butes to the high protein phosphatase activity at 4C From these data we conclude that the toxin OA is required for the expression of hsp70 at low temperature, and therefore contributes to the cold resistance of the sponge
Abbreviations
hsp70, heat shock protein-70; OA, okadaic acid; PP1, protein phosphatase-1; PP2A, protein phosphatase-2A.
Trang 2completely covered by ice; most organisms became
extinct during this period [1] As ‘living fossils’ [2],
spon-ges represent evolutionarily the oldest extant taxon, and
thus allow insight into the genome organization of
ani-mals that lived prior to the ‘Cambrian Explosion’ At
that time, sponges existed exclusively in the marine
envi-ronment, whereas later some taxa also occupied
fresh-water biotopes (during the Cenozoic period) From
cosmopolitan freshwater species, e.g Ephydatia
fluvia-tilis, endemic species branched off in ‘old lakes’ Lake
Baikal (Siberia), for example, harbors many prominent
endemic sponges [3] Major reasons for the recent rapid
evolution of the endemic sponge fauna in some areas,
that still continues, are: (a) successful adaptation to
environmental conditions, (b) dominance of sexual
reproduction over asexual reproduction in sponges, and
(c) differences in the habitats (littoral on rocks or on
calcifying algae, e.g Chara sp.) [4]
In Lake Baikal the dominant endemic sponge species
Lubomirskia baicalensis lives in a cold environment; in
March at an ambient temperature of)0.5 C and in
Sep-tember at around 17C [5] These animals, which grow
at depths of 1–20 m, maintain constant metabolic
activit-ies with pumping rates similar to those of specactivit-ies that live
at 15–20C [6,7] Surprisingly, L baicalensis produces
gametes and embryos in March when the lake is
com-pletely covered by 1 m of ice One major source of
essen-tial organic carbon for the animals during this season is
their ecological, symbiotic relationship with
chlorophyll-containing dinoflagellates [5] It has been reported
previ-ously that these symbiotic ‘Zoochlorellae’ exist in a 3-mm
thick external layer of the sponge [8] Field observations
revealed that, in the absence of light, the dinoflagellates
die and are removed from the sponge specimens which
likewise die (W E G Mu¨ller, University of Mainz,
unpublished results) In L baicalensis these protists,
which are closely related to Gymnodinium sanguineum,
produce glycerol and transfer this intermediate
metabo-lite via an aquaporin channel into the sponge cells
(W E G Mu¨ller, University of Mainz, submitted) This
raises two questions: how do the sponges live in the cold
environment; or, more specifically, (a) how do they solve
the problem of protein folding at low temperature, and
(b) how do they overcome the barrier of the required
acti-vation energy for the enzyme-mediated catalysis?
This study mainly addresses the first question It is
known that the growth of Escherichia coli at low
tem-perature is facilitated by chaperonins [9] The question
is, which sensor in these microorganisms regulates the
expression of the respective heat shock proteins? Here
we tested the hypothesis that secondary metabolites
produced by the symbiotic⁄ commensalic organisms in
sponges contribute to the cold stress response
The dinoflagellates of the taxon Gymnodinium identi-fied in L baicalensis are related to G sanguineum (Alve-olata; Dinophyceae; Gymnodiniales; Gymnodiniaceae; Gymnodinium), which has been described as a compo-nent of harmful algal blooms and has been found to be hemolytic and ichthyotoxic [10] One toxin often produced in these algae [11] is okadaic acid (OA), a polyether C38 fatty acid, originally isolated from Hali-chondria okadaii [12] The major targets of OA in all metazoans hitherto studied are the catalytic subunits of the proteins phosphatase-1 (PP1) and protein phos-phatase-2A (PP2A) which are sensitively inhibited at nanomolar concentrations (the 50% inhibitory con-centration for PP1 is 3–150 nm and that for PP2A is 0.03–0.2 nm) [11]
Here we show that OA is present in L baicalensis, where it is synthesized by the dinoflagellates In order
to perform functional studies between OA and PP1, the cDNA coding for PP1 had to be identified in L baical-ensis This polypeptide shares high sequence similarity with mammalian PP1 Antibodies against PP1 allowed its assessment during temperature-dependent expression
