Structure/function relationships in the minicollagen of Hydra nematocysts

The minicollagens found in the inner layer of the Hydra nematocyst walls are the smallest collagens known with 12-16 Gly-X-Y repeats. Minicollagen-1, the best characterized member of this protein family so far, consists of a central collagen triple helix of 12 nm in length flanked at both ends by a polyproline stretch and a conserved cysteine-rich domain. The cysteine-rich tails are proposed to function in the assembly of soluble minicollagen trimers to high molecular structures by a switch of the disulfide linkage from intramolecular to intermolecular bonds. In this study, we investigate the trimeric nature of minicollagen-1 and its capacity to form disulfide-linked polymers in vitro. A fusion protein of minicollagen-1 with maltose-binding protein is secreted as a soluble trimer with only intrachain and no interchain disulfide bridges as confirmed by melting the collagen triple helix under reducing and non-reducing conditions. The conversion of minicollagen-1 trimers to monomers takes place between 40 and 55 degrees C with the melting point being approximately 45 degrees C. Oxidative reshuffling of the minicollagen-1 trimers leads to the formation of high molecular aggregates, which upon reduction show distinct polytrimeric states. Minicollagen trimers in isolated nematocyst capsules proved to be sensitive to SDS and were engaged in polymeric structures with additional cross-links that were resistant to reducing agent.     

Rapid exocytosis of stenotele nematocysts in Hydra vulgaris

Each cnidarian nematocyte includes a vesicular organelle, called nematocyst, which discharges its content when the cell receives appropriate stimuli. Extracellular electrical stimuli induced discharge of in situ stenoteletype nematocysts in Hydra vulgaris when the apical membrane of nematocytes was depolarized by about 25 mV or more (threshold). Stimuli hyperpolarizing the apical membrane induced discharge only at high amplitudes, adding about 80 mV or more to the resting membrane potential of the nematocyte (resulting in a voltage that may permeabilize the apical membrane). In order to determine the speed of the initiating (exocytotic) process, the delay between stimulus and a clearly visible sign of discharge (i.e., protrusion of the nematocyst's stylets) was measured using video microscopy with triggered flash illumination. The minimal delay was 330–450 s and 230–350 s for depolarizing and large hyperpolarizing stimuli, respectively. With depolarizing stimuli, all discharges of stenoteles occurred between 330 and 950 s after the stimulus. The deviation was caused by differences in the physiological state of the animals tested rather than by variance in the responsiveness of different stenoteles in the same tentacle.Voltage dependence, short latency and Ca/Mg-antagonism are similar to those characterizing exocytosis of synaptic vesicles. This correspondence suggests that discharge of nematocysts is initiated by a similar exocytotic process preceding the ejection of the nematocyst's content.     

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.     

Organelle survival in a foreign organism: Hydra nematocysts in the flatworm Microstomum lineare

Nematocysts are characteristic organelles of the phylum cnidaria. They are designated kleptocnidae when sequestered in animals that feed on cnidaria. Kleptocnidae are known for more than a century. Nevertheless it is still enigmatic how selected nematocyst types survive in the predator and how they reach their final destination in the foreign body. In the free-living Platyhelminth Microstomum lineare the fate of nematocysts of the prey Hydra oligactis was analyzed at the ultrastructural level and by fluorescence microscopy using hydra polyps that had been stained in vivo with the fluorescent dyes TROMI and TRITC. M. lineare digested hydra tissue in its intestine within 30?min and all nematocyst types were phagocytosed without adherent cytoplasm by intestinal cnidophagocytes. Desmoneme and isorhiza nematocysts were digested whereas cnidophagocytes containing the venom-loaded stenotele nematocysts started to migrate out of the intestinal epithelia through the parenchyma to the epidermis thereby traversing the subintestinal and subepidermal muscle layer. Within one to two days, M. lineare began to form a muscle layer basolateral around epidermal cnidophagocytes. Epidermal stenoteles survived in M. lineare for at least four weeks. The ability of epidermal stenotele nematocysts to discharge suggest that this hydra organelle preserved its physiological properties in the new host.     

