Differential gene expression in the jellyfish Aurelia aurita

The body of Aurelia aurita, as well as other diploblasts, consists of two epithelial layers: ectodermal and gastral epithelium. These two tissues are separated by mesoglea, or extracellular matrix. In most coelenterates mesoglea is acellular. In A. aurita mesogleal cells are scattered in mesoglea. Differential display PCR was used to compare mRNA pools from ectodermal epithelium, gastral epithelium and mesoglea. 4 novel gene fragments were cloned and sequenced. According to RTPCR results, one of these fragments is differentially expressed in the ectodermal epithelium.     

Protein composition of mesoglea and mesogloeal cells of medusa Aurelia aurita

Protein composition of mesoglea of the scyphomedusa Aurelia aurita was revealed in SDS-PAGE. Some major bands are visible in mesoglea of a mature medusa: 30, 45-47, 85 kDa, three bands between 100-200 kDa, and several bands with molecular weights > 300 kDa. Polyclonal antisera RA45/47 against protein 45 kDa were raised. RA45/47 react with 45-47 kDa protein in mesogleal sample and protein 120 kDa in mesogleal cells on immunoblot. Immunohistochemical analysis of A. aurita histological sections of young and mature medusae showed antigen localization in mesogleal cell granules and in the apical part of ectodermal cells. In mature medusae, the antigen was localized also in elastic fibers. We can conclude that in A. aurita mesogleal cells, along with ectodermal cells, take part in the formation of extracellular matrix of mesoglea.     

Mesogleal cells of the jellyfish Aurelia aurita are involved in the formation of mesogleal fibres

The extracellular matrix of the jellyfish Aurelia aurita (Scyphozoa, Cnidaria), known as the mesoglea, is populated by numerous mesogleal cells (Mc). We determined the pattern of the Mc and the mesoglea, raised polyclonal antibodies (RA47) against the major mesogleal protein pA47 (47 kDa) and checked their specificity. In the mesoglea, RA47 stains pA47 itself. In immunoblots of Mc, RA47 stains bands of 120 kDa and 80 kDa; weaker staining is observed at pA47. The same staining pattern is seen on blots of jellyfish epidermal cells and of whole Hydra (Hydrozoa) or isolated mesoglea of Hydra. Our data indicate that pA47 is synthesized by Mc and epidermal cells as high molecular precursors. Using immunostaining techniques, we showed Mc to be involved in the formation of mesogleal non-collagenous (called "elastic" in classic morphological studies) fibres. The biochemical and morphological data suggest that Mc originate from the epidermis.     

Aurelin, a novel antimicrobial peptide from jellyfish Aurelia aurita with structural features of defensins and channel-blocking toxins

A novel 40-residue antimicrobial peptide, aurelin, exhibiting activity against Gram-positive and Gram-negative bacteria, was purified from the mesoglea of a scyphoid jellyfish Aurelia aurita by preparative gel electrophoresis and RP-HPLC. Molecular mass (4296.95 Da) and complete amino acid sequence of aurelin (AACSDRAHGHICESFKSFCKDSGRNGVKLRANCKKTCGLC) were determined. Aurelin has six cysteines forming three disulfide bonds. The total RNA was isolated from the jellyfish mesoglea, RT-PCR and cloning were performed, and cDNA was sequenced. A 84-residue preproaurelin contains a putative signal peptide (22 amino acids) and a propiece of the same size (22 amino acids). Aurelin has no structural homology with any previously identified antimicrobial peptides but reveals partial similarity both with defensins and K+ channel-blocking toxins of sea anemones and belongs to ShKT domain family.     

