| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 278, Issue 35, 32810-32817, August 29, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
||
From the
Institut für Physiologische Chemie,
Abteilung Angewandte Molekularbiologie, Universität Mainz, Duesbergweg 6,
D-55099 Mainz, Germany, the Institute of
International Health, Graduate School of Medicine, University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and the
¶Department of Molecular Genetics, Institute
Rudjer Boskovic, HR-10000 Zagreb, Croatia
Received for publication, April 18, 2003 , and in revised form, June 9, 2004.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Sponges have developed several strategies to cope with these threats. First, they possess an efficient chemical defense system to inhibit bacterial growth (see Ref. 4). Triggered by ecological constraints, Porifera developed a large variety of biologically highly active compounds (5) that were shaped during the last 1 billion years for optimal specificity and activity. Second, sponges are provided with efficient humoral and cellular defense/immune mechanisms that share, at the sequence level, surprisingly high similarity with the immune molecules found in humans (6). Third, a mechanism in these animals eliminates microorganisms by engulfing them through phagocytosis (reviewed in Ref. 7). An early illustration and functional analysis of phagocytosis in sponges were given by Metchnikoff (8). Molecular data indicate that receptors present on the cell surface might have the capacity to discriminate between self-self and non-self (6); examples are receptors that comprise both SRCR (scavenger receptor comprising cysteine-rich) group A domains (9) and/or SRCR group B domains (10). In addition, sponges are very rich in lysosomal enzymes, e.g. cathepsins (11).
Lately, our research has focused on those molecules that are involved in the recognition of bacteria and in the intracellular signaling pathway(s) activated after interaction of sponge cells with microorganisms. The search for the kinase pathways that are activated in response to bacterial infection was already successful. It has been demonstrated that, after bacterial infections or after addition of the model molecule for bacterial infection (lipopolysaccharide (LPS)1), the phosphorylation of the two MAPKs p38 and JNK strongly increases (12). Hence, it can be deduced that sponges react upon ligation to LPS/bacteria with activation of p38 and JNK MAPKs.
One goal of this study was to identify the potential receptor for bacteria on the surface of the sponge cells. The model system for bacterial infection in sponge, LPS-induced activation of the p38 kinase in primmorphs, was used. For this study, the demosponge Suberites domuncula was selected because a cell culture system has been established in this species (13, 14). Primmorphs are a special form of three-dimensional cell aggregates that contain both proliferating and differentiating cells. The spherical aggregates comprise, in the center, non-differentiated archaeocytes and, on the surface, in the pinacoderm, a layer of differentiated cells, the pinacocytes. Based on the functional analogy to the host defense lectins from the horseshoe crab Tachypleus tridentatus, we screened for a related molecule in S. domuncula. In T. tridentatus, a series of non-self-recognizing lectins have been described that were termed tachylectins or Tachypleus plasma lectins (see Ref. 15). A related molecule was cloned from the sponge cDNA library. The deduced protein shares high sequence similarity with the crab polypeptides, especially with the galactose-binding protein (GBP) from T. tridentatus (16); the S. domuncula molecule was named LEC_SUBDO. Based on the functional studies described here, we proposed that the S. domuncula lectin is also involved in non-self recognition.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Sponges and Primmorphs—Live specimens of S. domuncula (Porifera, Demospongiae, Hadromerida) were collected near Rovinj, Croatia. The sponges were kept in Mainz, Germany, for >4 months prior to the experiments. Where indicated, specimens were analyzed immediately after collection.
The procedure for the formation of primmorphs from single cells was applied as described previously (13, 14). Starting from single cells, primmorphs of 3–7 mm are formed after 5 days. For the experiments, 7-day-old primmorphs were used. They were cultivated in natural seawater supplemented with 0.2% RPMI 1640 medium.
Competition Experiments with LPS/D-GlcNAc—The primmorphs were treated with 10 µg/ml LPS for 12 h in the absence or presence of monosaccharides. After incubation, the primmorphs were immediately processed for Western blot analysis.
