The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra.

BACKGROUND: The Hydra head organizer acts as a signaling center that initiates and maintains the primary body axis in steady state polyps and during budding or regeneration. Wnt/beta-Catenin signaling functions as a primary cue controlling this process, but how Wnt ligand activity is locally restricted at the protein level is poorly understood. Here we report a proteomic analysis of Hydra head tissue leading to the identification of an astacin family proteinase as a Wnt processing factor. RESULTS: Hydra astacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone. HAS-7 siRNA knockdown abrogates HyWnt3 proteolysis in the head tissue and induces a robust double axis phenotype, which is rescued by simultaneous HyWnt3 knockdown. Accordingly, double axes are also observed in conditions of increased Wnt activity as in transgenic actin::HyWnt3 and HyDkk1/2/4 siRNA treated animals. HyWnt3-induced double axes in Xenopus embryos could be rescued by coinjection of HAS-7 mRNA. Mathematical modelling combined with experimental promotor analysis indicate an indirect regulation of HAS-7 by beta-Catenin, expanding the classical Turing-type activator-inhibitor model. CONCLUSIONS: We show the astacin family protease HAS-7 maintains a single head organizer through proteolysis of HyWnt3. Our data suggest a negative regulatory function of Wnt processing astacin proteinases in the global patterning of the oral-aboral axis in Hydra.

New Class of Crosslinker-Free Nanofiber Biomaterials from Hydra Nematocyst Proteins.

Nematocysts, the stinging organelles of cnidarians, have remarkable mechanical properties. Hydra nematocyst capsules undergo volume changes of 50% during their explosive exocytosis and withstand osmotic pressures of beyond 100 bar. Recently, two novel protein components building up the nematocyst capsule wall in Hydra were identified. The cnidarian proline-rich protein 1 (CPP-1) characterized by a "rigid" polyproline motif and the elastic Cnidoin possessing a silk-like domain were shown to be part of the capsule structure via short cysteine-rich domains that spontaneously crosslink the proteins via disulfide bonds. In this study, recombinant Cnidoin and CPP-1 are expressed in E. coli and the elastic modulus of spontaneously crosslinked bulk proteins is compared with that of isolated nematocysts. For the fabrication of uniform protein nanofibers by electrospinning, the preparative conditions are systematically optimized. Both fibers remain stable even after rigorous washing and immersion into bulk water owing to the simultaneous crosslinking of cysteine-rich domains. This makes our nanofibers clearly different from other protein nanofibers that are not stable without chemical crosslinkers. Following the quantitative assessment of mechanical properties, the potential of Cnidoin and CPP-1 nanofibers is examined towards the maintenance of human mesenchymal stem cells.

Emergence of a Thrombospondin Superfamily at the Origin of Metazoans.

Extracellular matrix (ECM) is considered central to the evolution of metazoan multicellularity; however, the repertoire of ECM proteins in nonbilaterians remains unclear. Thrombospondins (TSPs) are known to be well conserved from cnidarians to vertebrates, yet to date have been considered a unique family, principally studied for matricellular functions in vertebrates. Through searches utilizing the highly conserved C-terminal region of TSPs, we identify undisclosed new families of TSP-related proteins in metazoans, designated mega-TSP, sushi-TSP, and poriferan-TSP, each with a distinctive phylogenetic distribution. These proteins share the TSP C-terminal region domain architecture, as determined by domain composition and analysis of molecular models against known structures. Mega-TSPs, the only form identified in ctenophores, are typically >2,700 aa and are also characterized by N-terminal leucine-rich repeats and central cadherin/immunoglobulin domains. In cnidarians, which have a well-defined ECM, Mega-TSP was expressed throughout embryogenesis in Nematostella vectensis, with dynamic endodermal expression in larvae and primary polyps and widespread ectodermal expression in adult Nematostella vectensis and Hydra magnipapillata polyps. Hydra Mega-TSP was also expressed during regeneration and siRNA-silencing of Mega-TSP in Hydra caused specific blockade of head regeneration. Molecular phylogenetic analyses based on the conserved TSP C-terminal region identified each of the TSP-related groups to form clades distinct from the canonical TSPs. We discuss models for the evolution of the newly defined TSP superfamily by gene duplications, radiation, and gene losses from a debut in the last metazoan common ancestor. Together, the data provide new insight into the evolution of ECM and tissue organization in metazoans.

Wnt/PCP controls spreading of Wnt/ß-catenin signals by cytonemes in vertebrates.