Cytological studies of the nematocysts of Hydra. II. The stenoteles

Entire hydras or tentacles were prepared for electron microscopy as described in the preceding paper. The stenotele capsule has been observed to be composed of an external membrane, a thick chitinous or keratin layer, and an inner membrane. A sac-like extension of the capsular wall into the capsule bears spines and stylets on its inner surface and evagination of this structure occurs on discharge. Profiles of tubular or membranous structures often are seen within the capsules of resting stenoteles. These structures are presumably related to the external filament. The spines often reveal a flattened aspect which suggests that at least some of them might more accurately be called "vanes." A cnidocil has been found to accompany each stenotele. This study revealed several aspects of the developmental stages of stenoteles: A vacuole is formed which is nearly surrounded by the nematocyte nucleus. The vacuole content changes in density and a capsular wall is formed at the periphery of the vacuole. Tubules differentiate from the capsular matrix, and spines and stylets develop somewhat later. An operculum is formed from the nematocyte cytoplasm.     

Structure/function analysis of yeast ribosomal protein L2

Ribosomal protein L2 is a core element of the large subunit that is highly conserved among all three kingdoms. L2 contacts almost every domain of the large subunit rRNA and participates in an intersubunit bridge with the small subunit rRNA. It contains a solvent-accessible globular domain that interfaces with the solvent accessible side of the large subunit that is linked through a bridge to an extension domain that approaches the peptidyltransferase center. Here, screening of randomly generated library of yeast RPL2A alleles identified three translationally defective mutants, which could be grouped into two classes. The V48D and L125Q mutants map to the globular domain. They strongly affect ribosomal A-site associated functions, peptidyltransferase activity and subunit joining. H215Y, located at the tip of the extended domain interacts with Helix 93. This mutant specifically affects peptidyl-tRNA binding and peptidyltransferase activity. Both classes affect rRNA structure far away from the protein in the A-site of the peptidyltransferase center. These findings suggest that defective interactions with Helix 55 and with the Helix 65-66 structure may indicate a certain degree of flexibility in L2 in the neck region between the two other domains, and that this might help to coordinate tRNA-ribosome interactions.     

Cytological studies of the nematocysts of Hydra. I. Desmonemes,isorhizas, cnidocils,and supporting structures

Entire hydras or tentacles were fixed in OsO(4) or in KMnO(4) and thereafter washed, dehydrated, and embedded in a methacrylate mixture. Ultrathin sections were cut on an experimental model, thermal expansion type ultramicrotome or on a Porter-Blume microtome. The sections were examined in an RCA electron microscope. Type EMU-2 D. "Squash preparations" for light microscopy, were made from the hydra mouth region and the attached tentacles. These were observed with an AO Baker interference microscope. In the mature organism, three of the four types of nematocysts normally found in hydra could be positively identified with the electron microscope. The desmonemes, the smallest type, have a dense matrix and a thin capsule. The two different types of mature isorhizas could not be distinguished with certainty. They are intermediate in size between the desmonemes and stenoteles and have a capsule with a dense matrix. The cnidocil, or triggering hair, which is composed of a dense core and a fibrillar sheath has nine supporting elements arranged in a semi-circle near its base. Twenty "supporting structures" are arranged around the nematocyst capsule and interconnections between the supporting elements and these latter structures have been observed. Development of the nematocysts involves an increase in density of the matrix. Spines can be seen in the interior of tubular structures within the capsules of the holotrichous isorhizas.     