Amyloid Properties of the Mouse Egg Zona Pellucida

The zona pellucida (ZP) surrounding the oocyte is an extracellular fibrillar matrix that plays critical roles during fertilization including species-specific gamete recognition and protection from polyspermy. The mouse ZP is composed of three proteins, ZP1, ZP2, and ZP3, all of which have a ZP polymerization domain that directs protein fibril formation and assembly into the three-dimensional ZP matrix. Egg coats surrounding oocytes in nonmammalian vertebrates and in invertebrates are also fibrillar matrices and are composed of ZP domain-containing proteins suggesting the basic structure and function of the ZP/egg coat is highly conserved. However, sequence similarity between ZP domains is low across species and thus the mechanism for the conservation of ZP/egg coat structure and its function is not known. Using approaches classically used to identify amyloid including conformation-dependent antibodies and dyes, X-ray diffraction, and negative stain electron microscopy, our studies suggest the mouse ZP is a functional amyloid. Amyloids are cross-β sheet fibrillar structures that, while typically associated with neurodegenerative and prion diseases in mammals, can also carry out functional roles in normal cells without resulting pathology. An analysis of the ZP domain from mouse ZP3 and ZP3 homologs from five additional taxa using the algorithm AmylPred 2 to identify amyloidogenic sites, revealed in all taxa a remarkable conservation of regions that were predicted to form amyloid. This included a conserved amyloidogenic region that localized to a stretch of hydrophobic amino acids previously shown in mouse ZP3 to be essential for fibril assembly. Similarly, a domain in the yeast protein α-agglutinin/Sag 1p, that possesses ZP domain-like features and which is essential for mating, also had sites that were predicted to be amyloidogenic including a hydrophobic stretch that appeared analogous to the critical site in mouse ZP3. Together, these studies suggest that amyloidogenesis may be a conserved mechanism for ZP structure and function across billions of years of evolution.     

Primary structure and developmental expression of Dp ZP2, a vitelline envelope glycoprotein homolog of mouse ZP2, in Discoglossus pictus, one of the oldest living Anuran species

A glycoprotein of the Xenopus vitelline envelope, gp 69/64, which mediates sperm binding, is closely related to the components of ZPA family, such as the mouse zona pellucida ZP2. To test the generality of these findings, we studied Discoglossus pictus, a species evolutionary distant from Xenopus and identified as a protein of 63 kDa in the vitelline envelope. Preliminary studies suggest that this protein may bind sperm at fertilization. We found that the 63-kDa protein is glycosylated and contains both N- and O-linked chains. We have cloned the cDNA encoding the Discoglossus protein of 63 kDa (Dp ZP2) by screening a Discoglossus cDNA library using Xenopus gp 69/64 cDNA as a probe. Analysis of the deduced sequence of Discoglossus protein revealed 48% identity with Xenopus gp 69/64 and 37-40% identity with mouse ZP2. The sequence conservation included a ZP domain, a potential furin cleavage site and a putative transmembrane domain. The N-terminus region of Dp ZP2 was 40% identical to the corresponding region of Xenopus gp 69/64 which has been shown to be essential for sperm binding to the VE. Although, as of yet, there is no evidence for sperm binding at the Dp ZP2 N-terminus, it is interesting that in this region three potential O-glycosylation sites are conserved in both species, in contrast to N-glycosylation sites. It was found that the Dp ZP2 mRNA is expressed in stage 1 oocytes and in the follicle cells surrounding the oocyte. Similarly, in Xenopus oocytes, the gp 69/64m RNA, was found in the oocytes, as well as in the somatic cells. Mol. Reprod. Dev. 59:133-143, 2001.     

Structural conservation of mouse and rat zona pellucida glycoproteins. Probing the native rat zona pellucida proteome by mass spectrometry

The mammalian zona pellucida is an egg extracellular matrix to which sperm bind. Mouse zonae are composed of three glycoproteins (ZP1, ZP2, and ZP3), while rat zonae contain four (ZP1, ZP2, ZP3, and ZP4/ZPB). Mouse sperm bind to zonae comprised solely of mouse ZP2 and ZP3. In this report, we show that rat sperm also bind to these zonae, indicating that ZP2 and ZP3 contain a "minimum structure(s)" to which rodent sperm can bind, and ZP1 and ZP4/ZPB are dispensable in these two rodents. These data are consistent with our mass spectrometric analysis of the native rat zona pellucida proteome (defined as the fraction of the total rat proteome to which the zonae glycoproteins contribute) demonstrating that the rat zonae glycoproteins share a high degree of conservation of structural features with respect to their mouse counterparts. The primary sequences of the rat zonae proteins have been deduced from cDNA. Each zona protein undergoes extensive co- and post-translational modification prior to its secretion and incorporation into an extracellular zona matrix. Each has a predicted N-terminal signal peptide that is cleaved off once protein translation begins and an anchoring C-terminal transmembrane domain from which the mature protein is released. Mass spectrometric analysis with a limited amount of native material allowed determination of the mature N-termini of rat ZP1 and ZP3, both of which are characterized by cyclization of glutamine to pyroglutamate; the N-terminus of ZP2 was identified by Edman degradation. The mature C-termini of ZP1 and ZP3 end two amino acids upstream of a conserved dibasic residue that is part of, but distinct from, the consensus furin cleavage sequence, while the C-terminus of ZP2 was not determined. Each zona protein contains a "zona domain" with eight conserved cysteine residues that is thought to play a role in the polymerization of the zona proteins into matrix filaments. Partial disulfide bond assignment indicates that the intramolecular disulfide patterns in rat ZP1, ZP2, and ZP3 are identical to those of their corresponding mouse counterparts. Last, nearly all potential N-glycosylation sites are occupied in the rat zonae glycoproteins (three of three for ZP1, six or seven of seven for ZP2, and four or five of six for ZP3). In comparison, potential O-glycosylation sites are numerous (59-83 Ser/Thr residues), but only two regions were observed to carry O-glycans in rat ZP3.     