Samples were homogenized in lysis buffer (1x Tris-buffered saline (pH 7.5), 1 mM EDTA, 1% Nonidet P-40, 10 mM NaF, protease inhibitor mixture (one tablet/10 ml; Roche Applied Science, Mannheim, Germany), and 1 mM sodium orthovanadate) and centrifuged, and the supernatants were subjected to SDS-PAGE. The protein concentration in the samples was determined (19) and adjusted to 2 mg/ml. Total cell extracts (10 µg/lane) were subjected to electrophoresis on 12% polyacrylamide gels containing 0.1% SDS as described (12). Western blot experiments were performed using anti-human phospho-p38 antibody (1:2000 dilution) and anti-p38 MAPK antibody (1:2000). After incubation, the blots were incubated with peroxidase-coupled secondary IgG (1:10,000 dilution). Detection of the immunocomplex was carried out using the BM chemiluminescence blotting substrate kit (Roche Applied Science).
Isolation of the cDNA for the Suberites Lectin (LEC_SUBDO)—The cDNA encoding the potential LEC_SUBDO protein from S. domuncula was isolated from the cDNA library (17) by PCR. Primers were designed based on two highly conserved regions within T. tridentatus GBP (GenBankTM/EBI Data Bank accession number AAF74773 [GenBank] .1) (16) at amino acids 78–86 and 108–124. PCR was carried out by an initial denaturation at 95 °C for 4 min, followed by 30 amplification cycles at 94 °C for 30 s, 55 °C for 45 s, and 70 °C for 1.5 min and a final extension step at 70 °C for 10 min. The reaction mixture was as described previously (20). The fragments obtained were used to isolate the cDNA from the library (21–23) and resulted in one clone with a 775-nucleotide insert (excluding the poly(A) tail). The clone was termed SDLEC; it was sequenced using an automatic DNA sequenator (Li-Cor 4200).
Sequence Analysis—The sequence was analyzed using the computer programs BLAST2 and FASTA.3 Multiple alignments were performed with ClustalW Version 1.6 (24). Phylogenetic trees were constructed on the basis of amino acid sequence alignments by neighbor joining as implemented in the Neighbor program from the PHYLIP package (25). The distance matrices were calculated using the Dayhoff PAM matrix model as described (26). The degree of support for internal branches was further assessed by bootstrapping (25). The graphic presentations were prepared with GeneDoc.4
Recombinant LEC_SUBDO—The SDLEC sequence, which was
used for expression in E. coli, was isolated by PCR using forward
primer f1 (5'-GATATCAATTTCAACAGATAAGCAGAGATATG-3', with
the EcoRV restriction site underlined, nucleotides 67–93) and
reverse primer r1
(5'-CTCGAGATTTAAGCTCCGATAACATGTCGGAATGT-3', with the
XhoI restriction site underlined, nucleotides 730 to 703). The length
of the PCR product was 663 bp; it was used for expression in E. coli.
The PCR product was inserted into the bacterial glutathione
S-transferase (GST)/oligohistidine/S expression vector pET41a
(Novagen, Madison, WI) via the EcoRV (5'-end) and XhoI
(3'-end) restriction sites. E. coli strain BL21 was transformed
with this plasmid, and expression of the fusion protein was induced for 3 and
6 h at 37 °C with 1 mM
isopropyl-1-thio-
-D-galactopyranoside
(21). Bacteria from 500-ml
cultures were obtained by centrifugation; the fusion protein was extracted and
purified first using a His tag purification kit (Novagen) and subsequently
with a GST tag purification kit (Amersham Biosciences, Freiburg, Germany) as
recommended by the manufacturers.
The fusion protein was then cleaved with the recombinant enterokinase (5 units; Novagen) as recommended. The recombinant LEC_SUBDO molecule (tag-free) was purified in a batch procedure using the GST tag purification kit; recombinant LEC_SUBDO remained in the supernatant. Purified recombinant LEC_SUBDO was subjected to a refolding procedure using a protein refolding kit (Novagen). The protein was dialyzed against 25 mM Tris-HCl buffer (pH 7.2) supplemented with 10 mM DL-dithiothreitol. The purity of the material was checked on 10% polyacrylamide gels containing 0.1% SDS according to Laemmli (27, 28).