in vitro (cell culture) and in vivo (animals) At lower concentrations (< 100 nm), OA has no effect on the level and activity of the protein phosphatase(s) but induces the expression of heat shock protein-70 (hsp70) From earlier studies it is known that OA can trigger the expression of heat shock proteins in tissues [13] We applied the in vivo sponge cell culture system, the primmorphs [2], to demonstrate that in primmorphs
at 4C, OA upregulates both the expression of hsp70 transcripts and the amount of hsp70 protein to levels found at the ambient temperature of 17C Subsequent depletion experiments with antibodies against hsp70 showed that functionally active chaperon⁄ hsp70 mole-cules are required for full protein phosphatase activity
at low temperature From the data we conclude that at lower concentrations (< 100 nm) the secondary meta-bolite OA mediates⁄ controls in L baicalensis the cold stress defense, whereas higher concentrations are required to inhibit the protein phosphatase(s) It has been established that sponges, like Suberites domuncula [14] or L baicalensis, which contain symbiotic micro-organisms, display a stronger ‘resistance’ to OA; with regard to S domuncula only concentrations > 300 nm have a significant effect on protein synthesis
Results
L baicalensis specimens in winter and summer Animals were collected during September 2005 and March 2006 (Fig 1A,B) During these seasons, the
Trang 3animals have a bright green color, which is due to the
high abundance of dinoflagellates (Fig 1C,D) related
to the taxon G sanguineum (Alveolata; W E G Mu¨ller,
University of Mainz, submitted) Interestingly, during
winter the sponges form sexual propagation bodies,
reflecting an active metabolism Figure 1E shows one
spermatogenic cyst
Presence of OA in L baicalensis
The OA concentration in L baicalensis was determined
using HPLC⁄ MS analysis to be 83 ± 9 ngÆg)1 wet
weight (100 nm; March), whereas tissue from
speci-mens collected in September had a lower OA content
of 57 ± 6 ngÆg)1(70 nm) These values were confirmed
by competitive ELISA giving concentrations of
75 ngÆg)1(March) and 45 ngÆg)1(September)
Protein-coupled OA in L baicalensis
Protein extracts were prepared from specimen tissue
collected in March This preparation was subjected to
SDS⁄ PAGE (10% gel; Fig 2, lane a) After transfer,
blots were incubated with anti-OA sera (pAb-OA) This
revealed one prominent protein band of 14 kDa (Fig 2,
lane b) The specificity of the reaction was proven using
antibodies that had been adsorbed with OA bound to
the FID-33 peptide; under these conditions the
immu-noreaction of the 14 kDa band was strongly suppressed
(Fig 2, lane c) In contrast, the signal at 14 kDa was
of the same strength when membranes were incubated
with pAb-OA, which had been pretreated with FID-33
prior to use in the western blots (not shown)
Identification dinoflagellates in tissue using anti-OA sera
Slices of sponge tissue were prepared and reacted with pAb-OA The antibodies stained the dinoflagellates very brightly, whereas the sponge cells did not react
A
B
F
C
D
E
I
Fig 1 L baicalensis specimens during late summer (September) (A)
and the ice cover season (March) (B) In both seasons sponges
con-tain associated dinoflagellates (taxon Gymnodinium) Cross-sections
were prepared and examined using transmission electron
microsco-py (C, D) Sections through a branch from a September specimen
show the abundantly present dinoflagellates (d) that are assembled
at the rim of the green branches (E) Frequently the specimens
dur-ing the March season contain spermatogenic cysts (sc)
Identifica-tion of OA-producing dinoflagellates in L baicalensis (F–K) (F, I)
Slices were prepared from tissue of L baicalensis and the cells were
visualized Nomarsky interference contrast optics; dinoflagellates
(d) as well as sponge cells (s) are marked (G) In one series, the
slices were reacted with pAb-OA and then with Cy3-conjugated goat
anti-(rabbit IgG) and finally inspected by immunofluorescence
(wavelength ¼ 546 nm) (H) Autofluorescence of the chlorophyll in
the dinoflagellates was detected at a wavelength of 490 nm (J) In
parallel, the slices were reacted with pAb-OA, which had been
pretreated with OA, coupled to the FID-33 oligopeptide and then
with the labeled secondary antibodies (K) The same area was also
analyzed with a wave-length of 490 nm Scale bar ¼10 lm.