The attachment of nematocytes from the primitive invertebrate Hydra to fibronectin is specific and RGD-dependent.

The transient attachment of cells to components of the extracellular matrix is an important step in the complex molecular mechanisms involved in amoeboid cell locomotion. We have analyzed the attachment of nematocytes from the freshwater cnidarian Hydra to fibronectin which is a constituent of the mesoglea, the extracellular matrix, of the polyps. The percentage of attaching cells increased gradually in a concentration-dependent manner and reached a plateau value at a fibronectin concentration of 50 micrograms/ml. Attachment was inhibited by exposure of the fibronectin-coated surfaces to antibodies against the cell binding domain of fibronectin or by incubating the cells with peptides containing the recognition sequence Arg-Gly-Asp (RGD) known from vertebrate cells. This, together with data obtained by affinity chromatography, indicates that RGD-dependent binding to fibronectin, mediated by a receptor which possibly belongs to the integrin family, already occurs in Hydra, a member of an evolutionary low invertebrate phylum.     

Morphodynamics of the contract plate in the course of oocyte maturation in the scyphozoan Aurelia aurita (Cnidaria: Semaeostomae)

The structure forming in the area of contact between the oocyte and the germinal epithelium in the course of oocyte maturation of the scyphozoan Aurelia aurita is termed the contact plate. This study traces the successive stages of contact plate formation in the course of oocyte maturation at the light microscopic and ultrastructural levels. At early stages ofoocyte development, the appearance of granules is observed in the peripheral cytoplasm of the oocyte; these granules accumulate at the pole, which retains its connection with the germinal epithelium of the gonads. Two types of these granules are recognized: (1) granules with homogeneous content and (2) granules containing loose shapeless material in the form of thick cords. The transformation of type two granules into larger structures, as well as the consolidation of type one and type two granules at later stages of oocyte development, are probably the processes that lead to the formation of the characteristic structure and contact plate, visible in paraffin and semithin sections. It remains unclear where exactly the contact plate is localized at the moment of fertilization: inside or outside the oocyte. The content of granules and components of the plate specifically bind the antibodies (RA47) against mesoglein, the ZP domain-containing protein of the mesoglea of A. aurita. The contact plate, covering only the anomalous pole of the oocyte but detected by the presence of ZP domain-containing proteins, may prove to be the simplest egg membrane of the zona pellucida type.     

Fusion expression of DDR2 extracellular domain in insect cells and its purification and function characterization

