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Originally published In Press as doi:10.1074/jbc.M407613200 on October 4, 2004

J. Biol. Chem., Vol. 279, Issue 50, 52016-52023, December 10, 2004
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The Glycoprotein NOWA and Minicollagens Are Part of a Disulfidelinked Polymer That Forms the Cnidarian Nematocyst Wall*

Suat Özbek{ddagger}§, Elena Pokidysheva{ddagger}, Martine Schwager{ddagger}, Therese Schulthess{ddagger}, Naushaba Tariq{ddagger}, Dirk Barth¶, Alexander G. Milbradt¶, Luis Moroder¶, Jürgen Engel{ddagger}, and Thomas W. Holstein||

From the {ddagger}Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland, the Max-Planck-Institute for Biochemistry, D-82152 Martinsried, Germany, and the ||Institute of Zoology, Technical University of Darmstadt, Schnittpahnstrasse 10, D-64287 Darmstadt, Germany

Received for publication, July 7, 2004 , and in revised form, September 29, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The nematocyst is a unique extrusive organelle involved in the defense and capture of prey in cnidarians. Minicollagens and the glycoprotein NOWA are major components of the nematocyst capsule wall, which resists osmotic pressure of 15 MPa. Here we present the recombinant expression of NOWA, which spontaneously assembles to globular macromolecular particles that are sensitive to reduction as the native wall structure. Ultra-structural analysis showed that the Hydra nematocyst wall is composed of several layers of globular particles, which are interconnected via radiating rodlike protrusions. Evidence is presented that native wall particles contain NOWA and minicollagen, supposed to be linked via disulfide bonds between their homologous cysteine-rich domains. Our data suggest a continuous suprastructure of the nematocyst wall, assembled from wall proteins that share a common oligomerization motif.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The nematocyst is a unique subcellular organelle produced during a highly ordered secretion and assembly process of proteins into a giant post-Golgi vesicle (1). Nematocysts are of varying morphology and serve defensive or locomotory functions in cnidarians including hydroids, jellyfish, sea anemones, and corals. Nematocyst discharge, triggered by mechanical or chemical stimulation, is one of the fastest processes in biology driven by the extreme internal pressure of the capsule (15 MPa) and the gain of volume during exocytosis of the inverted tubule (2). The wall of the nematocyst, as an adaptation to this mechanical stress, is expected to combine both high resistance and flexibility. Investigation of the wall surface by atomic force microscopy (AFM)1 and field emission scanning electron microscopy (FESEM) revealed a dense globular structure that could be removed either mechanically by the cantilever tip or chemically by dithiothreitol treatment exposing a smooth layer with a fibrous, collagen-like appearance (3). A preliminary structural model was proposed in which collagen molecules are assembled to fibers forming the inner wall covered by an outer layer of globular particles (3, 4).

A major constituent of the capsule wall is a family of unusually short collagens, termed minicollagens (5). They comprise a central collagen triple helix with 12–16 Gly-X-Y repeats flanked by polyproline stretches and terminal cysteine-rich domains (MCRDs) with a conserved pattern of six closely set cysteines. Minicollagens are trimeric molecules that are expressed as soluble precursors, which during nematocyst maturation polymerize by a switch in the disulfide linkage from intramolecular to intermolecular connections (1, 6). This process is accompanied by a loss of minicollagen antibody reactivity in the head and tentacles regions of Hydra in mature nematocysts (1).

NOWA is a 90-kDa glycoprotein that has been described to be associated with the globular structure of the nematocyst outer surface (4, 5). Interestingly, the molecular architecture of NOWA comprises a C-terminal octarepeat of the minicollagen cysteine-rich domain, suggesting a possible disulfide-dependent heteroassembly of minicollagens and NOWA protein. During nematocyst morphogenesis NOWA appears at a very early stage and spontaneously assembles to the nematocyst membrane, whereas minicollagens are expressed later and homogenously fill the nematocyst matrix before condensing at the wall (4). It was suggested therefore that NOWA serves as a positional organizer of minicollagen assembly during the final stages of nematocyst maturation.