Trang 4(Fig 1G); using Nomarsky interference contrast optics
the granule-containing dinoflagellates could be
identi-fied (Fig 1F) As further evidence for the localization
of the dinoflagellates, slices were illuminated with
green fluorescent light (490 nm) to identify
dinoflagel-lates based on the autofluorescence of their chlorophyll
(Fig 1H) Again, the dinoflagellates were highlighted
in areas positive for pAb-OA In a parallel series,
sec-tions were treated with adsorbed antibodies against
OA; this preparation showed a slight signal only very
occasionally (Fig 1J) Dinoflagellates could again be
visualized by their autofluorescence (Fig 1K)
L baicalensis PP1 catalytic subunit
Complete cDNA encoding the L baicalensis PP1
pro-tein (LBPP1) was obtained from a cDNA library using
a degenerate primer against the Ser⁄ Thr-specific
pro-tein phosphatase signature of mammalian propro-tein
phosphatases The ORF between nucleotides 60–62
and 1057–1059(stop) codes for a 319 amino acid
polypeptide (PP1_LUBAI) with a predicted size of
36 802 Da (Fig 3A); the sequence was termed
PP1_LUBAI Like the related mammalian sequences,
the sponge protein comprises a characteristic Ser⁄
Thr-specific protein phosphatase signature (amino acids
121 and 126) and the conserved
matallophosphoest-erase (amino acids 57 and 252) The calcineurin-like
phosphoesterase (amino acids 57 and 252) overlaps
with the latter region Similarity between the sponge
molecule and other metazoan PP1 sequences is high;
the sponge PP1 shares 288 similar and 271 identical
amino acids with human PP1 (length: 323 amino acids), known to bind to OA (Fig 3A) The overall similarity⁄ homology to metazoan sequences is
80% ⁄ 70% For the alignment in Fig 3A, the human protein with the highest similarity score was used (‘Expect value [E]’ 2e-162) [15]; this phosphatase is known to function as PP1 (catalytic subunit, gamma isoforms)
A phylogenetic tree was computed after alignment
of the metazoan sequences with yeast and plant-related PP1 (Fig 3B) The tree was rooted with the highest similar phosphatases from Arabidopsis thaliana (pro-tein phosphatase-type 1; NP_181514.1) and Saccharo-myces cerevisiae (type 1 Ser⁄ Thr protein phosphatase; NP_011059.1) Among the metazoan proteins, the sponge phosphatase clusters together with that of Dro-sophila melanogaster (Ser⁄ Thr protein phosphatase; CAA49594.1), while the Caenorhabditis elegans protein (even-like phosphatases family member (NP_001022616.1) forms a separate branch together with the human sequence Both branches are separated only with low significance
Relative PP1 content Because of the high sequence similarity between L bai-calensis PP1 and the corresponding mammalian phos-phatases it was possible to use a commercial antibody for the western blots Extracts were size-separated by SDS⁄ PAGE and either stained with Coomassie Brilli-ant Blue (Fig 3C, lane a) or the proteins were trans-ferred to poly(vinylidene difluoride) membranes and reacted with antibodies against PP1, as described in Experimental procedures Extracts from animals obtained in both March (Fig 3, lane b) and September (Fig 3, lane c) show a strong signal at 37 kDa, cor-responding to the size of sponge PP1 In contrast, if the blots were reacted with adsorbed antibodies, the signals were strongly reduced (lanes d and e)
Phosphatase activity in the extract Extracts were prepared from sponge tissue specimens collected in March or September and subjected to the protein phosphatase assay described in Experimental procedures The specific enzyme activities in tissues from winter and summer animals were almost identical (22.6 ± 4.7 versus 23.4 ± 4.2 nmolÆmin)1Æmg)1) If OA was added, the reactions in the two series of experi-ments were inhibited dose-dependently; at 100 nm the activity differed from that seen in the controls (March, 17.3 ± 3.9 nmolÆmin)1Æmg)1; September, 19.2 ± 4.3 nmolÆmin)1Æmg)1) However, at 300 nm the activity
Fig 2 Identification of protein-coupled OA in L baicalensis.
Extracts were prepared and the protein size separated by
SDS ⁄ PAGE (10% gel) Lane a, the gel was stained with Coomassie
Brilliant Blue The proteins were blot transferred and the filters were
either incubated with anti-OA sera (lane b) or with the antibody
pre-paration, which had been preincubated with FID-33-OA which had
been adsorbed with FID-33-OA (lane c) M, marker proteins.