Discoidin domain receptor 2 (DDR2) is a kind of protein tyrosine kinases associated with cell proliferation and tumor metastasis, and collagen, a ligand for DDR2, up-regulates matrix metalloproteinase 1 (MMP-1) and MMP-2 expression in extracellular matrix (ECM). To investigate the role of DDR2 in cartilage destruction in rheumatoid arthritis (RA), we expressed the extracellular domain (ECD) of DDR2 (without signal peptide and transmembrane domain, designated DR) in insect cells, purified and characterized DR, hoping to use it as a specific antagonist of DDR2. By using Bac-To-Bac Expression System with a His tag, we successfully obtained the recombinant bacularvirus containing DDR2 ECD, purified it and characterized its function. The soluble fraction of DR was about 12% of the total fused protein. After chromatographic purification, DR with 92% purity was obtained. Competitive inhibition assay demonstrated that DR blocked the binding between DDR2 and natural DDR2 receptors on NIH3T3 and synovial cells. Results of RT-PCR, Western blotting, and gelatinase zymography showed that DR was capable of inhibiting MMP-1 and MMP-2 secretion from NIH3T3 and RA synoviocytes stimulated by collagen II. For MMP-1, inhibition was displayed at the levels of mRNA and protein, whereas for MMP-2 it was at the level of protein. These findings suggested that the expressed DR inhibited the activity of natural DDR2 and relevant MMP-1 and MMP-2 expression in RA synoviocytes and NIH3T3 cells provoked by collagen II.     

Molecular cloning and expression in Escherichia coli of cDNA encoding bonnet monkey (Macaca radiata) zona pellucida glycoprotein

Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for an immunocontraceptive vaccine. The efficacy of such a vaccine has to be evaluated in nonhuman primates, thus necessitating the characterization of their ZP glycoproteins. A bonnet monkey (Macaca radiata) ovarian cDNA λgt11 library was screened for ZP2 (bZP2) using full-length human ZP2 cDNA as a probe. Two identical full-length clones with an open reading frame of 2235 nt encoding a polypeptide of 745 aa residues were isolated. The deduced aa sequence of bZP2 revealed high sequence identity (94.2%) with human ZP2. The bZP2 cDNA (115–1914 nt, 1.8 kb), excluding sequences coding for N-terminal signal sequence and C-terminal transmembranelike domain, was PCR amplified and Sac1-Sal1 restricted fragment cloned in frame downstream of the T5 promoter under the lac operator control in a pQE-30 vector. Recombinant bZP2 (r-bZP2) was expressed as a polyhistidine fusion protein in Escherichia coli strain M15 [pREP4]. Immunoblot with rabbit polyclonal antibodies against bZP2 synthetic peptide (corresponding to aa residues 429–444; K₄₃₄ replaced by R and I₄₃₆ by V) revealed a major band of 68 kDa. Immunization of male rabbits with the r-bZP2 protein purified on Ni-NTA resin under denaturing conditions generated antibodies reactive with r-bZP2 in ELISA as well as with native protein as revealed by positive fluorescence of ZP of bonnet monkey ovary. The availability of r-bZP2 and its aa sequence will help in the development and evaluation of a contraceptive vaccine based on ZP2. Mol. Reprod. Dev. 50:229–239, 1998. © 1998 Wiley-Liss, Inc.     

Conserved furin cleavage site not essential for secretion and integration of ZP3 into the extracellular egg coat of transgenic mice

The extracellular zona pellucida surrounding mammalian eggs is formed by interactions of the ZP1, ZP2, and ZP3 glycoproteins. Female mice lacking ZP2 or ZP3 do not form a stable zona matrix and are sterile. The three zona proteins are synthesized in growing oocytes and secreted prior to incorporation into the zona pellucida. A well-conserved furin site upstream of a transmembrane domain near the carboxyl terminus of each has been implicated in the release of the zona ectodomains from oocytes. However, mutation of the furin site (RNRR --> ANAA) does not affect the intracellular trafficking or secretion of an enhanced green fluorescent protein (EGFP)-ZP3 fusion protein in heterologous somatic cells. After transient expression in growing oocytes, normal EGFP-ZP3 and mutant EGFP-ZP3 associate with the inner aspect of the zona pellucida, which is distinct from the plasma membrane. These in vitro results are confirmed in transgenic mice expressing EGFP-ZP3 with or without the mutant furin site. In each case, EGFP-ZP3 is incorporated throughout the width of the zona pellucida and the transgenic mice are fertile. These results indicate that the zona matrix accrues from the inside out and, unexpectedly, suggest that cleavage at the furin site is not required for formation of the extracellular zona pellucida surrounding mouse eggs.     