In the present study we demonstrate that recombinantly expressed NOWA and its C-terminal minicollagen cysteine-rich octarepeat domain (MCROD) undergo a disulfide-dependent self-assembly to globular particles similar to the ones found throughout the wall structure. Isolation of native wall particles from nematocysts and their biochemical and electron microscopic characterization indicate that they are composed of both NOWA and minicollagens, separable only by reduction. FESEM analysis of the mature nematocyst wall ruptured by sonication revealed a continuous structure built of six or seven layers of globular particles. A noncovalent preassembly of minicollagen molecules to NOWA particles is obviously mediated by the central C-type lectin domain of NOWA, which shows binding to the minicollagen-1 MCRD. Our data suggest a more precise model for the nematocyst wall suprastructure, contrasting the prevailing notion of a double-layered wall mainly stabilized by collagen fibers.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
cDNA Constructs and Expression of Recombinant Proteins—Sequences for full-length NOWA and its C-terminal MCROD comprising residues 464–749 were amplified by PCR using the NOWA pBluescript vector as a template. NheI and BamHI sites were introduced in the 5'- and 3'-primers, respectively, to enable convenient cloning into the corresponding sites of the mammalian expression vector pCEP-Pu. The primers used were: 5'-TGC GGC TAG CCA GAT CCT CAA CAG TAT GGT TGT TTT TAG CG-3' and 5'-CGG GAT CCT TAG GCT TTA CTT TGC TTT TTT CTT ACG GGA GG-3' for full-length NOWA; 5'-TGC GGC TAG CCC AAA TTA CTG GAA CAT GTC C-3' and 5'-TTT GGA TCC TTA CAT TCG TCC AAG ACT AC-3' for the cysteine-rich octarepeat domain. For stable transfection, 293 EBNA cells were kept in Dulbecco's modified Eagle's medium/Ham's F-12 medium supplemented with 10% fetal bovine serum, 1% Gln, and penicillin/streptomycin. The cells were grown to 80% confluence in 6-well plates and transfected overnight with 1 µg of vector DNA using 5 µl of LipofectAMINE reagent. The selection of positive clones was performed by culturing transfected cells with 2 µg/ml puromycin with frequent changes of medium until a resistant population appeared. All of the reagents were purchased from Invitrogen. For expression, stably transfected EBNA 293 cells were grown to high density in 125-ml cell culture flasks using complete medium and then switched to serum-free expression medium. The cell supernatants were harvested several times until the cells detached, pooled, and filtered. Recombinant proteins were then purified in native condition using nickel-Sepharose chromatography according to the manufacturer's instructions (Qiagen). Minicollagen-1-MBP fusion protein was expressed in 293 EBNA cells and purified via affinity chromatography using amylose-coupled Sepharose beads (NEB) as described before (1). The NOWA CTLD was expressed in Escherichia coli and refolded from inclusion bodies as described before (4).

SDS-PAGE and Western Blot Analysis—The samples were incubated in Laemmli buffer with or without {beta}-mercaptoethanol and separated on 12% SDS gels. For resolving higher aggregates gradient gels (3–10%) were applied with 2.5% stacker gels. Western blot analysis was performed using polyclonal antibodies (rabbit) raised against minicollagen-1 (1) and the CTLD or MCROD of NOWA. Primary antibody (1: 1000) was detected using an anti-rabbit horseradish peroxidase conjugate antibody (1:2000) and the ECL chemoluminiscence system (Amersham Biosciences).

Ultrasound Treatment of Nematocyst Capsules—Intact undischarged nematocysts were isolated from whole Hydra tissue as described previously (7). To induce wall ruptures ~2 x 106 nematocysts were suspended in 2 ml of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA and sonicated directly with a microtip probe (Branson) applying full intensity for 2 min. Overheating of the suspension was prevented by placing the sample tube into a precooled (–20 °C) metal block during sonication. Wall fragmentation was performed by prolonged sonication (10 min) with short bursts. For the dissociation of wall components while preserving the wall integrity, capsule suspensions were sonicated within an Eppendorf tube using a water bath sonicator (Elgasonic).

Transmission Electron Microscopy—Supernatants or pellets of sonicated capsule material as well as recombinant proteins were absorbed to freshly glow-discharged thin carbon films supported by thick perforated carbon layers and negatively stained with uranyl formiate following standard procedures (8).

FESEM Analysis—Approximately 1 x 105 capsules were suspended in PBS and set on glass cover slides treated with polylysine. The capsules were then fixed with PBS containing 0.2% glutaraldehyde and 2% formaldehyde for 10 min, subsequently rinsed for 10 min with 0.1 M phosphate buffer, pH 7.4, containing 2% bovine serum albumin, and washed with 0.02 M glycine in PBS. Fixation was then performed with 2.5% glutaraldehyde in PBS for 10 min. After several washing steps with PBS, the capsules were dehydrated stepwise with rising concentrations of ethanol (10–100%) before being subjected to critical point drying. FESEM analysis was performed in high vacuum mode (10–5–10–6 mBar) on a Phillips XL30 microscope. The samples were coated with carbon or sputtered with 5-nm platinum for higher resolution.