Trang 5decreased to 80% (March, 3.7 ± 1.9 nmolÆmin)1Æ
mg)1; September, 5.2 ± 1.6 nmolÆmin)1Æmg)1)
Expression level of hsp70 in animals and
primmorphs
The semiquantitative steady-state level of hsp70
tran-scripts in animals was in the same range, regardless of
whether the RNA had been isolated from specimens
collected in March or in September For these
Nor-thern blot studies the EST probe for hsp70 from
L baicalensis(LUBAIHSP70) was used (Fig 4A)
In order to assess the expression level under
con-trolled laboratory conditions, in vivo primmorphs were
incubated for 24 h at 4 and 17C Primmorphs were
incubated in the dark to suppress the metabolic
activ-ity of the remaining symbiotic algae Setting the
expression level at 4C to onefold, the amount of
hsp70 transcripts in the primmorph cells incubated at
17C was much higher (10-fold; Fig 4A) However,
if primmorphs were incubated at 4C together with
100 nm OA the amount of hsp70 transcripts was the same as that measured for cultures maintained at
17C The toxin had no strong effect on hsp70 expres-sion in cells at 17C (Fig 4A) In controls, a-tubulin expression was determined using the L baicalensis probe LUBAITUB in a parallel Northern blotting experiment Almost identical signal intensities were seen, confirming that the same amount of RNA was loaded onto the gels From these results, we conclude that OA induces hsp70 expression at the lower incuba-tion temperature
To support these studies, comparative Northern and western blot experiments were performed using hsp70 (LUBAIHSP70) or antibodies (mAb-HSP70) as the respective probes (Fig 4B) Again, the Northern blot
A
Fig 3 PP1 from L baicalensis (A, B) The PP1 sequence from L baicalensis (A) Alignment of the sponge PP1 protein (PP1_LUBAI) with the human PP1 which binds to OA (PP1_HUMAN; accession number 1JK7_A) Amino acids, identical in both sequences, are in inverted type and those similar in both sequences are shaded The characteristic Ser ⁄ Thr-specific protein phosphatase signature (S ⁄ Tp) and the conserved metallophosphoesterase (MetPhoEsterase) regions are marked (B) The phylogenetic tree is constructed from the two abovementioned sequences as well as the PP1 from D melanogaster (PP1_DROME; CAA49594.1), from C elegans (seven-like phosphatases family mem-ber) (PP1 CAEEL; NP_001022616.1), from S cerevisiae (PP1_YEAST; NP_011059.1) as well as from A thaliana (PP1_ARATH; NP_181514.1), which was used as outgroup to root the tree After alignment the tree was built Scale bar indicates an evolutionary distance
of 0.1 amino acid substitutions per position in the sequence (C) Identification of PP1 in tissue from L baicalensis In all lanes 10 lg of pro-tein were separated Lane a, the separated propro-teins were identified with Coomassie Brilliant Blue Western blot experiments: lanes b and c, the membranes were reacted with antibodies against PP1 (PcAb-PP1) and then with labelled secondary antibodies to visualize the immuno-complexes with the BM chemoluminescence substrate kit Samples from March (lane b) and September (lane c) were analyzed In parallel, membranes with the extracts were treated with adsorbed PcAb-PP1 (lanes d and e) The size markers are given.
Trang 6studies showed low expression of hsp70 in primmorphs
incubated at 4C or in the absence of OA, in
compar-ison with those incubated with OA or at 17C
(Fig 4B,a) Steady-state expression of the a-tubulin
gene is shown using the same amount of RNA for
ana-lysis The data from western blot experiments showed a
similar expression pattern; low levels of hsp70 in
cul-tures incubated at 4C and without OA, in comparison
with those incubated with the toxin and at higher
tem-perature (Fig 4B,b) From these data, we conclude that
the level of hsp70 is controlled in primmorphs at both
a transcriptional and translation level
Effect of depletion of hsp70 from extracts on the
activity of protein phosphatase(s)
An immunodepletion study was performed as described
in Experimental procedures Extracts were prepared
from animals collected in September and incubated at 4
or 17C Unexpectedly, enzyme activity at 4 C was
only 20% lower (18.1 ± 4.2 nmolÆmin)1Æmg)1) than that measured at 17C (22.7 ± 5.1 nmolÆmin)1Æmg)1) However, after incubation of the extracts for 60 min with antibodies against hsp70 the activity of the protein phosphatase was reduced at 4C to 5.3 ± 2.9 nmolÆ min)1Æmg)1, whereas the antibodies had no effect on activity at 17C (Fig 5) The adsorbed mAb-HSP70
A
b
Fig 4 Expression of heat shock protein hsp70 transcripts in ani-mals and primmorphs (A) Northern blot analysis RNA was extrac-ted both from animals, collecextrac-ted in March or September, and from primmorphs cultivated in vitro As indicated, the primmorphs were cultivated either 4 C or at 17 C for 24 h in the absence (–) or presence of 100 n M of OA (+) Then, total RNA was isolated and fractionated by electrophoresis, transferred to nylon membrane, and hybridized with the respective labeled probes; hsp70 (LUBAIHSP70) or a-tubulin (LUBAITUB) 2 lg of total RNA were loaded into each slot The relative degree of expression was corre-lated with that seen for the minimal expression in primmorphs at
4 C (set to onefold) (B) Comparison of the level of hsp70 tran-scripts and hsp70 protein in primmorphs, incubated at 4 or 17 C
in the absence (–) or presence of OA (+) As marked, the primmorphs were incubated at these two temperatures for 24 h and - ⁄ +OA Then extracts were prepared for Northern blotting (RNA) or western blotting (protein) (a) Northern blot: after size fraction and transfer the filter was hybridized with the hsp70 probe: N1, incubation at 4 C in the absence of OA; N2, at 4 C in the presence of OA; N3, incubation at 17 C in the absence of OA; N4, incubation at 17 C in the presence of OA In parallel, a filter was hybridized with a a-tubulin probe (b) From the same samples the proteins were extracted and subjected to western blot analysis Samples from cultures incubated at 4 C in the absence (lane W1) or presence of OA (W2) or at 17 C without (lane W3) or with OA (lane W3) are loaded onto the gel, and after separation and transfer probed with the antibodies mAb-HSP70 The relative expression levels, correlated to the values assessed for cultures at 4 C and without OA (set to onefold), are given.