Signaling filopodia, termed cytonemes, are dynamic actin-based membrane structures that regulate the exchange of signaling molecules and their receptors within tissues. However, how cytoneme formation is regulated remains unclear. Here, we show that Wnt/planar cell polarity (PCP) autocrine signaling controls the emergence of cytonemes, and that cytonemes subsequently control paracrine Wnt/ß-catenin signal activation. Upon binding of the Wnt family member Wnt8a, the receptor tyrosine kinase Ror2 becomes activated. Ror2/PCP signaling leads to the induction of cytonemes, which mediate the transport of Wnt8a to neighboring cells. In the Wnt-receiving cells, Wnt8a on cytonemes triggers Wnt/ß-catenin-dependent gene transcription and proliferation. We show that cytoneme-based Wnt transport operates in diverse processes, including zebrafish development, murine intestinal crypt and human cancer organoids, demonstrating that Wnt transport by cytonemes and its control via the Ror2 pathway is highly conserved in vertebrates.

Hydra Mesoglea Proteome Identifies Thrombospondin as a Conserved Component Active in Head Organizer Restriction.

Thrombospondins (TSPs) are multidomain glycoproteins with complex matricellular functions in tissue homeostasis and remodeling. We describe a novel role of TSP as a Wnt signaling target in the basal eumetazoan Hydra. Proteome analysis identified Hydra magnipapillata TSP (HmTSP) as a major component of the cnidarian mesoglea. In general, the domain organization of cnidarian TSPs is related to the pentameric TSPs of bilaterians, and in phylogenetic analyses cnidarian TSPs formed a separate clade of high sequence diversity. HmTSP expression in polyps was restricted to the hypostomal tip and tentacle bases that harbor Wnt-regulated organizer tissues. In the hypostome, HmTSP- and Wnt3-expressing cells were identical or in close vicinity to each other, and regions of ectopic tentacle formation induced by pharmacological ß-Catenin activation (Alsterpaullone) corresponded to foci of HmTSP expression. Chromatin immunoprecipitation (ChIP) confirmed binding of Hydra TCF to conserved elements in the HmTSP promotor region. Accordingly, ß-Catenin knockdown by siRNAs reduced normal HmTSP expression at the head organizer. In contrast, knockdown of HmTSP expression led to increased numbers of ectopic organizers in Alsterpaullone-treated animals, indicating a negative regulatory function. Our data suggest an unexpected role for HmTSP as a feedback inhibitor of Wnt signaling during Hydra body axis patterning and maintenance.

Spatially resolved photochemical coding of reversibly anchored cysteine-rich domains.

We present a novel methodology to generate recodable surfaces using cysteine-rich domains (CRD) via a combination of photolithography and reversible covalently peptide-driven disulfide formation. Therefore, two 21mer CRD peptide derivatives were synthesized, one bearing an electron deficient fumarate group for immobilization via nitrile imine-ene mediated cycloaddition (NITEC) to a tetrazole-functional surface. Secondly, a bromine moiety is introduced to the CRD for analytic labelling purposes to detect surface encoding. The photolithography is conducted by selectively passivating the surface with a polyethylene glycol (PEG)-fumarate via NITEC using a photomask in a dotted pattern. Consecutively, the CRD-fumarate is immobilized via NITEC adjacent to the PEG-functional areas to the unaffected tetrazole covered surface layer. Subsequently, the CRD-bromide is covalently linked to the CRD-fumarate by forming disulfide bonds under mild reoxidative conditions in a buffer solution. The CRD-bromide is released from the surface upon reduction to recover the prior state of the surface without the bromine marker. The analysis of the CRD precursors is based on electrospray ionization mass spectrometry (ESI-MS). The surface analytics were carried out via time-of-flight secondary ion mass spectrometry (ToF-SIMS), unambiguously verifying the successful immobilization as well as coding and decoding of the CRD-bromide on the surface based on dynamically reversible disulfide bond formation.

Minicollagen cysteine-rich domains encode distinct modes of polymerization to form stable nematocyst capsules.