Gel Filtration Analysis—A prepacked Superose 6 column (Amersham Biosciences) was equilibrated with running buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA. For the isolation of soluble capsule wall components, 1 ml of a suspension of ~2 x 106 nematocysts was sonicated in a water bath for 30 min, and the capsules were centrifuged in a microcentrifuge for 5 min at 6000 rpm. The supernatant was taken for size exclusion chromatography with a flow rate of 0.3 ml/min. 0.15-ml fractions were collected and analyzed by Western blotting.

Peptide Synthesis—The 6xStBu protected peptide Ac-PCPPVCVAQCVPTCPQYCCPAKRK-NH2 was synthesized on Fmoc-Rink-Amide PEGA resin by the Fmoc/tBu strategy using double couplings with Fmoc-Xaa-OH/HBTU/HOBt/n,n-diisopropylethylamine (4:4:4:8), intermediate Fmoc cleavage with 20% piperidine in dimethylformamide and acetic anhydride/n,n-diisopropylethylamine (4:8) for N-terminal acetylation. After resin cleavage/deprotection with trifluoroacetic acid/phenol/H2O/thioanisole/1,2-ethanedithiol (82.5:5:5:5:2.5) the product was isolated by reverse phase HPLC; yield, 6%; HPLC, tR = 12.5 min (>98%); electrospray ionization tandem mass spectrometry, m/z = 1055.0 [M+3H]3+; 1581.8 [M+2H]2+; Mr = 3162.31 calculated for C136H230N32O29S12. The Cys protecting groups were cleaved in trifluoroethanol/H2O with tributylphosphine (60 eq) at room temperature for 5 h, and the resulting fully deprotected peptide was oxidized at pH 8.0 in the presence of GSSG/GSH (9 eq, 10:1) under air atmosphere at 7 °C. The crude product was purified by preparative size exclusion column chromatography; yield, 20%; HPLC, tR = 12.3 min (>98%); electrospray ionization tandem mass spectrometry, m/z = 1314.8 [M+2H]2+, 876.4 [M+3H]3+; Mr = 2627.23 calculated for C112H176N32O29S6.

Co-precipitation—Noncovalent interaction of NOWA with minicollagen-1 was detected by precipitation of recombinant full-length NOWA with a minicollagen-1-MBP fusion protein that was expressed in 293 cells as described before (1). For precipitation, the complexes were incubated by continuous rotation with a suspension of amylose-coupled agarose beads (New England Biolabs) in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl at 4 °C and washed three times with PBS. Co-precipitated NOWA or CTLD protein was detected by Western blotting with anti-CTLD antibody. For the detection of the NOWA MCROD, a polyclonal antiserum was used raised against this protein. Blocking experiments were performed by incubating CTLD or MCROD protein with agarose-bound minicollagen-1-MBP together with an excess of the minicollagen-1 peptide.