Fig 5 Effect of antibodies against hsp70 on the activity of the pro-tein phosphatase in extracts from animals Extracts were prepared from animals, collected in summer and tested for protein phospha-tase activity at 4 or 17 C, as described in Experimental proce-dures Where indicated the samples were pretreated either with mAb-HSP70 or with adsorbed mAb-HSP70.
Trang 7preparation did not show a significant effect on the
enzyme activities From these results we conclude
that: (a) hsp70 proteins, which are supposedly
func-tionally active, are present in sponge extracts together
with the enzyme; and (b) hsp70 is required for the
full enzyme activity during incubation at lower
temperature
Discussion
OA is a secondary metabolite produced by free-living
microalgae, primarily by Prorocentrum lima [16] Other
dinoflagellates, e.g Gymnodinium sp., are also
consid-ered to be producers [17] Secondary metabolites are
surely not without metabolic function for the producer
or the host, because then they would have been
elimin-ated during evolution They have, however, no direct
role in the growth of the producing organism and are
considered not to play a key role in the maintenance
of cellular function but in defense [18] It remains
unexplained, however, why some secondary
metabo-lites, like OA, are synthesized not only by one taxon,
but by a whole range of microorganisms These
dinoflagellates are harbored in a series of hosts, e.g
mussels [11] or sponges such as H okadaii [12],
S domuncula [14] and Geodia cydonium (W E G
Mu¨ller, University of Mainz, unpublished results), or,
as shown here, in L baicalensis This latter finding is
surprising, because L baicalensis is a freshwater
sponge, in contrast to the others which are marine
ani-mals These findings suggest that OA has a crucial role
in the maintenance of a symbiotic relationship between
algae and host With S domuncula it has been shown
that OA augments the concentration-dependent
immune defense system against bacteria [14], and as
described recently, kills symbiotic⁄ parasitic annelids
[19] The dinoflagellates present in L baicalensis are
related to G sanguineum, a species that has been found
worldwide, especially in coastal waters [20]
We have shown that the dinoflagellates (G
sangui-neum) produce OA in L baicalensis For identification,
we applied antibodies raised against OA, which have
been previously qualified as specific for this secondary
metabolite [14,19] In cross-sections though L
baical-ensisthese antibodies reacted specifically with the
dino-flagellates; their signals could be suppressed by
adsorption with free OA Analytical measurements
revealed that the concentration of OA in L baicalensis
is 50 ngÆg)1 of tissue (70 nm), a level comparable
with that found in other sponges [14] In addition,
western blot analysis has shown that, like in extracts
prepared from S domuncula and L baicalensis, OA
exists in a covalent linkage with a protein of 14 kDa
This finding, first described in S domuncula [19], can
be explained as a depot⁄ storage form of the free OA
It is not known whether OA is released from the sponge into the surrounding aqueous habitat Previous studies with S domuncula have shown that in this sponge OA accumulates in the epithelial layers of the aquiferous system within the animals, suggesting that
OA is involved in defense against microbial invaders [21] Because L baicalensis ingests⁄ feeds on microor-ganisms and plankton [7] it is very likely that OA is accumulated in the aquiferous canal system and acts
as a protecting metabolite
Based on existing data, it is increasingly evident that
OA in the symbiotic bacteria also affects the primary cell metabolism of the host Previously, the main focus
of research has been on the effect of OA on attacking
or commensalic organisms, via inhibition of enzymes (protein phosphatases) [11] In view of the data, the corresponding cDNA for one of these enzymes (PP1) needed to be identified first cDNA coding for PP1 was completely isolated and the corresponding protein deduced This polypeptide contains all the characteris-tic domains of other enzymes in this group, e.g the characteristic Ser⁄ Thr-specific protein phosphatase signature and the conserved metallophosphoesterase region Based on the high sequence similarity between the sponge protein phosphatase and mammalian enzymes an antibody against the latter could be used here Signals obtained by western blot analysis,
37 kDa, matched the expected size Enzyme activity in the prepared extract was 20 nmol inorganic phos-phate released per min and mg of protein in tissue extracts Inhibition studies with OA were performed which revealed that the toxin blocks the enzyme(s) significantly at OA concentrations > 300 nm; at