The stinging capsules of cnidarians, nematocysts, function as harpoon-like organelles with unusual biomechanical properties. The nanosecond discharge of the nematocyst requires a dense protein network of the capsule structure withstanding an internal pressure of up to 150 bar. Main components of the capsule are short collagens, so-called minicollagens, that form extended polymers by disulfide reshuffling of their cysteine-rich domains (CRDs). Although CRDs have identical cysteine patterns, they exhibit different structures and disulfide connectivity at minicollagen N and C-termini. We show that the structurally divergent CRDs have different cross-linking potentials in vitro and in vivo. While the C-CRD can participate in several simultaneous intermolecular disulfides and functions as a cystine knot after minicollagen synthesis, the N-CRD is monovalent. Our combined experimental and computational analyses reveal the cysteines in the C-CRD fold to exhibit a higher structural propensity for disulfide bonding and a faster kinetics of polymerization. During nematocyst maturation, the highly reactive C-CRD is instrumental in efficient cross-linking of minicollagens to form pressure resistant capsules. The higher ratio of C-CRD folding types evidenced in the medusozoan lineage might have fostered the evolution of novel, predatory nematocyst types in cnidarians with a free-swimming medusa stage.

Secreted Frizzled-related Protein 2 (sFRP2) Redirects Non-canonical Wnt Signaling from Fz7 to Ror2 during Vertebrate Gastrulation.

Convergent extension movements during vertebrate gastrulation require a balanced activity of non-canonical Wnt signaling pathways, but the factors regulating this interplay on the molecular level are poorly characterized. Here we show that sFRP2, a member of the secreted frizzled-related protein (sFRP) family, is required for morphogenesis and papc expression during Xenopus gastrulation. We further provide evidence that sFRP2 redirects non-canonical Wnt signaling from Frizzled 7 (Fz7) to the receptor tyrosine kinase-like orphan receptor 2 (Ror2). During this process, sFRP2 promotes Ror2 signal transduction by stabilizing Wnt5a-Ror2 complexes at the membrane, whereas it inhibits Fz7 signaling, probably by blocking Fz7 receptor endocytosis. The cysteine-rich domain of sFRP2 is sufficient for Ror2 activation, and related sFRPs can substitute for this function. Notably, direct interaction of the two receptors via their cysteine-rich domains also promotes Ror2-mediated papc expression but inhibits Fz7 signaling. We propose that sFRPs can act as a molecular switch, channeling the signal input for different non-canonical Wnt pathways during vertebrate gastrulation.

A Comprehensive Transcriptomic and Proteomic Analysis of Hydra Head Regeneration.

The cnidarian freshwater polyp Hydra sp. exhibits an unparalleled regeneration capacity in the animal kingdom. Using an integrative transcriptomic and stable isotope labeling by amino acids in cell culture proteomic/phosphoproteomic approach, we studied stem cell-based regeneration in Hydra polyps. As major contributors to head regeneration, we identified diverse signaling pathways adopted for the regeneration response as well as enriched novel genes. Our global analysis reveals two distinct molecular cascades: an early injury response and a subsequent, signaling driven patterning of the regenerating tissue. A key factor of the initial injury response is a general stabilization of proteins and a net upregulation of transcripts, which is followed by a subsequent activation cascade of signaling molecules including Wnts and transforming growth factor (TGF) beta-related factors. We observed moderate overlap between the factors contributing to proteomic and transcriptomic responses suggesting a decoupled regulation between the transcriptional and translational levels. Our data also indicate that interstitial stem cells and their derivatives (e.g., neurons) have no major role in Hydra head regeneration. Remarkably, we found an enrichment of evolutionarily more recent genes in the early regeneration response, whereas conserved genes are more enriched in the late phase. In addition, genes specific to the early injury response were enriched in transposon insertions. Genetic dynamicity and taxon-specific factors might therefore play a hitherto underestimated role in Hydra regeneration.

Nodal signalling determines biradial asymmetry in Hydra.

In bilaterians, three orthogonal body axes define the animal form, with distinct anterior-posterior, dorsal-ventral and left-right asymmetries. The key signalling factors are Wnt family proteins for the anterior-posterior axis, Bmp family proteins for the dorsal-ventral axis and Nodal for the left-right axis. Cnidarians, the sister group to bilaterians, are characterized by one oral-aboral body axis, which exhibits a distinct biradiality of unknown molecular nature. Here we analysed the biradial growth pattern in the radially symmetrical cnidarian polyp Hydra, and we report evidence of Nodal in a pre-bilaterian clade. We identified a Nodal-related gene (Ndr) in Hydra magnipapillata, and this gene is essential for setting up an axial asymmetry along the main body axis. This asymmetry defines a lateral signalling centre, inducing a new body axis of a budding polyp orthogonal to the mother polyp's axis. Ndr is expressed exclusively in the lateral bud anlage and induces Pitx, which encodes an evolutionarily conserved transcription factor that functions downstream of Nodal. Reminiscent of its function in vertebrates, Nodal acts downstream of ß-Catenin signalling. Our data support an evolutionary scenario in which a 'core-signalling cassette' consisting of ß-Catenin, Nodal and Pitx pre-dated the cnidarian-bilaterian split. We presume that this cassette was co-opted for various modes of axial patterning: for example, for lateral branching in cnidarians and left-right patterning in bilaterians.

