Sequence-structure and structure-function analysis in cysteine-rich domains forming the ultrastable nematocyst wall

The nematocyst wall of cnidarians is a unique biomaterial that withstands extreme osmotic pressures, allowing an ultrafast discharge of the nematocyst capsules. Assembly of the highly robust nematocyst wall is achieved by covalent linkage of cysteine-rich domains (CRDs) from two main protein components, minicollagens and nematocyst outer wall antigen (NOWA). The bipolar minicollagens have different disulfide patterns and topologies in their N and C-terminal CRDs. The functional significance of this polarity has been elusive. Here, we show by NMR structural analysis that all representative cysteine-rich domains of NOWA are structurally related to N-terminal minicollagen domains. Natural sequence insertions in NOWA CRDs have very little effect on the tightly knit domain structures, nor do they preclude the efficient folding to a single native conformation. The different folds in NOWA CRDs and the atypical C-terminal minicollagen domain on the other hand can be directly related to different conformational preferences in the reduced states. Ultrastructural analysis in conjunction with aggregation studies argues for an association between the similar NOWA and N-terminal minicollagen domains in early stages of the nematocyst wall assembly, which is followed by the controlled association between the unusual structures of C-terminal minicollagen domains.     

A novel minicollagen gene links cnidarians and myxozoans

Myxozoans are enigmatic endoparasitic organisms sharing morphological features with bilateria, protists and cnidarians. This, coupled with their highly divergent gene sequences, has greatly obscured their phylogenetic affinities. Here we report the sequencing and characterization of a minicollagen homologue (designated Tb-Ncol-1) in the myxozoan Tetracapsuloides bryosalmonae. Minicollagens are phylum-specific genes encoding cnidarian nematocyst proteins. Sequence analysis revealed a cysteine-rich domain (CRD) architecture and genomic organization similar to group 1 minicollagens. Homology modelling predicted similar three-dimensional structures to Hydra CRDs despite deviations from the canonical pattern of group 1 minicollagens. The discovery of this minicollagen gene strongly supports myxozoans as cnidarians that have radiated as endoparasites of freshwater, marine and terrestrial hosts. It also reveals novel protein sequence variation of relevance to understanding the evolution of nematocyst complexity, and indicates a molecular/morphological link between myxozoan polar capsules and cnidarian nematocysts. Our study is the first to illustrate the power of using genes related to a taxon-specific novelty for phylogenetic inference within the Metazoa, and it exemplifies how the evolutionary relationships of other metazoans characterized by extreme sequence divergence could be similarly resolved.     

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.     

The structure of the Cys-rich terminal domain of Hydra minicollagen, which is involved in disulfide networks of the nematocyst wall

The minicollagens found in the nematocysts of Hydra constitute a family of invertebrate collagens with unusual properties. They share a common modular architecture with a central collagen sequence ranging from 14 to 16 Gly-X-Y repeats flanked by polyproline/hydroxyproline stretches and short terminal domains that show a conserved cysteine pattern (CXXXCXXXCXXX-CXXXCC). The minicollagen cysteine-rich domains are believed to function in a switch of the disulfide connectivity from intra- to intermolecular bonds during maturation of the capsule wall. The solution structure of the C-terminal fragment including a minicollagen cysteine-rich domain of minicollagen-1 was determined in two independent groups by 1H NMR. The corresponding peptide comprising the last 24 residues of the molecule was produced synthetically and refolded by oxidation under low protein concentrations. Both presented structures are identical in their fold and disulfide connections (Cys2-Cys18, Cys6-Cys14, and Cys10-Cys19) revealing a robust structural motif that is supposed to serve as the polymerization module of the nematocyst capsule.     

Structure/function analysis of spinalin, a spine protein of Hydra nematocysts

The nematocyst capsules of the cnidarians are specialized explosive organelles that withstand high osmotic pressures of approximately 15 MPa (150 bar). A tight disulfide network involving cysteine-rich capsule wall proteins, like minicollagens and nematocyst outer wall antigen, characterizes their molecular composition. Nematocyst discharge leads to the expulsion of a long inverted tubule that was coiled inside the capsule matrix before activation. Spinalin has been characterized as a glycine-rich, histidine-rich protein associated with spine structures on the surface of everted tubules. Here, we show that full-length Hydra spinalin can be expressed recombinantly in HEK293 cells and has the property to form disulfide-linked oligomers, reflecting its state in mature capsules. Furthermore, spinalin showed a high tendency to associate into dimers in vitro and in vivo. Our data, which show incomplete disulfide connectivity in recombinant spinalin, suggest a possible mechanism by which the spine structure may be linked to the overall capsule polymer.     