The attachment of nematocytes from the primitive invertebrate Hydra to fibronectin is specific and RDG-dependent

The transient attachment of cells to components of the extracellular matrix is an important step in the complex molecular mechanisms involved in amoeboid cell locomotion. We have analyzed the attachment of nematocytes from the freshwater cnidarian Hydra to flbronectin which is a constituent of the mesoglea, the extracellular matrix, of the polyps. The percentage of attaching cells increased gradually in a concentration-dependent manner and reached a plateau value at a fibronectin concentration of 50 μg/ml. Attachment was inhibited by exposure of the fibronectin-coated surfaces to antibodies against the cell binding domain of fibronectin or by incubating the cells with peptides containing the recognition sequence Arg-Gly-Asp (RGD) known from vertebrate cells. This, together with data obtained by affinity chromatography, indicates that RGD-dependent binding to fibronectin, mediated by a receptor which possibly belongs to the integrin family, already occurs in Hydra, a member of an evolutionary low invertebrate phylum.     

Identification of a Novel TGF-β-Binding Site in the Zona Pellucida C-terminal (ZP-C) Domain of TGF-β-Receptor-3 (TGFR-3)

The zona pellucida (ZP) domain is present in extracellular proteins such as the zona pellucida proteins and tectorins and participates in the formation of polymeric protein networks. However, the ZP domain also occurs in the cytokine signaling co-receptor transforming growth factor β (TGF-β) receptor type 3 (TGFR-3, also known as betaglycan) where it contributes to cytokine ligand recognition. Currently it is unclear how the ZP domain architecture enables this dual functionality. Here, we identify a novel major TGF-β-binding site in the FG loop of the C-terminal subdomain of the murine TGFR-3 ZP domain (ZP-C) using protein crystallography, limited proteolysis experiments, surface plasmon resonance measurements and synthetic peptides. In the murine 2.7 Å crystal structure that we are presenting here, the FG-loop is disordered, however, well-ordered in a recently reported homologous rat ZP-C structure. Surprisingly, the adjacent external hydrophobic patch (EHP) segment is registered differently in the rat and murine structures suggesting that this segment only loosely associates with the remaining ZP-C fold. Such a flexible and temporarily-modulated association of the EHP segment with the ZP domain has been proposed to control the polymerization of ZP domain-containing proteins. Our findings suggest that this flexibility also extends to the ZP domain of TGFR-3 and might facilitate co-receptor ligand interaction and presentation via the adjacent FG-loop. This hints that a similar C-terminal region of the ZP domain architecture possibly regulates both the polymerization of extracellular matrix proteins and cytokine ligand recognition of TGFR-3.     

Structural characterization of native mouse zona pellucida proteins using mass spectrometry

The zona pellucida is an extracellular matrix consisting of three glycoproteins that surrounds mammalian eggs and mediates fertilization. The primary structures of mouse ZP1, ZP2, and ZP3 have been deduced from cDNA. Each has a predicted signal peptide and a transmembrane domain from which an ectodomain must be released. All three zona proteins undergo extensive co- and post-translational modifications important for secretion and assembly of the zona matrix. In this report, native zonae pellucidae were isolated and structural features of individual zona proteins within the mixture were determined by high resolution electrospray mass spectrometry. Complete coverage of the primary structure of native ZP3, 96% of ZP2, and 56% of ZP1, the least abundant zona protein, was obtained. Partial disulfide bond assignments were made for each zona protein, and the size of the processed, native protein was determined. The N termini of ZP1 and ZP3, but not ZP2, were blocked by cyclization of glutamine to pyroglutamate. The C termini of ZP1, ZP2, and ZP3 lie upstream of a dibasic motif, which is part of, but distinct from, a proprotein convertase cleavage site. The zona proteins are highly glycosylated and 4/4 potential N-linkage sites on ZP1, 6/6 on ZP2, and 5/6 on ZP3 are occupied. Potential O-linked carbohydrate sites are more ubiquitous, but less utilized.     