RNA Preparation and Expression Studies in Intact Specimens—RNA was extracted from liquid nitrogen-pulverized primmorphs with TRIzol reagent (Invitrogen). Reverse transcription-PCR was then performed as follows using commercial components (Invitrogen). cDNA was prepared using 10x PCR buffer, MgCl2, dNTP mixture, dithiothreitol, Super-Script reverse transcriptase, and oligo(dT) as described in the Invitrogen protocol. RNA was digested with RNase, and a cDNA segment was amplified (initial denaturation at 95 °C for 3 min, followed by 35 amplification cycles at 95 °C for 30 s, 54 °C for 45 s, and 61 °C for 1.5 min and a final extension step at 74 °C for 10 min) with the following two primer pairs: first, with fe1 (5'-TGGCCAATTTCAACAGATAAGC-3', nucleotides 63–84) and re1 (5'-TATCTCACATGTTTACGGTTCTCAG-3', nucleotides 853 to 829); and second, with fe2 (5'-AGGCCCTGCAACGGTAAGTG-3', nucleotides 163–182) and re2 (5'-TGCCCTCCATCTGTTCCCATTCT-3', nucleotides 649–627). The size of the product obtained with primers fe1 and re1 was 791 bp, and the size of the product obtained with primers fe2 and re2 was 487 bp. The products were size-separated on agarose, followed by staining with ethidium bromide.
Expression of SDLEC Analyzed by Northern Blotting—Primmorphs were incubated either in the absence or presence of 10 µg/ml LPS for 0–36 h. In one series of experiments, the assay with LPS contained also 10 mM D-GlcNAc. RNA was extracted, and 5 µg of total RNA was electrophoresed through a 1% formaldehyde-agarose gel and blotted onto a Hybond-Nylon membrane (Amersham Biosciences, Little Chal-font, Buckinghamshire, UK) following the manufacturer's instructions (20). Hybridization was performed with a 600-bp part of the corresponding SDLEC cDNA. The probe was labeled with the PCR digoxigenin probe synthesis kit (Roche Applied Science) according to the instruction manual. After washing, digoxigenin-labeled nucleic acid was detected with anti-digoxigenin Fab fragments (conjugated to alkaline phosphatase; 1:10,000 dilution) and visualized by the chemiluminescence technique using CDP according to the instructions of Roche. The screens were scanned with a Bio-Rad GS-525 molecular imager.
Purification of LEC_SUBDO—The procedure recently described for GBPs in plasma from Tachypleus trunculus (16) was applied with modifications. 200 g of S. domuncula tissue was used for the procedure immediately after collection. The material was homogenized in 3 volumes of buffer A (100 mM Tris-HCl (pH 8.3), 2 M NaCl, 10 mM CaCl2, and 1% (v/v) Triton X-100) and stirred overnight at 4 °C. The debris was then centrifuged at 10,000 x g for 30 min, and the supernatant was collected. The supernatant was brought to 0.70 saturation with solid ammonium sulfate under stirring. After 12 h, a precipitate was formed, which was collected by centrifugation at 10,000 x g for 30 min. The pellet was dissolved in 30 ml of buffer B (100 mM Tris-HCl (pH 8.3), 0.2 M NaCl, and 10 mM CaCl2) and dialyzed exhaustively overnight against the same buffer. Finally, the fraction was loaded onto an LPS-Sepharose CL-4B column. This matrix was prepared from LPS and CNBractivated Sepharose CL-4B (Amersham Biosciences) as recommended in the instruction manual. A column (2 x 10 cm) was prepared and equilibrated with buffer B, and the sample (20 ml) was loaded onto the column. After washing the column with buffer B, the lectin was eluted from the LPS matrix with buffer C (100 mM Tris-HCl (pH 8.3), 2 M NaCl, 20 mM EDTA, and 100 mM D-GlcNAc). Fractions of 2 ml were collected, and the pooled fractions (fractions 66–68) were dialyzed against Ca2+-and Mg2+-free artificial seawater (29).