100 nm no inhibition was seen The sensitivity of the enzyme to OA was in the range published previously [11,14] In the plant Medicago sativa it could be shown that at low temperature hyperphosphorylation of proteins occurs and this is the result of inhibition of protein phosphatase(s) [22] It has been established that OA is a more sensitive inhibitor of PP2 than of PP1 [11] Therefore, we screened our EST database from L baicalensis, which comprises over 4000 sequences, and also performed extensive screening studies using degenerate primers, designed against the conserved regions within the PP2 nucleotide sequence,
to identify PP2 transcripts in the cDNA library from
L baicalensis However, these attempts were without success Therefore, we focused our studies at the protein and cellular level on PP1 only Nevertheless, the data presented here do not exclude that PP2 is involved in thermoregulation in L baicalensis
Trang 8As outlined earlier, L baicalensis lives in a biotope
with ambient temperatures between )0.5 C (March)
and 17C (August–September) To date, no data have
been available that could help in understanding which
protection system allows these animals to maintain
high metabolic activity during these extreme situations
The first series of experiments now demonstrates that
the relative level of enzyme(s) (protein phosphatase)
in the animals is the same in March or in September
Also, the sensitivities of the enzymes towards OA are
very similar Based on these results, we conclude that
OA has an inhibitory effect in the animals during both
seasons at concentrations > 300 nm
In multicellular organisms one major protection
sys-tem against sys-temperature stress is provided by the heat
shock proteins [23], with hsp70 being the most
thor-oughly studied example In contrast to the related
con-stitutively expressed cognate hsc70, which changes its
level only slightly upon differing stresses, hsp70 is
strongly upregulated upon exposure to stress [24] For
the marine sponges S domuncula or G cydonium we
were able to show that at both the gene-expression level
[25] and the protein level [26] expression of hsp70
increa-ses strongly after temperature change and also after
exposure to xenobiotics [27] The induction of the gene
with respect to the stressors proceeds with the same
kin-etics in the sponge and in fish [28] Focusing on Lake
Baikal sponges, hsp70 proved to be a suitable biomarker
for xenobiotics and temperature stress [29]
Few publications are available that describe the
effect of OA on the expression of heat shock proteins
[13,30] These authors demonstrated that injection of
300 ng of OA into 250 g rats resulted in notable
expression of hsp70⁄ 72 after an incubation of 72 h
More importantly, Joyeux et al [30] showed that OA
treatment results in a potentiation of hsp72 mRNA
expression In this study, we tested whether OA
chan-ges the level of hsp70 expression at both the gene
expression level and the protein level For these
stud-ies, the primmorph system was used, which allowed
the study of this toxin under controlled laboratory
conditions In earlier studies, primmorphs have proved
to be suitable for measuring these effects [31]
A hsp70 cDNA probe was used to measure
semiquan-titatively the steady-state expression hsp70 in animals
collected in March and September; there were no
signifi-cant changes As outlined, the sponges contain OA in
March and September However, if primmorphs, kept in
the dark to suppress the photosynthetic activity of
the algae, were cultivated at 4C the level of hsp70
transcripts was very low compared with primmorphs
cultivated at 17C, or animals in the biotope,
irrespect-ive of whether they were collected at )0.5 C or at
12C If primmorphs were cultivated at 4 C together with 100 nm OA the hsp70 level reached values seen in animals or primmorphs incubated at 17C Interest-ingly, the level of hsp70 protein also followed this pat-tern From these results we conclude that OA causes, at both the gene- and the protein level, increased expres-sion of this heat shock protein in primmorphs at 4 C Interestingly, expression of a-tubulin in primmorphs is low during incubation at 4C, and is upregulated in the presence of OA or at higher temperatures This suggests that OA plays an inducer role for hsp70 and tubulin in primmorphs under cold stress conditions
A set of immunodepletion experiments was per-formed Extracts from animals collected during the summer were prepared that, according to the above-mentioned data, contained greater amounts of hsp70 protein They were subjected to protein phosphatase activity determination in the presence or absence of antibodies raised against hsp70 Hsp70 is known to bind to target protein(s) in the presence of ATP [23,32,33], which was therefore added If the extracts were assayed for protein phosphatase(s) activity it was found that at lower temperatures (4C) enzyme activ-ity was close to that seen after incubation at 17 C However, if antibodies were added to the mixture and incubation was performed at 4C the activity was almost completely abolished At 17C the antibodies had no effect on the high expression level of hsp70 These results strongly suggest that native hsp70 binds