The glycoprotein NOWA and minicollagens are part of a disulfide-linked polymer that forms the cnidarian nematocyst wall

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.     

The cnidarian nematocyst: a miniature extracellular matrix within a secretory vesicle

Nematocysts are the taxon-defining features of all cnidarians including jellyfish, sea anemones, and corals. They are highly sophisticated organelles used for the capture of prey and defense. The nematocyst capsule is produced within a giant post-Golgi vesicle, which is continuously fed by proteins from the secretory pathway. Mature nematocysts consist of a hollow capsule body in which a long tubule is coiled up that, upon discharge, is expelled in a harpoon-like fashion. This is accompanied by the release of a toxin cocktail stored in the capsule matrix. Nematocyst discharge, which is one of the fastest processes in biology, is driven by an extreme osmotic pressure of about 150 bar. The molecular analysis of the nematocyst has from the beginning indicated a collagenous nature of the capsule structure. In particular, a large family of unusual minicollagens has been demonstrated to form the highly resistant scaffold of the capsule. Recent findings on the molecular composition of Hydra nematocysts have confirmed the notion of a specialized extracellular matrix, which is assembled during an intracellular secretion process to form the most complex predatory apparatus at the cellular level.     

A Non-sulfated Chondroitin Stabilizes Membrane Tubulation in Cnidarian Organelles

Membrane tubulation is generally associated with rearrangements of the cytoskeleton and other cytoplasmic factors. Little is known about the contribution of extracellular matrix components to this process. Here, we demonstrate an essential role of proteoglycans in the tubulation of the cnidarian nematocyst vesicle. The morphogenesis of this extrusive organelle takes place inside a giant post-Golgi vesicle, which topologically represents extracellular space. This process includes the formation of a complex collagenous capsule structure that elongates into a long tubule, which invaginates after its completion. We show that a non-sulfated chondroitin appears as a scaffold in early morphogenesis of all nematocyst types in Hydra and Nematostella. It accompanies the tubulation of the vesicle membrane forming a provisional tubule structure, which after invagination matures by collagen incorporation. Inhibition of chondroitin synthesis by β-xylosides arrests nematocyst morphogenesis at different stages of tubule outgrowth resulting in retention of tubule material and a depletion of mature capsules in the tentacles of hydra. Our data suggest a conserved role of proteoglycans in the stabilization of a membrane protrusion as an essential step in organelle morphogenesis.     

Expression profiling and cellular localization of myxozoan minicollagens during nematocyst formation and sporogenesis

In free-living cnidarians, minicollagens are major structural components in the biogenesis of nematocysts. Recent sequence mining and proteomic analysis demonstrate that minicollagens are also expressed by myxozoans, a group of evolutionarily ancient cnidarian endoparasites. Nonetheless, the presence and abundance of nematocyst-associated genes/proteins in nematocyst morphogenesis have never been studied in Myxozoa. Here, we report the gene expression profiles of three myxozoan minicollagens, ncol-1, ncol-3, and the recently identified noncanonical ncol-5, during the intrapiscine development of Myxidium lieberkuehni, the myxozoan parasite of the northern pike, Esox lucius. Moreover, we localized the myxozoan-specific minicollagen Ncol-5 in the developing myxosporean stages by Western blotting, immunofluorescence, and immunogold electron microscopy. We found that expression of minicollagens was spatiotemporally restricted to developing nematocysts within the myxospores during sporogenesis. Intriguingly, Ncol-5 is localized in the walls of nematocysts and predominantly in nematocyst tubules. Overall, we demonstrate that despite being significantly reduced in morphology, myxozoans retain structural components associated with nematocyst development in free-living cnidarians. Furthermore, our findings have practical implications for future functional and comparative studies as minicollagens are useful markers of the developmental phase of myxozoan parasites.     

A switch in disulfide linkage during minicollagen assembly in Hydra nematocysts.