Radioligand Binding Assay—For radioligand binding assay, iodination of minicollagen-MBP was performed as described previously (9). Recombinant NOWA-CTLD was diluted to 10 µg/ml with PBS and immobilized on microtiter plates (Falcon) by overnight incubation at 4 °C. Blocking was carried out for 1 h with PBS, pH 7.4, containing 1 mg/ml bovine serum albumin and 2% fetal calf serum. After three washes with PBS, the unlabeled Minicollagen-1-MBP or oxidized MCRD was added as competitor in PBS containing 3% bovine serum albumin, followed by the addition of 10 nM iodinated Minicollagen-1-MBP. After washing three times with PBS, the radioactivity in each well was measured in a {gamma} counter.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Recombinant Expression of NOWA and Its MCROD—We have shown previously that the MCRD in minicollagens is responsible for intermolecular disulfide links during formation of the nematocyst (10). To assess a possible similar function for the NOWA MCRDs, we decided to design constructs both for full-length NOWA and its MCROD (residues 464–749) (Fig. 1A). Both proteins were recombinantly expressed in HEK293 cells together with an N-terminal polyhistidine tag for purification via nickel-Sepharose chromatography. In reducing gels, NOWA and the MCROD showed apparent molecular masses of ~92 and ~45 kDa, respectively (Fig. 1B). The difference to the calculated molecular mass observed for the MCROD protein (32.6 kDa) can be explained by a more extended shape of the molecule caused by reduction. We have previously reported a similar behavior for minicollagen molecules (1). In nonreducing SDS-PAGE both protein bands were dramatically reduced, indicating that they formed cysteine-linked oligomers during expression. To assess the oligomeric state of the purified proteins, they were analyzed in the presence or absence of reducing agent using 3–10% gradient gels. As shown in Fig. 1C (left panel) NOWA almost exclusively existed as a distinct oligomeric fraction, hardly entering the separating gel without prior reduction. By addition of a reducing agent, unheated samples displayed a ladder of lower oligomeric bands beside the monomeric 92-kDa protein. Heat denaturation alone did not lead to dissociation of the aggregate, indicating that the molecules were linked by multiple disulfide bonds. By applying both heat and reducing agent, the oligomeric fraction was almost quantitatively converted to monomers. A similar experiment performed with the purified MCROD (Fig. 1C, middle panel) revealed comparable behavior with the exception that reduction alone did not yield a band pattern of different oligomers and that in the unreduced state the high molecular mass aggregates were not detectable in the separating gel. This suggests that the other domains of NOWA on the one hand contribute to self-assembly by providing noncovalent interactions and on the other hand restrict the assembly process to a certain extent because the MCROD alone appears to form larger aggregates than the full-length protein. Thus, recombinant NOWA undergoes a spontaneous disulfide-dependent self-assembly process that is predominantly a feature of its C-terminal MCROD. Western blot analysis of NOWA protein in native nematocyst capsules revealed that it was completely engaged in disulfide-linked oligomers that could be dissociated already by mild reduction (Fig. 1C, right panel).



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  		FIG. 1.
Recombinant expression of NOWA and NOWA MCROD. A, schematic representation of the cDNA constructs for full-length NOWA comprising an N-terminal rodent sperm-coating glycoprotein (SCP) domain, a central CTLD, and a C-terminal octarepeat of the MCROD and for the MCROD alone. B, SDS-PAGE and Western blotting analysis of purified NOWA and MCROD proteins. Western blots were performed using anti-CTLD or anti-MCROD antibody, respectively. C, (left panel, analysis of purified NOWA protein by gradient SDS-PAGE (3–10%) under different conditions as indicated. Middle panel, the same experiment performed with the purified NOWA MCROD that shows an apparent molecular mass of 45 kDa. Note that heat denaturation alone in this case did not yield a clear protein band but instead a smear that might represent degradation products. Right panel, Western blot analysis of isolated nematocyst capsules using anti-NOWA (CTLD) antibody. The capsule samples were treated for 5 min with dithiothreitol (DTT) at concentrations indicated prior to SDS-PAGE. {beta}-ME, {beta}-mercaptoethanol.

 
Electron Microscopic Analysis of Recombinant NOWA and Isolated Nematocyst Wall Particles—Electron micrographs of negatively stained recombinant NOWA protein showed macromolecular particles of globular shape with an average diameter of 23 ± 1.8 nm (Fig. 2, A and B). Interestingly, the NOWA MCROD formed similar particles with slightly reduced diameters (14 ± 2.7 nm), confirming its essential role in NOWA self-assembly (Fig. 2C). Also, the NOWA MCROD particles appeared to form large clusters, which explains their behavior in nonreducing SDS-PAGE.



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  		FIG. 2.
Electron micrographs of negatively stained nematocyst wall components and recombinantly expressed NOWA or NOWA MCROD. A, overview of full-length NOWA protein. Bar, 200 nm. B, close view of full-length NOWA protein. Bar, 20 nm. C, NOWA MCROD. Bar, 50 nm. D, soluble fraction of capsules after strong sonication showing isolated wall particles. Bar, 100 nm. E, close view on wall particles with halos of smaller particles detected in the soluble fraction of capsules obtained by weak sonication in a water bath. Bar, 20 nm. F, part of a nematocyst wall fragment in the insoluble fraction of strongly sonicated capsules. Note that the contrast was inverted to visualize the rodlike connections between the capsulomers. Bar, 100 nm. Inset, part of F in which the network was visualized by a stronger contrast. Bar, 40 nm.