at lower temperature to the enzyme(s) and restored the activity to values seen at higher temperature
OA is a toxin present in dinoflagellates that coexist with marine animals⁄ sponges and, as described here, in freshwater sponges Quantitative determinations showed that the level of this toxin in the freshwater sponge L baicalensis was as high as in the marine sponge S domuncula As reported here, in these fresh-water animals OA is involved in processes that result
in a high steady-state expression of the chaperon– hsp70 system In addition, the data suggest that the hsp system contributes to the cold thermotolerance seen in L baicalensis, because the secondary metabo-lite OA functions as an inducer for hsp70 Therefore,
it can be postulated that OA contributes markedly to the survival strategies of these animals during unfavo-rable environmental conditions This view is outlined
in Fig 6 In animals, collected during winter and summer the level of hsp70 is high If extracts from these specimens were used for immunodepletion experiments the ‘activating’ effect of hsp70 on protein phosphatase activity could be demonstrated in extracts prepared from animals collected during the cold season This suggests that at low temperature
Trang 9ATP-dependent hsp70 molecules are required for the
pro-motion of folding, transport and⁄ or assembly of target
proteins participating in the primary metabolism or,
perhaps also, in the composition of the fluid
mem-branes Energetically, the dinoflagellates supply their
host with primary metabolites via their photosynthetic
activity, glycerol being the major compound We
recently identified that the dinoflagellates synthesize
glycerol which is then taken up by cells of L baicalensis
through the aquaporin channel (W E G Mu¨ller,
manuscript submitted) In conclusion, our data suggest
that OA causes induction of hsp70 at low⁄ cold
tem-perature stress; in turn, hsp70 contributes to the proper
activity of the protein phosphatase at low temperature
Experimental procedures
Chemicals and enzymes
Restriction enzymes, SNAP ‘Total RNA Isolation Kit’,
Superscript II and reagents for RACE procedure were
pur-chased from Invitrogen (Carlsbad, CA), TriplEx2 vector
from BD (Palo Alto, CA), TRIzol Reagent from
Gib-coBRL (Grand Island, NY), Hybond-N+nylon membrane
from Amersham (Little Chalfont, UK), mAb against hsp70
(bovine; H 5147) was obtained from Sigma (St Louis, MO),
polyclonal antibodies raised against PP1 were from Santa Cruz Biotechnology (Santa Cruz, CA), CDP from Roche (Mannheim; Germany), Technovit 8100 from Heraeus Kul-zer (Wehrheim, Germany), Sephadex G-20 from Pharmacia (Uppsala, Sweden), Lake Baikal water was obtained from
‘Lake’ Comp (Irkutsk; Russia), and the protein phospha-tase assay kit from Upstate Biotechnology (Lake Placid, NY) Okadaic acid was purchased from Alexis Biochemi-cals (Gru¨nberg; Germany), the toxin was dissolved in dimethylsulfoxide
Sponges, cells/primmorphs and cDNA libraries Specimens of L baicalensis (Porifera, Demospongiae, Haplo-sclerida) were collected in Lake Baikal (Russia) near the village Bolshiye Koty (5158 N, 10521¢E) from depths between 7 and 12 m during September 2005 and March 2006 Primmorphs were prepared by immerging sponge tissue into natural Lake Baikal water, supplemented with 50 mm EDTA Lake Baikal water was used to guarantee a suitable mineral composition [34] After gentle squeezing and subse-quent shaking for 30 min at 16C on a rotatory shaker, the solution was discarded and new Baikal water⁄ EDTA was added After 40 min the supernatant was collected and filtered through a 40-lm mesh nylon net; shaking in Baikal water⁄ EDTA and filtration were repeated once Single cells
Fig 6 Proposed function of OA in L baicalensis The toxin OA is produced by the dinoflagellates from the taxon Gymnodinium In both win-ter and summer the dinoflagellates produce primary metabolites via their photosynthetic activity; the metabolite is taken up by the sponge cells Using the primmorph system it could be shown that the level of hsp70 is low at the lower temperature (in primmorphs at 4 C) How-ever, the abundance of hsp70 increases in response to low concentrations of OA (100 n M ) to levels which are seen in primmorphs incubated
at a higher temperature (17 C) or in animals collected during March and September It is postulated that a sufficiently high level of hsp70 is one prerequisite for the promotion of folding, transport and⁄ or assembly of target proteins participating in the primary metabolism or in the composition of the fluid membranes during unfavorable environmental conditions (in nature, at )0.5 C) Only at higher concentrations (> 300 n M ) does OA inhibit protein phosphatase(s).