The smallest known collagens with only 14 Gly-X-Y repeats referred to as minicollagens are the main constituents of the capsule wall of nematocysts. These are explosive organelles found in Hydra, jellyfish, corals and other Cnidaria. Minicollagen-1 of Hydra recombinantly expressed in mammalian 293 cells contains disulfide bonds within its N- and C-terminal Cys-rich domains but no interchain cross-links. It is soluble and self-associates through non-covalent interactions to form 25-nm-long trimeric helical rod-like molecules. We have used a polyclonal antibody prepared against the recombinant protein to follow the maturation of minicollagens from soluble precursors present in the endoplasmic reticulum and post-Golgi vacuoles to the disulfide-linked insoluble assembly form of the wall. The switch from intra- to intermolecular disulfide bonds is associated with 'hardening' of the capsule wall and provides an explanation for its high tensile strength and elasticity. The process is comparable to disulfide reshuffling between the NC1 domains of collagen IV in mammalian basement membranes.     

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.     

Ultrastructure of a novel eurytele nematocyst of Carybdea alata Reynaud (Cubozoa,Cnidaria)

The ultrastructural characteristics of nematocysts from the cubozoan Carybdea alata Reynaud, 1830 (Hawaiian box jellyfish) were examined using light, scanning and transmission electron microscopy. We reclassified the predominant nematocyst in C. alata tentacles as a heterotrichous microbasic eurytele, based on spine, tubule and capsule measurements. These nematocysts exhibited a prominent and singular stylet, herein referred to as the lancet. Discharged nematocysts from fixed tentacle preparations displayed the following structures: a smooth shaft base, lamellae, a hemicircumferential fissure demarking the proximal end of a stratified lancet, and a gradually tapering tubule densely covered with large triangularly shaped spines. The lancet remained partially adjoined to the shaft base in a hinge-like fashion in rapidly fixed, whole-tentacle preparations. In contrast, this structure was not observed in discharged nematocyst preparations which involved multiple transfer steps prior to fixation. Various approaches were designed to detect this structure in the absence of fixative. Detached lancets were located in proximity to discharged tubules in undisturbed coverslip preparations of fresh tentacles. In addition, examination of embedded nematocysts from fresh tentacles laid on polyacrylamide gels revealed still-attached lancets. To examine the function of this structure in prey capture, Artemia sp. laden tentacles were prepared for scanning electron microscopy. While carapace exteriors exhibited structures proximal to the lancet, i.e., the nematocyst capsule and shaft base, neither tubule nor lancet structures were visible. Taken together, the morphological data suggested a series of events involved in the discharge of a novel eurytele from C. alata.     

Structure of the leech protein saratin and characterization of its binding to collagen

The leech protein Saratin from Hirudo medicinalis prevents thrombocyte aggregation by interfering with the first binding step of the thrombocytes to collagen by binding to collagen. We solved the three-dimensional structure of the leech protein Saratin in solution and identified its collagen binding site by NMR titration experiments. The NMR structure of Saratin consists of one α-helix and a five-stranded β-sheet arranged in the topology ββαβββ. The C-terminal region, of about 20 amino acids in length, adopts no regular structure. NMR titration experiments with collagen peptides show that the collagen interaction of Saratin takes place in a kind of notch that is formed by the end of the α-helix and the β-sheet. NMR data-driven docking experiments to collagen model peptides were used to elucidate the putative binding mode of Saratin and collagen. Mainly, parts of the first and the end of the fifth β-strand, the loop connecting the α-helix and the third β-strand, and a short part of the loop connecting the fourth and fifth β-strand participate in binding.     

Remodelling during the development of nematocysts in a siphonophore

Remodelling of a smoothly tapering internal tubule to form the enlarged shaft of microbasic mastigophores of the siphonophore Rosacea takes place very late in development. It occurs after deployment of the nematocyst in its final position, and after the pleats of the internal tubule have been completely obliterated in the shaft region by the growth of spine material. The optical and mechanical properties of the internal tubule at the earlier (bedspring) stage give no hint that a shaft will develop. The possible ways in which remodelling might occur inside the capsule and remote from the cytoplasm are discussed.     

Recognition and Detoxification of the Insecticide DDT by Drosophila melanogaster Glutathione S-Transferase D1

GSTD1 is one of several insect glutathione S-transferases capable of metabolizing the insecticide DDT. Here we use crystallography and NMR to elucidate the binding of DDT and glutathione to GSTD1. The crystal structure of Drosophila melanogaster GSTD1 has been determined to 1.1 Å resolution, which reveals that the enzyme adopts the canonical GST fold but with a partially occluded active site caused by the packing of a C-terminal helix against one wall of the binding site for substrates. This helix would need to unwind or be displaced to enable catalysis. When the C-terminal helix is removed from the model of the crystal structure, DDT can be computationally docked into the active site in an orientation favoring catalysis. Two-dimensional ¹H,¹⁵N heteronuclear single-quantum coherence NMR experiments of GSTD1 indicate that conformational changes occur upon glutathione and DDT binding and the residues that broaden upon DDT binding support the predicted binding site. We also show that the ancestral GSTD1 is likely to have possessed DDT dehydrochlorinase activity because both GSTD1 from D. melanogaster and its sibling species, Drosophila simulans, have this activity.     