Antibacterial Activity—The assay was performed as described (30). E. coli strain BL21 was grown in LB broth overnight. 20 µl of bacterial suspension was added to 80 µl of lectin (either the recombinant or the natural protein) and incubated for 60 min at room temperature. The lectin samples were dialyzed against Ca2+- and Mg2+-free artificial seawater. Antibacterial activity was assayed, and the reduction of colony-forming units was determined after serial dilution of the assay samples and overnight incubation. The percent inhibition was calculated as follows: (number of control and test colonies/number of control colonies) x 100 (n = 5). In one series of experiments, the lectin samples (100 µg/ml) were preincubated with LPS (10 µg/ml) for 12 h at 4 °C and then applied.
Antibodies against LEC_SUBDO—Polyclonal antibodies were raised against the recombinant lectin in New Zealand White female rabbits as described (31). In 4-weeks intervals, three boosts (10 µg of protein each) were injected. The serum was collected, and the polyclonal antibodies were termed pAb-LEC_SUBDO. In control experiments, 100 µl of pAb-LEC_SUBDO was adsorbed to 20 µg of recombinant lectin (30 min, 4 °C) prior to use.
Inhibition of LEC_SUBDO-LPS Interaction Using an Enzyme-linked Immunosorbent Assay System—Enzyme-linked immunosorbent assays were performed at 20 °C as described (32). 96-well polystyrene plates were coated with LPS (10 µg/ml) and incubated for 5 h. Excess protein-binding sites on the microtiter plates were blocked with 3% bovine serum albumin and washed three times. The wells were incubated with increasing concentrations of D-GlcNAc together with 10 µg/ml natural S. domuncula LEC_SUBDO. After an additional wash, the wells were incubated with pAb-LEC_SUBDO (1:500 dilution) for 30 min. The immunocomplexes were visualized using horseradish peroxidase-coupled secondary antibodies (1:1000) under application of o-phenylenediamine as substrate. The plates were read at 492 nm. The minimal inhibitory concentration of D-GlcNAc was determined.
Western Blotting—Gel electrophoresis of the protein extracts was performed on 10 or 15% polyacrylamide gels containing 0.1% SDS according to Laemmli (28). Protein samples were subjected to gel electrophoresis in the presence of 2-mercaptoethanol and stained with Coomassie Brilliant Blue. Semidry electrotransfer was performed according to Kyhse-Andersen (33) onto Immobilon membranes (polyvinylidene difluoride). Membranes were processed (34) and incubated with pAb-LEC_SUBDO (1:500 dilution) for 90 min at room temperature. After blocking the membranes with 5% bovine serum albumin, the immunocomplexes were visualized by incubation with alkaline phosphatase-conjugated anti-mouse IgG, followed by staining with 4-chloro-1-naphthol.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
In contrast, when the primmorphs were incubated with LPS, a strong increase in the phosphorylation of the kinase was seen (Fig. 1, lower panels). In the absence of LPS, no immunoreaction occurred with anti-phospho-p38 antibody. A strong reduction of p38 phosphorylation could be recorded only in the presence of D-GlcNAc among the different monosaccharides tested (Fig. 1); no effect was seen in the assays with D-Gal and D-GalNAc (Fig. 1) and D-Glc, D-GlcNH2, and D-GalNH2 (data not shown). From these results, we draw the conclusion that the interaction between LPS and the primmorphs occurs via a lectin-related reaction.