 
Analysis of the soluble fraction of harshly sonicated (10 min) capsules by negative staining showed nests of globular particles of more heterogenous size ranging between 15 and 45 nm with an average diameter of 27.1 ± 6.7 nm (Fig. 2D). The isolated particles were often grouped to large irregular clusters indicating a tendency for aggregation. This observation was additionally emphasized by our attempts to obtain a sedimentation coefficient for isolated native particles resulting in values above 8000 S, which is at least 2 orders of magnitude higher than would be expected from the calculated molecular mass of a single particle. The insoluble fraction of the sonicated nematocyst sample contained wall fragments of differing size, of which some were small enough to be absorbed on carbon films for negative staining. In Fig. 2F a part of an outer wall surface is visualized. As compared with Fig. 2D the contrast is inverted for better demonstration of the rodlike connections between the globular particles. The orientation of the fragment was deduced from transparent patches of additional staining that we interpreted as fragments of the capsule membrane covering the wall on the outside surface (not shown). At the wall surface the particles were apparently distributed without direct contact to each other. They were quite heterogenous in size comparable with the particles in Fig. 2D and showed sometimes even larger diameters. The space between them was filled with numerous smaller globes sometimes radiating from them by rodlike connections. Higher contrast revealed a dense network of thin protrusions that attach to the particles from various directions (Fig. 2F, inset). Solubilization of wall particles by mild sonication in a water bath yielded similar particles as in Fig. 2D that in this case were sometimes surrounded by a halo of smaller globes with a diameter of 2–4 nm connected to the central body in a distance of 5–6 nm (Fig. 2E). In electron micrographs of recombinantly expressed full-length NOWA, particles with radiating arms comparable with those in Fig. 2E were never observed, indicating that the protrusions originate from molecules not present in the recombinant material.

Molecular Composition of Native Wall Particles—Because we had demonstrated in previous studies that minicollagen molecules show rodlike shapes in transmission electron microscopy (1), we supposed that the isolated wall particles, which apparently contained rodlike structures, might be composed of both molecule species, NOWA and minicollagens, probably in a disulfide-linked manner. To confirm this hypothesis we treated nematocysts by mild sonication using a water bath to prevent heating and to preserve native complexes. The supernatant of sonicated capsules was then submitted to size exclusion chromatography on a Superose 6 column. By the assumption of a spherical shape for the wall particles with an average diameter of 27.1 ± 6.7 nm (Fig. 2D), we estimated an average molecular mass of about 8.0 MDa per sphere. Gel filtration under nonreducing conditions yielded two major peaks with the first peak eluting within the void volume of the column (exclusion limit for globular proteins = ~40 MDa) (Fig. 3A), which indicates that the individual particles were associated to higher aggregates. The second pronounced peak eluted in the molecular mass range of 5–15 kDa and showed a smear of low molecular mass bands in SDS-PAGE, which were not positive for NOWA or minicollagen-1 in Western blot (not shown). Western blot analysis of the peak fractions eluting in the high molecular mass range showed that they contained both minicollagen-1 and NOWA (Fig. 3B). Minor peaks did not contain NOWA protein, but several were positive for minicollagen (not shown), implying a certain amount of soluble minicollagen that is not part of the wall structure. This is in agreement with Western blot analysis of isolated capsules published previously (10). Gel filtration of the solubilized wall particles under reducing conditions (10 mM dithiothreitol) led to dissociation of the high molecular mass peak yielding instead several fractions in the molecular mass range between 50 and 200 kDa (Fig. 3C). Western blot analysis of the whole elution profile (Fig. 3D) showed that the major peak (fraction 16) contained minicollagen, whereas monomeric NOWA eluted earlier (fractions 14–15). There was some overlap of the two signals in a higher molecular mass region (fraction 11), indicating that the proteins partially interact noncovalently after reduction.



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  		FIG. 3.
Detection of NOWA and minicollagen-1 in isolated wall particles. A, elution profile of solubilized wall particles separated on a Superose 6 column under nonreducing conditions. The void volume peak containing solubilized wall particles is marked by an arrow. DTT, dithiothreitol. B, detection of NOWA and minicollagen-1 in fractions of the void volume peak obtained under nonreducing conditions by Western blotting with anti-minicollagen-1 or anti-NOWA (anti-CTLD) antibody. Both protein species show a slightly heterogeneous band pattern because of glycosylation or cross-reaction with other members of the protein family in the case of minicollagen-1. C, elution profile of solubilized wall particles separated under reducing conditions. D, detection of NOWA and minicollagen-1 in all fractions obtained under reducing conditions.