Formation of the rabbit zona pellucida and its relationship to ovarian follicular development

The zona pellucida is the unique extracellular glycoprotein matrix which is assembled during growth of the mammalian oocyte. The present studies were carried out to examine the formation of this structure in relation to the differentiation of ovarian cell types during follicular development. Specific antibodies were developed against total rabbit ZP proteins as well as against ZP proteins electrophoretically purified by high-resolution two-dimensional polyacrylamide electrophoresis gels (2D-PAGE). Antibodies were characterized by (a) immunoelectrophoresis, (b) a Staphylococcus aureus protein A binding assay, and (c) immunoblotting following 2D-PAGE separation of ZP proteins. Immunoperoxidase localization with these antibodies was used to determine the stage of ovarian follicular development at which ZP antigens first appear as well as to evaluate the cellular and extracellular distribution of these proteins throughout folliculogenesis. The ZP proteins were first observed in the cytoplasm and at the periphery of the oocytes surrounded by a thin squamous follicular cell layer. No staining was observed in the cytoplasm of follicle cells during early folliculogenesis. As the ZP matrix was assembled extracellularly, the intensity of staining of the outer and inner regions could be distinguished. This differentiation of the matrix coincided with the differentiation of the follicular cells into a multilayer cell complex. At this stage, specific ZP proteins are localized within the cytoplasm of the inner layers of these follicular cells. The staining is then diminished in cells of preantral follicles. These studies demonstrate that the formation of the ZP is an excellent model system to study the early stages of follicular development and cell differentiation.     

Egg extracellular coat proteins: from fish to mammals

The extracellular coat surrounding fish (vitelline envelope; VE) and mammalian (zona pellucida; ZP) eggs is composed of long, interconnected filaments. Fish VE and mammalian ZP proteins that make up the filaments are highly conserved groups of proteins that are related to each other, as well as to their amphibian and avian egg counterparts. The rainbow trout (O. mykiss) egg VE is composed of 3 proteins, called VEalpha (approximately 58 kDa), VEbeta (approximately 54 kDa), and VEgamma (approximately 47 kDa). The mouse (M. musculus) egg ZP also is composed of 3 proteins, called ZP1 (approximately 200 kDa), ZP2 (approximately 120 kDa), and ZP3 (approximately 83 kDa). Overall, trout VE and mouse ZP proteins share approximately 25% sequence identity and have features in common; these include an N-terminal signal sequence, a ZP domain, a consensus furin cleavage-site, and a C-terminal tail. VEalpha, VEbeta, and ZP1 also have a trefoil or P-type domain upstream of the ZP domain. VEalpha and VEbeta are very similar in sequence (approximately 65% sequence identity) and are related to ZP1 and ZP2, whereas VEgamma is related to ZP3 (approximately 25% sequence identity). Mouse ZP proteins are synthesized and secreted exclusively by growing oocytes in the ovary. Trout VE proteins are synthesized by the liver under hormonal control and transported in the bloodstream to growing oocytes in the ovary. The trout VE is assembled from VEalpha/gamma and VEbeta/gamma heterodimers. The mouse ZP is assembled from ZP2/3 heterodimers and crosslinked by ZP1. Despite approximately 400 million years separating the appearance of trout and mice, and the change from external to internal fertilization and development, trout VE and mouse ZP proteins have many common structural features; as do avian and amphibian egg VE proteins. However, the site of synthesis of trout and mouse egg extracellular coat proteins has changed over time from the liver to the ovary, necessitating some changes in the C-terminal region of the polypeptides that regulates processing, secretion, and assembly of the proteins.     

Extracellular matrix (mesoglea) of Hydra vulgaris. I. Isolation and characterization.

Hydrozoans such as Hydra vulgaris, as with all classes of Cnidaria, are characterized by having their body wall organized as an epithelial bilayer with an intervening acellular layer termed the mesoglea. The present study was undertaken to determine what extracellular matrix (ECM) components are associated with Hydra mesoglea. Using polyclonal antibodies generated from vertebrate ECM molecules, initial light and electron microscopic immunocytochemical studies indicated the presence of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin immunoreactive components in Hydra mesoglea. These immunocytochemical observations were in part supported by biochemical analyses of isolated Hydra mesoglea which indicated the presence of fibronectin and laminin based on Western blot analysis. Amino acid analysis of total mesoglea and some of its isolated components confirmed the presence of collagen molecules in mesoglea. Additional studies indicated the presence of (1) a gelatin binding protein in Hydra which was immunoreactive with antibodies raised to human plasma fibronectin and (2) a noncollagen fragment extracted from mesoglea which was immunoreactive to antibodies raised to the NC1 domain (alpha 1 subunit) of bovine glomerular basement membrane type IV collagen. These observations indicate that Hydra mesoglea is evolutionarily a primitive basement membrane that has retained some properties of interstitial ECM.