Cloning of the cDNA Encoding the S. domuncula Lectin— The S. domuncula lectin protein (LEC_SUBDO) was obtained from the cDNA library by PCR cloning as described under "Materials and Methods." The cDNA obtained (SDLEC) comprises an open reading frame that ranges from nucleotides 15/17 to 729/731 (stop codon). The deduced protein, termed LEC_SUBDO, comprises 238 amino acids residues with a calculated size of 25,908 Da. A potential transmembrane region was identified (35) ranging from amino acids 1 to 18 (Fig. 2A).
|
Three disulfide linkages have been assigned for the most closely related
protein, T. tridentatus lectin L6
(36). The respective Cys
residues exist in the S. domuncula sequence at identical positions;
the first disulfide bridge is located between Cys49 and
Cys53, the second between Cys124 and Cys128,
and the third between Cys200 and Cys204. As for T.
tridentatus lectin L6, the characteristic feature of the six internal
tandem repeats (36) is also
found in the sponge molecule. They range in S. domuncula LEC_SUBDO
from amino acids 19 to 55 (repeat 1), 56 to 90 (repeat 2), 91 to 129 (repeat
3), 130 to 166 (repeat 4), 167 to 205 (repeat 5), and 206 to 236 (repeat 6)
(Fig. 2A).
Phylogenetic Analysis—The sponge LEC_SUBDO polypeptide shares
the highest sequence similarity with the lectins from the horseshoe crab
T. tridentatus. These lectins (tachylectin-P, lectin L6, and
tachylectin-1) show 
50% amino acid identity and 65% amino acid similarity
(with respect to the physicochemical properties) to sponge LEC_SUBDO. The
similarity is lower to the deuterostomian sequences from the fish
Carassius auratus putative membrane protein and the potential GBP
from Danio rerio (22% identity and 40% similarity) as well as to the
two tectonins from the Mycetozoa Physarum polycephalum (20% identity
and 38% similarity). There is no considerable similarity to the
protostomian/nematode putative proteins in the data base.
This relationship is also reflected by the phylogenetic tree, which was rooted with one predicted open reading frame from Saccharomyces cerevisiae. This tree shows that the S. domuncula protein clusters with the tachylectins (see Ref. 37) in one branch, from which the vertebrate lectins (38) and the tectonin sequences (39) are separated (Fig. 2B). The tachylectins are located extracellularly (37), whereas the P. polycephalum tectonins are present in the nucleus (39). No (potential) function is known for the vertebrate molecules (38).
Hydropathicity plot analysis was performed to narrow down the relationship of the molecules mentioned above. The plots show that, in all proteins from metazoans, one N-terminal transmembrane region could be predicted according to the described method (35). In the S. domuncula sequence, the transmembrane helix was predicted to be between amino acids 1 and 18 (Figs. 2A and 3); in the C. auratus sequence, this region is found at amino acids 2–21; and in the GBP from T. tridentatus, it is between amino acids 1 and 24. In contrast, such a region is absent in P. polycephalum tectonin-1 (Fig. 3). The metazoan molecules comprise the six characteristic hydrophilic regions, which correspond to the six tandem repeats; they are absent in tectonin.
|
Recombinant LEC_SUBDO—Starting with the cDNA encoding sponge
LEC_SUBDO, the recombinant protein was synthesized from amino acid 19 to the
end of the deduced protein at amino acid 238; hence, the transmembrane segment
was omitted. The fusion protein was obtained in E. coli BL21 and
contained, in addition to the sponge protein (24,059 Da), the bacterial GST
tag (26 kDa), the oligohistidine tag (0.9 kDa), and the S tag (S-peptide
fragment of RNase A) (1.8 kDa) (Fig.
4, lanes a and b). The fusion protein (termed
recombinant LEC_SUBDO) was extracted and purified sequentially using the His
tag and subsequently the GST tag affinity purification procedure; the apparent
size was 60 kDa (Fig. 4,
lanes b and c). The fusion protein was cleaved with the
enterokinase, and the recombinant LEC_SUBDO molecule (tag-free, 24 kDa) was
purified as described under "Materials and Methods" (data not
shown).