 
FESEM Analysis of Sonicated Capsules—Motivated by our previous notion of a double-layered wall of the nematocyst, we attempted the visualization of the wall fine structure in profile. To do so, isolated undischarged capsules were sonicated to induce wall ruptures. FESEM was then performed with carbon or platinum-covered samples fixed on glass covers. Most of the nematocysts were still intact after a short (2 min) ultrasound treatment, but some showed a rupture predominantly perpendicular to the length axis of the capsule body where the tubule had then discharged without eversion. Fig. 4A shows a total view of a stenotele displaying a typical rupture. The operculum at its narrow end, which opens to release the tubule when nematocyst discharge is triggered, is still closed, and the stylet apparatus used for puncturing the cuticle of a prey organism is folded back inside the capsule body and wrapped by the base of the tubule. A high resolution imaging of the wall surfaces by platinum covering revealed a composition of the wall profile consisting of several layers (six or seven layers) of globular wall particles with a diameter of ~20 nm (Fig. 4, B and C). The wall showed a thickness of ~150 nm, and in contrast to our earlier model we did not observe two distinct wall layers but a homogenous globular appearance throughout the whole cross-section.



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  		FIG. 4.
FESEM Analysis of sonicated capsules with carbon (A) or platinum shadowing (B–E). A, total view of a stenotele with a characteristic wall rupture induced by ultrasound treatment. Bar, 3 µm. B and C, profile of ruptured wall shown from different angles with a view on the outside or inside surface of the capsule. Bars, 100 nm. D–E, close view of the outer and inner surface of the ruptured capsule. Bars, 100 nm.

 
On the outer surface the wall particles were arranged in parallel lines resembling the "fibrous" structures observed before on dithiothreitol-treated capsules (Fig. 4D) (3). The appearance of globular units at the outside of the capsule was also displayed for the inner wall surface (Fig. 4E) with an apparently more dense and smooth packing. Both on the outside and the inside surface, the capsule wall was covered by larger aggregates that might represent loosely attached redundant wall material. Although applying different sonication treatments, we were not able to confirm the existence of isolated collagen fibers as suggested by our earlier AFM data (3).

Binding of NOWA to Minicollagen-1—Because the polymerization process of the wall proteins takes place in the final stage of maturation, we supposed that a noncovalent preassembly of wall components like NOWA and minicollagen might precede the intermolecular reshuffling. To analyze which domains in NOWA were responsible for minicollagen binding, we performed co-precipitation experiments using recombinant minicollagen-1-MBP fusion protein. Binding of full-length NOWA to minicollagen-1 was confirmed by adsorbing minicollagen-1-MBP to amylose-coupled agarose beads and incubation with recombinant NOWA protein. Co-precipitated NOWA was detected by Western blotting with anti-CTLD antibody. NOWA-minicollagen interactions proved to be resistant to washing with PBS but were removed by washing with a 1 M NaCl solution, indicating that binding was of a noncovalent nature (Fig. 5A). To analyze the roles of the different domains of NOWA in the noncovalent interaction with minicollagen-1, we performed similar co-precipitation experiments using either the NOWA CTLD or MCROD. As shown in Fig. 5B, the CTLD bound strongly to minicollagen-1 MBP, whereas the MCROD showed no binding activity. Interestingly, the 18-kDa CTLD protein co-precipitated by minicollagen-1-MBP displayed an SDS-stable dimer band, which probably points to a structural prerequisite for minicollagen binding. Binding of the NOWA CTLD to minicollagen-1-MBP could be blocked in a dose-dependent manner by a synthetically produced and reoxidized minicollagen-1 peptide corresponding to the C-terminal MCRD (PCPPVCVAQCVPTCPQYCCPAKRK) (11). This demonstrates that NOWA-minicollagen interactions are mediated by the terminal MCRDs of the minicollagen molecules and not by the collagen triple helix. The affinity of the minicollagen-1 CTLD interaction (kD = 30 nM) was determined by radioligand assay using unlabeled minicollagen-1-MBP or MCRD peptide as competitors. The affinity of the CTLD-MCRD interaction proved to be significantly weaker (kD = 0.12 µM), probably implying that a definite orientation of the minicollagen MCRD improves its interaction with NOWA.