Structure,expression,and developmental function of early divergent forms of metalloproteinases in Hydra

Metalloproteinases have a critical role in a broad spectrum of cellular processesranging from the break-down of extracellular matrix to the processing of signaltransduction-related proteins.These hydrolytic functions underlie a variety of mechanisms related to developmental processes as well as disease states.Structural analysis of metalloproteinases from both invertebrate and vertebrate species indicates that these enzymes are highly conserved and arose early during metazoan evolution.In this regard,studies from vari-ous laboratories have reported that a number of classes of metalloproteinases are found in hydra,a member of Cnidaria,the second oldest of existing animal phyla.These studies demonstrate that the hydra genome contains at least three classes of metalloproteinases to include members of the 1)astacin class,2)matrix met-alloproteinase class,and 3)neprilysin class.Functional studies indicate that these metalloproteinases play diverse and important roles in hydra morphogenesis and cell differentiation as well as specialized functions in adult polyps.This article will review the structure,expression,and function of these metalloproteinases in hydra.     

Structure, expression, and developmental function of early divergent forms of metalloproteinases in Hydra

Metalloproteinases have a critical role in a broad spectrum of cellular processes ranging from the breakdown of extracellular matrix to the processing of signal transduction-related proteins. These hydrolytic functions underlie a variety of mechanisms related to developmental processes as well as disease states. Structural analysis of metalloproteinases from both invertebrate and vertebrate species indicates that these enzymes are highly conserved and arose early during metazoan evolution. In this regard, studies from various laboratories have reported that a number of classes of metalloproteinases are found in hydra, a member of Cnidaria, the second oldest of existing animal phyla. These studies demonstrate that the hydra genome contains at least three classes of metalloproteinases to include members of the 1) astacin class, 2) matrix met-alloproteinase class, and 3) neprilysin class. Functional studies indicate that these metalloproteinases play diverse and important roles in hydra morphogenesis and ce     

Structure, expression, and developmental function of early divergent forms of metalloproteinases in Hydra

Metalloproteinases have a critical role in a broad spectrum of cellular processes ranging from the break-down of extracellulax matrix to the processing of signal transduction-related proteins. These hydrolyticfunctions underlie a variety of mechanisms related to developmental processes as well as disease states.Structural analysis of metalloproteinases from both invertebrate and vertebrate species indicates that theseenzymes are highly conserved and arose early during metazoan evolution. In this regard, studies from vari-ous laboratories have reported that a number of classes of metalloproteinases are found in hydra, a memberof Cnidaria, the second oldest of existing animal phyla. These studies demonstrate that the hydra genomecontains at least three classes of metalloproteinases to include members of the 1) astacin class, 2) matrix met-alloproteinase class, and 3) neprilysin class. Functional studies indicate that these metalloproteinases playdiverse and important roles in hydra morphogenesis and cell differentiation as well as specialized functionsin adult polyps. This article will review the structure, expression, and function of these metalloproteinasesin hydra.     

Some physical and chemical properties of purified nematocysts of Hydra attenuata pall. (Hydrozoa,cnidaria)

• 1.1. A method for purifying undischarged nematocysts from Hydra and other cnidarians is described.
• 2.2. Isolated cysts (relative densities 1.22–1.24) evaginate their tubular content even after previous dehydration.
• 3.3. The cyst wall is permeable to dyes of mol. wts up to 600,000.
• 4.4. Approximately two-thirds of the cyst's dry wt are soluble proteins. Eighty per cent of them are of low mol. wt and highly anionic, presumably serving as binding sites for Ca²⁺ and Mg²⁺.
• 5.5. The other 20% includes 30 different proteins amongst them toxins and enzymes (phospholipase and little proteases but no collagenase, chitinase or hyaluronidase).

     

Poly(gamma-glutamic acid)s are the major constituents of nematocysts in Hydra (Hydrozoa, Cnidaria).