|
Gene Expression of the Lectin in Different Specimens—To clarify whether different specimens express the SDLEC gene, S. domuncula specimens were collected from different areas around Rovinj at distances of 15–20 km. RNA was prepared immediately after collection and subjected to reverse transcription-PCR. As described under "Materials and Methods," two primer sets were applied: primers fe1 and re1, resulting in 791-bp products; and primers fe2 and re2, resulting in 487-bp products. The reactions were performed with 10 specimens; all of them expressed the SDLEC gene as seen by the formation of the two PCR products; the expression patterns from animals 1–6 are shown in Fig. 5.
|
Induction of the Lectin Gene in Response to LPS—To determine whether the steady-state level of expression of SDLEC changes in response to LPS, primmorphs were incubated with the endotoxin. The cells that were used for the preparation of the primmorphs came from animals that had been kept in an aquarium for 5 months. From such animals, it is known that they are exposed to only a minute level of bacteria in the surrounding seawater (40). The assays, which were performed as described under "Materials and Methods," remained either non-treated or were supplemented with 10 µg/ml LPS. Primmorphs were taken at time 0 and after 24 or 36 h of incubation. In one series of experiments, the assay with LPS contained the competitor sugar D-GlcNAc. It is obvious that, in the absence of LPS, the expression of the lectin gene remained at a very low level (Fig. 6, upper panel). In contrast, if LPS was added, a strong increase in expression was seen, which amounted to severalfold after an incubation period of 36 h (Fig. 6, middle panel). This strong induction caused by LPS could be reduced if the primmorphs were co-incubated with LPS and D-GlcNAc; under such conditions, only a low steady-state level of expression of the lectin gene was seen (Fig. 6, lower panel).
|
Purification of LEC_SUBDO from Sponge Tissue—The tissue was homogenized in buffer A, and the supernatant (1.5 mg/ml protein, 550 ml; Fraction I) was fractionated by ammonium sulfate precipitation as described under "Materials and Methods." The precipitate (after 0.70 saturation) was dissolved (6.5 mg/ml protein, 30 ml; Fraction II) and subjected to affinity chromatography using an LPS-Sepharose CL-4B column (Fig. 7). After adsorption of the protein with buffer B, the lectin was eluted with buffer C. Fractions 66–68 were pooled (total of 7.5 mg; Fraction III) and dialyzed against Ca2+- and Mg2+-free artificial seawater. The protein patterns from SDS-PAGE analysis of the crude extracts from S. domuncula and of the purified fraction are shown (Fig. 8A, lanes a and b).
|
|
Antibacterial Activity—The antibacterial activity of sponge LEC_SUBDO was against the Gram-negative bacteria E. coli and the Gram-positive bacteria Staphylococcus aureus. The lectin was added to the bacteria and incubated. The antibacterial activity was determined in colony-forming units and expressed in percent inhibition as described under "Materials and Methods."
The results are summarized in Table I and show that the natural lectin caused, even at a low concentration of 10 µg/ml, a significant antibacterial activity (16%) against E. coli, which increased with increasing concentrations; at 300 µg/ml, the inhibition was 81%. The recombinant lectin was also effective; at 300 µg/ml, a 36% inhibition was recorded. The significant antibacterial activity was reduced for both the natural (reduced to 43% at 300 µg/ml) and recombinant (reduced to 15% at 300 µg/ml) lectins when they were preincubated with LPS.
|
The inhibitory effect of the lectin on the Gram-positive bacteria S. aureus was comparatively low. At a concentration of 300 µg/ml natural lectin, an inhibition of only 15% was seen; for the recombinant protein, the activity was even lower (8%).
Immunological Cross-reactivity—To demonstrate that recombinant LEC_SUBDO shares identical/similar epitopes with the natural protein, antibodies were raised against the recombinant protein. The purified natural as well as recombinant lectins were size-separated by SDS-PAGE and, after blot transfer, reacted with the polyclonal antibody. First, the size-separated proteins were stained with Coomassie Brilliant Blue; the patterns of the crude extract and the purified natural protein are shown (Fig. 8A, lanes a and b). Western blot analysis was performed with the purified protein and pAb-LEC_SUBDO; a distinct band was found (Fig. 8B, lane a), which corresponded to the band of the purified natural protein of 27 kDa (Fig. 8A, lane b). Likewise, the antibody stained recombinant LEC_SUBDO within the same size range (Fig. 8B, lane c). Preimmune serum and serum adsorbed with the recombinant protein did not react with the proteins on the blot (data not shown). In a further attempt to demonstrate the specificity of the antibody, flow-through fractions from the affinity purification procedure (fractions 18–20) (Fig. 7) were analyzed and were found not to react with pAb-LEC_SUBDO on the Western blot (Fig. 8B, lane b).