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  		FIG. 5.
Co-precipitation of NOWA with minicollagen-1-MBP. A, minicollagen-1-MBP fusion protein was adsorbed to amylose beads that were washed with PBS and subsequently incubated with recombinant full-length NOWA at 100 nM for 4 h at 4 °C. Precipitation of complexes was performed after washing either with PBS or 1 M NaCl solution. Bound NOWA protein was detected by anti-CTLD antibody after Western blotting. B, minicollagen-1-MBP bound to amylose beads was incubated with NOWA CTLD (100 µg/ml) or NOWA MCROD (90 µg/ml) with the addition of blocking minicollagen-1 peptide comprising the C-terminal MCRD (PCPPVCVAQCVPTCPQYCCPAKRK) at concentrations as indicated. Complexes were washed with PBS, and bound protein was detected by Western blotting with anti-CTLD or anti-MCROD antibodies. C, binding of minicollagen-1-MBP to the NOWA-CTLD as measured by radioligand assay. MOWA-CTLD was immobilized on enzyme-linked immunosorbent assay plates at 10 ng/µl. Binding of radiolabeled minicollagen-1-MBP was competed with minicollagen-1-MBP or MCRD peptide at different concentrations. The bars indicate standard deviations of three experiments. Curve fitting was performed using Prism 2.0 software (Graphpad).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The wall of cnidarian nematocysts is a unique pressure-resistant structure. Different from other biological polymers including cell walls of plants and bacteria, it encloses a matrix where no metabolic processes take place, and it accomplishes a purely structural function. The composition of the wall is relatively simple and contains two main protein species, minicollagens (1, 5) and NOWA (4). A sequential secretion process of these proteins, which are accumulating in a growing post-Golgi vesicle, characterizes the formation of the nematocyst capsule. The organization of the wall and tube structures from these proteins is mainly directed by a membrane-associated assembly and disulfide reshuffling process resulting in a polymer stabilized by disulfide bonds.

It has been shown in several publications that the capsule architecture is highly sensitive to reducing agents and that Hydra minicollagens are only solubilized by reduction (1, 12–14). We demonstrated recently that polymerization of recombinantly expressed minicollagen-1 can be triggered in vitro by oxidative reshuffling of the initially soluble collagen trimers (10). Because cysteine residues are constricted to the short terminal domains of the minicollagen molecules, we proposed a model in which the cysteine-rich domains function as linkers of a collagen polymer by an intermolecular exchange of disulfide linkage (6).

NOWA contains homologous cysteine-rich domains as a C-terminal octarepeat suggesting a similar assembly process and a possibility to link to minicollagens (4). Recombinant expression of NOWA and the isolated MCROD showed now that these proteins spontaneously assemble to disulfide-linked homo-oligomers with a globular shape (Figs. 1 and 2). Consequently, the homo-oligomerization process of NOWA is mainly defined by its C-terminal cysteine-rich domain.

It was proposed that NOWA proteins regulate the assembly of the capsule wall (4). We found that NOWA particles show a high tendency for aggregation (Fig. 2A). Interestingly, recombinant NOWA particles can also associate with a lipid bilayer, probably by ionic interactions.2 This suggests that a membrane-bound layer is formed, which then might serve as a scaffold for minicollagen polymerization. The very early expression of NOWA concomitant with capsule formation emphasizes its regulatory function in nematocyst morphogenesis (4).

Electron micrographs of negatively stained wall fragments revealed a dense network of rodlike molecules filling the space between the globular wall particles and apparently connected to them from various directions (Fig. 2F). Isolation of wall particles by mild sonication sometimes preserved these native complexes showing spheres with a halo of smaller globular domains surrounding them in a distance of ca. 6 nm (Fig. 2E). We have shown earlier that minicollagen-1 molecules visualized by rotary shadowing display short rodlike structures of about 15 nm terminated by globular extensions at both ends (1). Our model for NOWA-minicollagen heteroassembly would therefore propose an interlinking of NOWA molecules via the bipolar minicollagen MCRDs, which would then facilitate elongation of a polymer in all three dimensions.

We cannot rule out, however, that this assembly process follows a preferred directionality resulting in the linear patterns observed on the capsule surface. In electron micrographs of recombinant minicollagen-1 published earlier, we have shown that minicollagens exhibit a high tendency for lateral aggregation (1). This feature of the minicollagen molecules might therefore result in a supramolecular structure that follows the elongated shape of the nematocyst and thus can act as an abutment for the high internal pressure.