Trang 10were harvested by centrifugation (500 g for 5 min,
Eppen-dorf centrifuge 5702 with rotor A-8-17) and washed once
The cells of this pellet were resuspended in Baikal water,
supplemented with 5 lgÆmL)1 penicillin and 100 lgÆmL)1
streptomycin A cell suspension of 107 cells was added to
6 mL (final volume) of medium in 10 mL flasks (Nuclon
surface; #136196; Nunc Wiesbaden, Germany) Primmorphs
were obtained from these single cells; they reached sizes of
3–7 mm after two days in the dark The primmorphs were
incubated at 4 or 17C; during the summer season the
ambient water environment of L baicalensis can reach a
temperature of up 22C [35]
The cDNA library from L baicalensis was prepared in
TriplEx2vector
Preparation of antibodies against OA
Polyclonal antibodies against OA (pAb-OA) were raised in
female rabbits (White New Zealand) as described previously
[14] The antigen (OA) was covalently coupled via its
C-ter-minus to the FID-33 oligopeptide (sequence:
NH2-FIDA-VWKCVTPFIDAVWKTKFICVTPFIDAVWK-COOH),
using EDC
(1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide; Sigma) as described previously [14] This OA
conju-gate was injected at 4-week intervals into the animals; after
three boosts serum was collected and the antibodies were
prepared [36] The titer of the antibodies was determined to
be 1 : 2000 Where indicated the antibodies (pAb-OA;
0.3 mL of undiluted serum) were adsorbed with FID-33
coupled to OA (1 mg) for 30 min at room temperature
The studies with animals (antibody production) have been
approved by the respective state authorities
Histological analysis
Tissue was fixed in paraformaldehyde, embedded in
Tech-novit 8100 and sectioned [37] The 4-lm thick slices were
incubated with pAb-OA (1 : 250 dilution) overnight Then
the slides were treated with Cy3-conjugated goat
anti-(rab-bit IgG) for 2 h Subsequently, the sections were inspected
by immunofluorescence with an Olympus AHBT3
micro-scope, using an excitation light wavelength of 546 nm In
addition, the slices were illuminated with light of a
wave-length of 490 nm, which detects the autofluorescence of
chlorophyll in the dinoflagellates In parallel, the slices were
inspected directly using Nomarsky interference contrast
optics In controls, the pAb-OA (10 lg) were preincubated
with OA, coupled to the FID-33 oligopeptide (10 lg)
Competitive ELISA
The ELISA was performed similar to the procedure
des-cribed earlier [14] OA, coupled to the FID-33 oligopeptide
was linked to 96-well plates (Covalink-primary amine;
Nunc) [14] After three washing steps with NaCl⁄ Pi (containing 0.05% Tween-20) the plates were blocked prior
to use with 3% of fatty acids-free bovine serum albumin in NaCl⁄ Pi Then 100 lL of pAb-OA were added at different dilutions to each well for 90 min Subsequently, the immu-nocomplexes were visualized using secondary antibodies, coupled to horseradish peroxidase (1 : 1000; Sigma, Dei-senhofen, Germany) under application of o-phenylenedi-amine as substrate The plates were read at 492 nm In the competitive ELISA procedure, OA was added at different concentrations (1 ngÆmL)1 to 1 lgÆmL)1 NaCl⁄ Pi) from a stock solution of 1 mgÆmL)1, dissolved in methanol Within the range of 10 ngÆmL)1 to 1 lgÆmL)1 the change of the absorbance was linear (logarithmic plot) For the determin-ation in the tissue of the sponge, extracts were prepared from the tissue with 80% methanol The values for the absorbance were extrapolated using the calibration curve obtained with the free toxin
Identification of OA-bound protein Extracts were prepared from tissue samples; they were homogenized in lysis buffer (1· Tris-buffered saline,
pH 7.5, 1 mm EDTA, 10 mm NaF, 0.1 lm aprotinin, 1 mm sodium orthovanadate) Total cell extracts (10 lgÆlane)1) were subjected to electrophoresis in 10% polyacrylamide gels containing 0.1% SDS⁄ PAGE as described previously [37] The gels were stained with Coomassie Brilliant Blue Subsequently, western-blotting experiments were performed after transfer of the proteins onto poly(vinylidene difluo-ride) membranes (Millipore-Roth) using pAb-OA (1 : 300 dilution) After incubation for 3 h, the blots were incubated with goat anti-(rabbit IgG), peroxidase-coupled (1 : 5000 dilution; New England Biolabs) Detection of the immuno-complex was carried out using the BM Chemoluminescence Blotting Substrate kit Where indicated the pAb-OA had been adsorbed with OA coupled to the FID-33 oligo-peptide
Relative PP1 content (western blotting) The relative content of PP1 in the extracts was determined
by western blotting First, the tissue extracts (see previous section) were size separated by electrophoresis (SDS⁄ PAGE⁄ 12% polyacrylamide); samples of 10 lg protein extract were loaded onto the gels In addition, the proteins were blot transferred and reacted with polyclonal antibodies raised against PP1 (PcAb-PP1; 1 : 2000 dilution) After incu-bation for 3 h, blots were incubated with peroxidase-coupled goat anti-(rabbit IgG); and the immunocomplexes were visualized using the BM Chemoluminescence Blotting Substrate kit In control experiments 10 lL of undiluted PcAb-PP1 was adsorbed with 10 lL of cell extract (1 mgÆmL)1; 30 min; 4C) prior to the use onto the blots