Inhibition of LEC_SUBDO-LPS Interaction—An enzyme-linked immunosorbent assay system was used to determine the minimal inhibitor concentration of D-GlcNAc. Under the conditions used, concentrations as low as 3 µM D-GlcNAc resulted in inhibition of the binding between LEC_SUBDO and LPS. No effect could be seen with D-Glc, D-Gal, D-GalNAc, D-GlcNH2, or D-GalNH2 even at concentrations >10 mM.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Exposure of primmorphs to LPS results in strong phosphorylation of p38 MAPK (see also Ref. 2). This is an indication that an inflammation-related process occurs after addition of this toxin to these 3D cell aggregates (see Ref. 42). p38 MAPK is known to be phosphorylated in response to LPS in mammalian systems (43). To clarify whether lectins are also involved in the host defense in sponges, primmorphs were incubated with LPS in the absence or presence of different sugars; the monosaccharide D-GlcNAc turned out to be the most effective competitor molecule.
By application of the PCR cloning approach and screening a potential cDNA encoding a horseshoe crab-related lectin was identified. The horseshoe crab lectins have been demonstrated to be effective defense molecules (see Ref. 15). In S. domuncula, a cDNA was found whose deduced polypeptide has significant sequence similarity to the tachylectins. The most closely related proteins are the GBP from T. tridentatus (16), tachylectin-P (44), and lectin L6/tachylectin-1 (36). Like the crab molecules, sponge LEC_SUBDO comprises a predicted transmembrane region, suggesting that the lectin is cell surface-associated. In addition, six distinct hydrophilic regions are present in the sponge molecule, corresponding to six tandem repeats in the tachylectins; these are absent in tectonins, related molecules found in the Mycetozoa P. polycephalum (39). To rule out the possibility that the cloned LEC_SUBDO is not of sponge origin and might have been introduced into the cDNA library from commensal animals, different S. domuncula specimens were analyzed by reverse transcription-PCR. In all cases examined, a strong expression of the SDLEC transcript could be identified.
The described sequence similarity suggests that the sponge lectin is also, as the horseshoe crab lectins, involved in the bacterial defense mechanism of S. domuncula. It has been proposed that the horseshoe crab lectins, molecules that are present already before infection and are of a relative specificity, are key elements of the innate/natural immunity (see Ref. 15). In horseshoe crabs, the lectins are associated with specialized cells, the hemocytes, and/or the hemolymph; lectin L6/tachylectin-1 from T. trunculus is associated with the cell membrane of hemocytes (36), whereas the GBP from T. tridentatus (16) and tachylectin-P from T. tridentatus (44) exist in the hemolymph. Like the crab molecules, the sponge LEC_SUBDO comprises a predicted transmembrane region, suggesting that the lectin might be associated with immune cells. The most likely candidates in sponges, which are devoid of a circulation system, to act as immune cells are the archaeocytes and perhaps also the choanocytes (45, 46).
To approach the question of whether the lectin is involved in the host antibacterial defense in the sponge S. domuncula, its expression was determined. It was found that SDLEC expression was strongly up-regulated after exposure of primmorphs to LPS. The conclusion that this gene is under the control of LPS was supported by competition experiments using the monosaccharide D-GlcNAc; this sugar almost completely abolished the effect of LPS.