In the present study we also show by FESEM analysis that the mature nematocyst wall displays a rather uniform appearance in cross-sections accessed by ultrasound-induced ruptures. This was a surprising result because previous transmission electron microscopy studies on developing capsules revealed a distinct double-layered wall structure (15, 16). Evidence for a double-layered wall came also from immunogold electron microscopy showing a precise localization of the NOWA protein on the outer side of the capsular wall (4). Furthermore, also AFM on intact capsules seemed to confirm the existence of a double-layered wall, because a layer of outer globular particles was only loosely attached to a hard wall structure with a fibrous substructure (3). The globular structures identified by AFM at low forces correspond roughly in their dimensions to the globular structures resolved by FESEM. They probably represent the same globular wall particles. However, our biochemical data and FESEM imaging of the inner side of the wall clearly indicate that globular shaped particles represent the dominating structural units composing the wall. We therefore presume that the fibrous structure revealed by the AFM approach reveals a subtle feature in the arrangement of wall particles, which is not detectable in fixed capsules prepared for FESEM. During the fixation and dehydration procedure, capsules contract to 50% of their initial size (2). Under those conditions a fiber-like supramolecular arrangement of the wall particles may be no longer visible. Further support for a uniform wall structure comes from light and transmission electron microscopy. In transmission electron microscopy of mature capsules there is only one major wall structure visible, and Nomarsky interference contrast microscopy of living differentiating capsules also reveals only one major wall structure. This suggests that the manifestation of a double-layered wall in developing capsules is an artifact of the preparation (fixation) procedure.

Finally it should be mentioned that cross-linking and "hardening" of the wall at the end of capsule morphogenesis is a comparatively fast event (1–2 h) (15, 17). This suggests that NOWA-minicollagen interactions are initially of a noncovalent nature and that polymer formation is only triggered when the components of the network are fully assembled. It is yet unknown whether the final disulfide reshuffling process is spontaneous or enzymatically controlled. An enzymatic catalysis of the disulfide reshuffling process must be a "self-limiting process," because the diffusion-range of an activated enzyme is impeded by the progression of the wall cross-linking. Such a competitive mechanism could explain why wall particles at the outside of the wall are only loosely cross-linked to the rest of the wall (3).

Our binding studies indicate that the initial NOWA-minicollagen interactions are maintained between the central C-type lectin domain of NOWA and the terminal Cys-rich domains of minicollagens. Because the MCRDs in minicollagens and NOWA probably share the same structure, we suppose that NOWA homoassembly is directed by a similar process. The CTLD of NOWA exhibits a high tendency for dimerization both in SDS-PAGE (Fig. 5B) and in ultracentrifugation studies (data not shown). Dimer formation has been shown to occur in several CTLDs by a subdomain exchange, which may additionally include a disulfide interlinking (18). Binding of minicollagen-1 to NOWA could be competed by addition of a synthetic peptide corresponding to the C-terminal Cys-rich domain of minicollagen-1, indicating that it had been successfully reoxidized. Recently, we have reported the NMR structure of this peptide, which showed an unambiguous disulfide pattern and suggested disulfide bridges involving cysteines in N- and C-terminal positions as likely candidates for intermolecular links (11). Furthermore, we could show that the oxidation kinetics of the minicollagen-1 MCRD is extremely fast and lies well within the time limit proposed for the wall hardening process. It remains to be shown whether the MCRDs in NOWA show an identical fold and disulfide pattern and how the reshuffling process takes place on the molecular level.


   FOOTNOTES
 
* This work was supported by Swiss National Science Foundation Grant 3100-049281.96 and Deutsche Forschungsgemeinschaft Grant SFB-269 (to T. W. H.). 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. Back

§ To whom correspondence should be addressed. Tel.: 41612672204; Fax: 41612672189; E-mail: Suat.Oezbek{at}unibas.ch.

1 The abbreviations used are: AFM, atomic force microscopy; FESEM, field emission scanning electron microscopy; MCRD, minicollagen cysteine-rich domain; MCROD, minicollagen cysteine-rich octarepeat domain; PBS, phosphate-buffered saline; Fmoc, N-(9-fluorenyl)methoxy-carbonyl; HPLC, high pressure liquid chromatography; CTLD, C-type lectin domain; MBP, maltose-binding protein. Back

2 E. Pokidysheva, C. Wurm, J. Engel, T. W. Holstein, and S. Ozbek, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Daniel Mathys (Zentrum für Mikroskopie Universität Basel) for performing the FESEM analysis.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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