Molecular dissection of Wnt3a-Frizzled8 interaction reveals essential and modulatory determinants of Wnt signaling activity.

BACKGROUND: Wnt proteins are a family of secreted signaling molecules that regulate key developmental processes in metazoans. The molecular basis of Wnt binding to Frizzled and LRP5/6 co-receptors has long been unknown due to the lack of structural data on Wnt ligands. Only recently, the crystal structure of the Wnt8-Frizzled8-cysteine-rich-domain (CRD) complex was solved, but the significance of interaction sites that influence Wnt signaling has not been assessed. RESULTS: Here, we present an extensive structure-function analysis of mouse Wnt3a in vitro and in vivo. We provide evidence for the essential role of serine 209, glycine 210 (site 1) and tryptophan 333 (site 2) in Fz binding. Importantly, we discovered that valine 337 in the site 2 binding loop is critical for signaling without contributing to binding. Mutations in the presumptive second CRD binding site (site 3) partly abolished Wnt binding. Intriguingly, most site 3 mutations increased Wnt signaling, probably by inhibiting Wnt-CRD oligomerization. In accordance, increasing amounts of soluble Frizzled8-CRD protein modulated Wnt3a signaling in a biphasic manner. CONCLUSIONS: We propose a concentration-dependent switch in Wnt-CRD complex formation from an inactive aggregation state to an activated high mobility state as a possible modulatory mechanism in Wnt signaling gradients.

Proteome of Hydra nematocyst.

Stinging cells or nematocytes of jellyfish and other cnidarians represent one of the most poisonous and sophisticated cellular inventions in animal evolution. This ancient cell type is unique in containing a giant secretory vesicle derived from the Golgi apparatus. The organelle structure within the vesicle comprises an elastically stretched capsule (nematocyst) to which a long tubule is attached. During exocytosis, the barbed part of the tubule is accelerated with >5 million g in <700 ns, enabling a harpoon-like discharge (Nüchter, T., Benoit, M., Engel, U., Ozbek, S., and Holstein, T. W. (2006) Curr. Biol. 16, R316-R318). Hitherto, the molecular components responsible for the organelle's biomechanical properties were largely unknown. Here, we describe the proteome of nematocysts from the freshwater polyp Hydra magnipapillata. Our analysis revealed an unexpectedly complex secretome of 410 proteins with venomous and lytic but also adhesive or fibrous properties. In particular, the insoluble fraction of the nematocyst represents a functional extracellular matrix structure of collagenous and elastic nature. This finding suggests an evolutionary scenario in which exocytic vesicles harboring a venomous secretome assembled a sophisticated predatory structure from extracellular matrix motif proteins.

In vivo imaging of basement membrane movement: ECM patterning shapes Hydra polyps.

Growth and morphogenesis during embryonic development, asexual reproduction and regeneration require extensive remodeling of the extracellular matrix (ECM). We used the simple metazoan Hydra to examine the fate of ECM during tissue morphogenesis and asexual budding. In growing Hydra, epithelial cells constantly move towards the extremities of the animal and into outgrowing buds. It is not known, whether these tissue movements involve epithelial migration relative to the underlying matrix or whether cells and ECM are displaced as a composite structure. Furthermore, it is unclear, how the ECM is remodeled to adapt to the shape of developing buds and tentacles. To address these questions, we used a new in vivo labeling technique for Hydra collagen-1 and laminin, and tracked the fate of ECM in all body regions of the animal. Our results reveal that Hydra 'tissue movements' are largely displacements of epithelial cells together with associated ECM. By contrast, during the evagination of buds and tentacles, extensive movement of epithelial cells relative to the matrix is observed, together with local ECM remodeling. These findings provide new insights into the nature of growth and morphogenesis in epithelial tissues.

Expression of pancreatitis-associated protein after traumatic brain injury: a mechanism potentially contributing to neuroprotection in human brain.