The 2.80 +/- 0.20 mumol of anions found/mg of isolated and purified dry nematocysts (capsular secretory products of stinging cells) from Hydra make up the majority of the soluble capsular content. They are, in cooperation with corresponding cations, responsible for the generation and regulation of an internal osmotic pressure that amounts up to 150 bar (Weber, J. (1989) Eur. J. Biochem. 184, 465-476). The anions are organized as linear homopolymers of L-glutamic acids which are linked by gamma-carboxyl-alpha-amino amide bonds; the degree of polymerization is heterogeneous and dependent on the particular type of nematocyst. In situ the intracapsular glutamic acid monomer concentration is as high as 2 M. This is the first time that poly(gamma-glutamic acid)s, which are known to occur in some selected bacteria, are reported for eucaryotes. It is suggested that they may also be present as predominant components in nematocysts of other cnidarian species and thus might represent a class of compounds which is characteristic for a whole phylum of the animal kingdom.     

Structure,function, and protein taxonomy

This paper considers two recent arguments that structure should not be regarded as the fundamental individuating property of proteins. By clarifying both what it might mean for certain properties to play a fundamental role in a classification scheme and the extent to which structure plays such a role in protein classification, I argue that both arguments are unsound. Because of its robustness, its importance in laboratory practice, and its explanatory centrality, primary structure should be regarded as the fundamental distinguishing characteristic of protein taxonomy.     

肌动蛋白相关蛋白2/3复合体的结构、功能与调节

微丝参与了细胞形态维持及细胞运动等多种重要的细胞过程。微丝由肌动蛋白单体组装而成 ,肌动蛋白相关蛋白 2 / 3(Arp2 /Arp3,Arp2 / 3)复合体在微丝形成过程中起重要作用。Arp2 / 3复合体由 7个亚单位组成 ,在细胞内受到多种核化促进因子的调节 ,并与这些因子协同作用来调节肌动蛋白的核化。Arp2 / 3复合体结构、功能及调节的研究对于阐明微丝形成机制及细胞骨架与某些信号分子的关系有重要意义。     

Structure,function, and pathology of protein O-glucosyltransferases

Protein O-glucosylation is a crucial form of O-glycosylation, which involves glucose (Glc) addition to a serine residue within a consensus sequence of epidermal growth factor epidermal growth factor (EGF)-like repeats found in several proteins, including Notch. Glc provides stability to EGF-like repeats, is required for S2 cleavage of Notch, and serves to regulate the trafficking of Notch, crumbs2, and Eyes shut proteins to the cell surface. Genetic and biochemical studies have shown a link between aberrant protein O-glucosylation and human diseases. The main players of protein O-glucosylation, protein O-glucosyltransferases (POGLUTs), use uridine diphosphate (UDP)-Glc as a substrate to modify EGF repeats and reside in the endoplasmic reticulum via C-terminal KDEL-like signals. In addition to O-glucosylation activity, POGLUTs can also perform protein O-xylosylation function, i.e., adding xylose (Xyl) from UDP-Xyl; however, both activities rely on residues of EGF repeats, active-site conformations of POGLUTs and sugar substrate concentrations in the ER. Impaired expression of POGLUTs has been associated with initiation and progression of human diseases such as limb-girdle muscular dystrophy, Dowling–Degos disease 4, acute myeloid leukemia, and hepatocytes and pancreatic dysfunction. POGLUTs have been found to alter the expression of cyclin-dependent kinase inhibitors (CDKIs), by affecting Notch or transforming growth factor-β1 signaling, and cause cell proliferation inhibition or induction depending on the particular cell types, which characterizes POGLUT’s cell-dependent dual role. Except for a few downstream elements, the precise mechanisms whereby aberrant protein O-glucosylation causes diseases are largely unknown, leaving behind many questions that need to be addressed. This systemic review comprehensively covers literature to understand the O-glucosyltransferases with a focus on POGLUT1 structure and function, and their role in health and diseases. Moreover, this study also raises unanswered issues for future research in cancer biology, cell communications, muscular diseases, etc.Subject terms: Glycosylation, Oncogenes     

The structure and function of the nematocysts of Chironex fleckeri Southcott, 1956

Cell and Tissue Research - Microbasic p-mastigophores, euryteles of two size groups, holotrichous isorhizas and atrichous isorhizas, comprise the cnidom of Chironex fleckeri, a cubozoan that has...     