Length Is Associated with Pain: Jellyfish with Painful Sting Have Longer Nematocyst Tubules than Harmless Jellyfish

A large number of humans are stung by jellyfish all over the world. The stings cause acute pain followed by persistent pain and local inflammation. Harmful jellyfish species typically cause strong pain, whereas harmless jellyfish cause subtle or no pain. Jellyfish sting humans by injecting a tubule, contained in the nematocyst, the stinging organ of jellyfish. The tubule penetrates into the skin leading to venom injection. The detailed morphology of the nematocyst tubule and molecular structure of the venom in the nematocyst has been reported; however, the mechanism responsible for the difference in pain that is caused by harmful and harmless jellyfish sting has not yet been explored or explained. Therefore, we hypothesized that differences in the length of the nematocyst tubule leads to different degrees of epithelial damage. The initial acute pain might be generated by penetration of the tubule, which stimulates pain receptor neurons, whilst persistent pain might be caused by injection of venom into the epithelium. To test this hypothesis we compared the lengths of discharged nematocyst tubules from harmful and harmless jellyfish species and evaluated their ability to penetrate human skin. The results showed that the harmful jellyfish species, Chrysaora pacifica, Carybdea brevipedalia, and Chironex yamaguchii, causing moderate to severe pain, have nematocyst tubules longer than 200 μm, compared with a jellyfish species that cause little or no pain, Aurelia aurita. The majority of the tubules of harmful jellyfishes, C. yamaguchii and C. brevipedalia, were sufficiently long to penetrate the human epidermis and physically stimulate the free nerve endings of Aδ pain receptor fibers around plexuses to cause acute pain and inject the venom into the human skin epithelium to cause persistent pain and inflammation.     

Structures of a Bifunctional Cell Wall Hydrolase CwlT Containing a Novel Bacterial Lysozyme and an NlpC/P60 dl-Endopeptidase

Tn916-like conjugative transposons carrying antibiotic resistance genes are found in a diverse range of bacteria. Orf14 within the conjugation module encodes a bifunctional cell wall hydrolase CwlT that consists of an N-terminal bacterial lysozyme domain (N-acetylmuramidase, bLysG) and a C-terminal NlpC/P60 domain (γ-d-glutamyl-l-diamino acid endopeptidase) and is expected to play an important role in the spread of the transposons. We determined the crystal structures of CwlT from two pathogens, Staphylococcus aureus Mu50 (SaCwlT) and Clostridium difficile 630 (CdCwlT). These structures reveal that NlpC/P60 and LysG domains are compact and conserved modules, connected by a short flexible linker. The LysG domain represents a novel family of widely distributed bacterial lysozymes. The overall structure and the active site of bLysG bear significant similarity to other members of the glycoside hydrolase family 23 (GH23), such as the g-type lysozyme (LysG) and Escherichia coli lytic transglycosylase MltE. The active site of bLysG contains a unique structural and sequence signature (DxxQSSES + S) that is important for coordinating a catalytic water. Molecular modeling suggests that the bLysG domain may recognize glycan in a similar manner to MltE. The C-terminal NlpC/P60 domain contains a conserved active site (Cys-His-His-Tyr) that appears to be specific to murein tetrapeptide. Access to the active site is likely regulated by isomerism of a side chain atop the catalytic cysteine, allowing substrate entry or product release (open state), or catalysis (closed state).     

ELECTRON MICROSCOPE OBSERVATIONS ON THE STRUCTURE AND DISCHARGE OF THE STENOTELE OF HYDRA

Sections of the stenotele type of nematocyst of Chlorohydra hadleyi have revealed that the stenotele, upon firing, completely everts its stylets and spines and the long, thin tubule, much as the eversion of the tubule of the nematocyst of the jewel anemone (Picken, 1953; Robson, 1953). Alternative mechanisms for supplying the energy necessary to forcefully discharge the stenotele contents are discussed as well as the possible significance of several regions containing highly ordered periodic structure. The origin of nematocysts as kinetosomal derivatives is discussed as a possibility suggested by the symmetry of the stenotele contents and the structure, location, and function of the cnidocil.