Neuronal cell death after severe traumatic brain injury (TBI) is caused by a complex interplay of pathological mechanisms including excitotoxicity, oxidative stress, mitochondrial dysfunction, extensive neuroinflammation, and ischemia-reperfusion injury. Pancreatitis-associated protein I (PAP I/reg2) was reported to be a survival factor for peripheral neurons, particularly sensory and motor neurons. In rat brains, by experimental TBI as well as by kainic acid induced brain seizure, PAP I and PAP III were found to be up-regulated in central neurons. In this study, we performed immunohistochemical staining in postmortem human brain from patients who died after severe TBI to demonstrate PAP expression on protein level in cerebellar Purkinje cells, pyramidal and granular neurons in cerebral cortex, and cortical neurons in the fore- and mid-brain. In primary cultures of rat brain cortical, hippocampal, and cerebellar neurons, we found neuroprotective effects for PAP I on H(2)O(2)-induced oxidative stress. Moreover, serum K(+)-deprivation induces apoptotic cell death in 55% of cerebellar granule neurons (CGN), whereas upon treatment with PAP I only 32% of CGN are apoptotic. Using Western blot analyses, we compared protein phosphorylation in neuronal signaling pathways activated by PAP I versus Interleukin-6 (IL-6). We found a rapid activation of Akt-kinase phosphorylation by PAP I with a peak at 15 min, whereas IL-6 induces Akt-phosphorylation lasting longer than 30 min. Phosphorylation of MAP-42/44 kinases is stimulated in a comparable fashion. Both, IL-6 and PAP I increase phosphorylation of NFκB for activation of gene transcription, whereas only IL-6 recruits STAT3 phosphorylation, indicating that STAT3 is not a target of PAP I transcription activation in brain neurons. Application of the Akt-inhibitor Wortmanin reveals only a partial inhibition of PAP I-dependent protection of CGN from H(2)O(2)-induced oxidative stress. Based on our findings, we suggest that PAP I is a long lasting neurotrophic signal for central neurons. The neuroprotective effects parallel those that have been described for effects of PAP I in ciliary neurotrophic factor (CNTF)-mediated survival of sensory and motor neurons. PAP I may act in autocrine and/or paracrine fashion and thus may contribute to endogenous protective mechanisms relevant under harmful conditions like oxidative stress, brain injury, or neurodegeneration.

The dynamic genome of Hydra.

The freshwater cnidarian Hydra was first described in 1702 and has been the object of study for 300 years. Experimental studies of Hydra between 1736 and 1744 culminated in the discovery of asexual reproduction of an animal by budding, the first description of regeneration in an animal, and successful transplantation of tissue between animals. Today, Hydra is an important model for studies of axial patterning, stem cell biology and regeneration. Here we report the genome of Hydra magnipapillata and compare it to the genomes of the anthozoan Nematostella vectensis and other animals. The Hydra genome has been shaped by bursts of transposable element expansion, horizontal gene transfer, trans-splicing, and simplification of gene structure and gene content that parallel simplification of the Hydra life cycle. We also report the sequence of the genome of a novel bacterium stably associated with H. magnipapillata. Comparisons of the Hydra genome to the genomes of other animals shed light on the evolution of epithelia, contractile tissues, developmentally regulated transcription factors, the Spemann-Mangold organizer, pluripotency genes and the neuromuscular junction.

Utilization of a right-handed coiled-coil protein from archaebacterium Staphylothermus marinus as a carrier for cisplatin.

BACKGROUND: The nano-sized right-handed coiled-coil (RHCC) protein, originating from the archaebacterium Staphylothermus marinus, is stable at high salt concentrations, high temperatures, high pressures and extremes of pH. Its crystal structure reveals four hydrophobic cavities which can incorporate heavy metals. Nano-sized compounds have been used to carry cytotoxic drugs to tumours, avoiding delivery to healthy tissue, in part due to enhanced permeability in tumour blood vessels (enhanced permeability and retention effect). MATERIALS AND METHODS: The ability of RHCC to carry the platinum-containing chemotherapeutic drug cisplatin to cells, while retaining the cytotoxic potential was tested both in vitro and in vivo. RESULTS: RHCC was able to bind and enter cells in vitro and was not severely toxic or immunogenic in mice. Moreover, RHCC incorporated cisplatin, without inhibiting the cytotoxic potential of the drug against tumour cell lines in vitro or in vivo. CONCLUSION: RHCC can be used as a carrier of cisplatin without abrogating the effect of the drug.

Multiple Wnts are involved in Hydra organizer formation and regeneration.