Sequestration of nematocysts by divergent cnidarian predators: mechanism,function, and evolution

Animals have evolved diverse mechanisms to protect themselves from predators. Although such defenses are typically generated endogenously, some species have evolved the ability to acquire defenses by sequestering defensive chemicals or structures from other species. Chemical sequestration is widespread among animals, but the ability to sequester entire structures, such as organelles, appears to be rare. Here, we review information on the sequestration of functional nematocysts, the stinging organelles produced by Cnidaria, by divergent predators. Nematocyst sequestration has evolved multiple times, having been documented in Ctenophora, Acoelomorpha, Platyhelminthes, and Mollusca. For each of these phyla, we review the phylogenetic distribution, mechanisms, and possible functions of nematocyst sequestration. We estimate that nematocyst sequestration has evolved 9–17 times across these four phyla. Although data on the mechanism of sequestration remain limited, similarities across several groups are evident. For example, in multiple groups, nematocysts are transported within cells from the gut to peripheral tissues, and certain types of nematocysts are selectively sequestered over others, suggesting convergent evolution in some aspects of the sequestration process across phyla. Similarly, although the function of nematocyst sequestration has not been well documented, several studies do suggest that the nematocysts sequestered by these groups are effective for defense. We highlight several traits that are common to Ctenophora, Acoelomorpha, Platyhelminthes, and Mollusca and suggest hypotheses for how these traits could have played a role in the evolution of nematocyst sequestration. Finally, we propose a generalized working model for the steps that may lead to the evolution of nematocyst sequestration and discuss important areas for future research.     

拟南芥SRO蛋白家族的结构及功能分析

许多生物及非生物胁迫都会引起植物的氧化胁迫, 参与植物氧化胁迫反应组分的鉴定备受人们的关注。拟南芥SRO家族成员包括AtRCD1、AtSRO1、AtSRO5等, 调节植物对氧化胁迫的反应。AtSROs参与植物正常的生长发育, 同时在植物应对干旱、盐、重金属等胁迫反应中扮演重要角色。AtSROs存在保守的PARP、RST等特殊功能区, 推测其可能具备蛋白的转录、调节、修饰等功能。文章就拟南芥SRO家族成员的基本状况, 在植物生长发育及应对非生物胁迫反应中的作用进行概述, 为进一步研究AtSROs的生物学功能提供理论基础。     

Structure and function of a paramyxovirus fusion protein

Paramyxoviruses initiate infection by attaching to cell surface receptors and fusing viral and cell membranes. Viral attachment proteins, hemagglutinin-neuraminidase (HN), hemagglutinin (HA), or glycoprotein (G), bind receptors while fusion (F) proteins direct membrane fusion. Because paramyxovirus fusion is pH independent, virus entry occurs at host cell plasma membranes. Paramyxovirus fusion also usually requires co-expression of both the attachment protein and the fusion (F) protein. Newcastle disease virus (NDV) has assumed increased importance as a prototype paramyxovirus because crystal structures of both the NDV F protein and the attachment protein (HN) have been determined. Furthermore, analysis of structure and function of both viral glycoproteins by mutation, reactivity of antibody, and peptides have defined domains of the NDV F protein important for virus fusion. These domains include the fusion peptide, the cytoplasmic domain, as well as heptad repeat (HR) domains. Peptides with sequences from HR domains inhibit fusion, and characterization of the mechanism of this inhibition provides evidence for conformational changes in the F protein upon activation of fusion. Both proteolytic cleavage of the F protein and interactions with the attachment protein are required for fusion activation in most systems. Subsequent steps in membrane merger directed by F protein are poorly understood.