After determining that the SDLEC gene is modulated in response to exposure of primmorphs to LPS, it was pressing to determine the potential antibacterial effect of LEC_SUBDO. Therefore, the lectin was isolated from the tissue and prepared in a recombinant way. By application of specific antibodies raised against the recombinant protein, it was confirmed that both forms of protein comprise the same antigenic sites, suggesting that they are the same species of molecules. The antimicrobial assays confirmed that the sponge lectin displays a strong antibacterial effect against Gram-negative bacteria E. coli and, to a lower extent, also against the Gram-positive bacteria S. aureus. Also in this respect, the sponge molecule shares a striking functional similarity with T. trunculus lectin L6/tachylectin-1; this molecule also displays a strong effect against Gram-negative bacteria and a lower effect against Gram-positive bacteria (36).
Taken together, the results summarized in this work indicate that a natural immune system exists also in sponges, as determined here with LEC_SUBDO as an antibacterial molecule. This finding contributes not only an important aspect to the complexity of the immune competence in sponges in general, but also interesting facts on the evolution of the immune system in particular. LEC_SUBDO identified in S. domuncula indicates that these animals can defend themselves against bacterial invaders by a (probably cell membrane-associated) antibacterial lectin. In view of the fact that a series of antimicrobial lectins exist in horseshoe crabs (reviewed in Ref. 15), it might be predicted that such a diversity occurs also in sponges. Screening studies by PCR cloning using degenerate primers directed against the conserved regions are under way. Likewise, studies to localize the immune cells by in situ hybridization are in progress. Furthermore, the finding sheds new light on the origin of the immune system in Metazoa. In previous studies, the immune molecules have been documented to have emerged during the transition to Urmetazoa (6). The data presented indicate that the immune system in sponges comprises also basic molecules that are reminiscent of molecules that are present in more ancestral multicellular organisms such as the slime mold P. polycephalum (tectonins, representing nuclear matrix molecules). Moreover, we have demonstrated that some potential immune molecules exist in sponges up to horseshoe crabs, the tachylectins from Tachypleus and the molecule now found also in the sponge S. domuncula, that have been lost later in the evolution to the crown taxa of Deuterostomia (animals characterized by the process of the blastopore becoming the anus in the adult) and of Protostomia (animals characterized by the process of the mouth arising from the blastopore).
|
FOOTNOTES |
|---|
* This work was supported by Deutsche Forschungsgemeinschaft Grant
Mü/14-3, the Bundesministerium für Bildung und Forschung, Germany
(Project: Center of Excellence BIOTECmarin), and International Human Frontier
Science Program Grant RG-333/96-M. The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked "advertisement" in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact. 
|| To whom correspondence should be addressed. Tel.: 61-31-392-5910; Fax: 61-31-392-5243; E-mail: wmueller{at}mail.uni-mainz.de.
1 The abbreviations used are: LPS, lipopolysaccharide; MAPK,
mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; GBP,
galactose-binding protein; GST, glutathione S-transferase.
2 Available at
www.ncbi.nlm.nih.gov/blast/blast.cgi.
3 Available at
www.ncbi.nlm.nih.gov/BLAST/fasta.html.
4 Available at
cris.com/~ketchup/genedoc.shtml.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  H. C. Schroder, H. J. Breter, E. Fattorusso, H. Ushijima, M. Wiens, R. Steffen, R. Batel, and W. E. G. Muller Okadaic Acid, an Apoptogenic Toxin for Symbiotic/Parasitic Annelids in the Demosponge Suberites domuncula. Appl. Envir. Microbiol., July 1, 2006; 72(7): 4907 - 4916. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Wiens, M. Korzhev, A. Krasko, N. L. Thakur, S. Perovic-Ottstadt, H. J. Breter, H. Ushijima, B. Diehl-Seifert, I. M. Muller, and W. E. G. Muller Innate Immune Defense of the Sponge Suberites domuncula against Bacteria Involves a MyD88-dependent Signaling Pathway: INDUCTION OF A PERFORIN-LIKE MOLECULE J. Biol. Chem., July 29, 2005; 280(30): 27949 - 27959. [Abstract] [Full Text] |