Wnt genes and beta-catenin signaling are involved in axial patterning processes in vertebrate embryogenesis in setting up the Spemann-Mangold organizer in amphibian embryos. An organizer with a similar function is present in the hypostome of an adult Hydra polyp. Previously, a Hydra ortholog of Wnt3 (HyWnt3), which is expressed in the hypostome, has been described. Here, ten additional Hydra Wnt genes have been identified. Of these, six (HyWnt1, -7, -9/10a, -9/10c, -11, and -16) are expressed in the adult hypostome. And, as is HyWnt3, these six Wnt genes are also expressed when a new head organizer is formed during head regeneration and bud formation. The kinetics of Wnt gene expressions during head regeneration suggests that a cascade of consecutive Wnt activation accompanies regeneration, and HyWnt3 begins this cascade. Recombinant HyWnt3 protein induced body column tissue to undergo head formation. It also increased the head formation capacity in the head regeneration-deficient mutant strain reg-16 to that of wild-type strains. In addition our data reveal striking similarities in the molecular basis of the organizer in Hydra and axis polarization in chordates (e.g. Spemann's organizer) as well as it's role in regeneration suggesting a conserved function of Wnt signaling in setting up this ancient metazoan signaling center.

Wnt/beta-catenin and noncanonical Wnt signaling interact in tissue evagination in the simple eumetazoan Hydra.

In and evaginations of 2D cell sheets are major shape generating processes in animal development. They result from directed movement and intercalation of polarized cells associated with cell shape changes. Work on several bilaterian model organisms has emphasized the role of noncanonical Wnt signaling in cell polarization and movement. However, the molecular processes responsible for generating tissue and body shape in ancestral, prebilaterian animals are unknown. We show that noncanonical Wnt signaling acts in mass tissue movements during bud and tentacle evagination and regeneration in the cnidarian polyp Hydra. The wnt5, wnt8, frizzled2 (fz2), and dishevelled-expressing cell clusters define the positions, where bud and tentacle evaginations are initiated; wnt8, fz2, and dishevelled remain up-regulated in those epithelial cells, undergoing cell shape changes during the entire evagination process. Downstream of wnt and dsh expression, JNK activity is required for the evagination process. Multiple ectopic wnt5, wnt8, fz2, and dishevelled-expressing centers and the subsequent evagination of ectopic tentacles are induced throughout the body column by activation of Wnt/beta-Catenin signaling. Our results indicate that integration of axial patterning and tissue morphogenesis by the coordinated action of canonical and noncanonical Wnt pathways was crucial for the evolution of eumetazoan body plans.

Evolution of complex structures: minicollagens shape the cnidarian nematocyst.

The generation of biological complexity by the acquisition of novel modular units is an emerging concept in evolutionary dynamics. Here, we review the coordinate evolution of cnidarian nematocysts, secretory organelles used for capture of prey, and of minicollagens, proteins constituting the nematocyst capsule. Within the Cnidaria there is an increase in nematocyst complexity from Anthozoa to Medusozoa and a parallel increase in the number and complexity of minicollagen proteins. This complexity is primarily manifest in a diversification of N- and C-terminal cysteine-rich domains (CRDs) involved in minicollagen polymerization. We hypothesize that novel CRD motifs alter minicollagen networks, leading to novel capsule structures and nematocyst types.

Minicollagen-15, a novel minicollagen isolated from Hydra, forms tubule structures in nematocysts.

Minicollagens constitute a family of unusually short collagen molecules isolated from cnidarians. They are restricted to the nematocyst, a cylindrical explosive organelle serving in defense and capture of prey. The nematocyst capsule contains a long tubule inside of its matrix, which is expelled and everted during an ultrafast discharge process. Here, we report the cloning and characterization of a novel minicollagen in Hydra, designated minicollagen-15 (NCol-15). NCol-15, like NCol-3 and NCol-4, shows deviations from the canonical cysteine pattern in its terminal cysteine-rich domains (CRDs). Minicollagens share common domain architectures with a central collagen sequence flanked by polyproline stretches and short N- and C-terminal CRDs. The CRDs are involved in the formation of a highly resistant cysteine network, which constitutes the basic structure of the nematocyst capsule. Unlike NCol-1, which is part of the capsule wall, NCol-15 is localized to tubules, arguing for a functional differentiation of minicollagens within the nematocyst architecture. NMR analysis of the altered C-terminal CRD of NCol-15 showed a novel disulfide-linked structure within the cysteine-containing region exhibiting similar folding kinetics and stability as the canonical CRDs. Our data provide evidence for evolutionary diversification among minicollagens, which probably facilitated alterations in the morphology of the nematocyst wall and tubule.