RESUMEN
It has been reported that GCS1 (Generative Cell Specific 1) is a transmembrane protein that is exclusively expressed in sperm cells and is essential for gamete fusion in flowering plants. The GCS1 gene is present not only in angiosperms but also in unicellular organisms and animals, implying the occurrence of a common or ancestral mechanism of GCS1-mediated gamete fusion. In order to elucidate the common mechanism, we investigated the role of GCS1 in animal fertilization using a sea anemone (Cnidaria), Nematostella vectensis. Although the existence of the GCS1 gene in N. vectensis has been reported, the expression of GCS1 in sperm and the role of GCS1 in fertilization are not known. In this study, we showed that the GCS1 gene is expressed in the testis and that GCS1 protein exists in sperm by in situ hybridization and proteomic analysis, respectively. Then we made four peptide antibodies against the N-terminal extracellular region of NvGCS1. These antibodies specifically reacted to NvGCS1 among sperm proteins on the basis of Western analysis and potently inhibited fertilization in a concentration-dependent manner. These results indicate that sperm GCS1 plays a pivotal role in fertilization, most probably in sperm-egg fusion, in a starlet sea anemone, suggesting a common gamete-fusion mechanism shared by eukaryotic organisms.
Asunto(s)
Fertilización/fisiología , Hormonas de Invertebrados/fisiología , Proteínas de la Membrana/fisiología , Interacciones Espermatozoide-Óvulo/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Masculino , Plantas/genética , Anémonas de Mar , Alineación de Secuencia , Espermatozoides/metabolismoRESUMEN
Ascidians (primitive chordates) are hermaphroditic animals that release spermatozoa and eggs almost simultaneously, but some species, including Halocynthia roretzi, show strict self-sterility. In H. roretzi, a 70-kDa vitelline coat (VC) protein consisting of 12 EGF-like repeats, named HrVC70, appears to be a promising candidate for the self/nonself-recognition (or allorecognition) system during gamete interaction. After spermatozoon recognizes the VC as nonself, sperm 700-kDa extracellular ubiquitin-conjugating enzyme complex appears to ubiquitinate Lys234 of HrVC70, and the ubiquitinated HrVC70 is degraded by the sperm 26S proteasome that is located on the sperm head surface. This novel ubiquitin-proteasome system enables spermatozoa to penetrate through the VC. Sperm trypsin-like proteases, acrosin and spermosin, also participate in fertilization, probably as sperm-side 'movable' binding proteins to the VC.
Asunto(s)
Fertilización , Péptido Hidrolasas/fisiología , Complejo de la Endopetidasa Proteasomal/fisiología , Erizos de Mar/fisiología , Espermatozoides/enzimología , Ubiquitina/fisiología , Urocordados/fisiología , Animales , Proteínas del Huevo/fisiología , Espacio Extracelular/metabolismo , MasculinoRESUMEN
Vitellogenin is a precursor of yolk protein that is necessary for embryonic development. This protein is a large multi-domain protein consisting of a signal peptide, a heavy-chain lipovitellin, a phosvitin, a light-chain lipovitellin, a von Willebrand factor type D domain (vWF-D), and a C-terminal coding region (CT), which are processed to respective domains after uptake into oocytes. It is currently believed that only lipovitellin and phosvitin domains are necessary for nutrient supply to oocytes. Thus, molecular species of vitellogenin lacking these domains are not known. Here, we show that two novel isoforms of vitellogenin, both of which possess vWF-D and CT domains but not a lipovitellin or phosvitin domain, are expressed in the gonad of the ascidian Halocynthia roretzi. In situ hybridization revealed that mRNAs of these proteins are specifically expressed in oocytes and test cells, accessory cells in the perivitelline space of ascidian eggs. Immunohistochemistry showed that these proteins are localized around the surface of test cells in immature oocytes. Immunoelectron microscopy revealed that vitellogenin associates with vesicles located beneath the vitelline coat (VC) before fertilization but that it dissociates from the VC after fertilization. These results, together with our previous results showing that vWF-D and CT domains are capable of binding to the two sperm proteases HrProacrosin and HrSpermosin, led us to propose that novel isoforms of vitellogenin, which are expressed in oocytes and test cells and released to the perivitelline space during oocyte maturation, may participate in gamete interaction upon fertilization.
Asunto(s)
Óvulo/metabolismo , Isoformas de Proteínas/metabolismo , Urocordados/metabolismo , Vitelogeninas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Vitelogeninas/genéticaRESUMEN
Sperm trypsin-like proteases are known to play important roles in fertilization, but their detailed functions are still unknown. We previously explored the binding partners of sperm trypsin-like proteases, HrProacrosin and HrSpermosin, in the ascidian Halocynthia roretzi, and we isolated several candidate proteins on the vitelline coat. We found that some of these proteins are identical to the C-terminal coding region (CT) and von Willebrand factor type D (vWF-D) domain of vitellogenin. We also found that CT on the vitelline coat disappears after fertilization. Vitellogenin is a large lipid transfer protein that is enzymatically processed during vitellogenesis. Although the processed domains including phosvitin and lipovitellin are known to function as yolk nutrient proteins, the roles of the CT and vWF-D domain remain elusive. Our results showed that the CT and vWF-D domain of vitellogenin are processed and attached to the vitelline coat, which in turn participate in fertilization as the binding partners of sperm proteases.
Asunto(s)
Acrosina/metabolismo , Precursores Enzimáticos/metabolismo , Fertilización , Serina Endopeptidasas/metabolismo , Espermatozoides/enzimología , Urocordados/fisiología , Vitelogeninas/metabolismo , Acrosina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Precursores Enzimáticos/genética , Femenino , Masculino , Datos de Secuencia Molecular , Óvulo/fisiología , Estructura Terciaria de Proteína , Serina Endopeptidasas/genética , Urocordados/metabolismo , Vitelogeninas/genéticaRESUMEN
Snake venom metalloproteases (SVMPs) are members of the a disintegrin and metalloprotease (ADAM) family of proteins, as they possess similar domains. SVMPs are known to elicit snake venom-induced haemorrhage; however, the target proteins and cleavage sites are not known. In this work, we identified a target protein of vascular apoptosis-inducing protein 1 (VAP1), an SVMP, relevant to its ability to induce haemorrhage. VAP1 disrupted cell-cell adhesions by relocating VE-cadherin and γ-catenin from the cell-cell junction to the cytosol, without inducing proteolysis of VE-cadherin. The Wnt receptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) are known to promote catenin relocation, and are rendered constitutively active in Wnt signalling by truncation. Thus, we examined whether VAP1 cleaves LRP5/6 to induce catenin relocation. Indeed, we found that VAP1 cleaved the extracellular region of LRP6 and LRP5. This cleavage removes four inhibitory ß-propeller structures, resulting in activation of LRP5/6. Recombinant human ADAM8 and ADAM12 also cleaved LRP6 at the same site. An antibody against a peptide including the LRP6-cleavage site inhibited VAP1-induced VE-cadherin relocation and disruption of cell-cell adhesions in cultured cells, and blocked haemorrhage in mice in vivo. Intriguingly, animals resistant to the effects of haemorrhagic snake venom express variants of LRP5/6 that lack the VAP1-cleavage site, or low-density lipoprotein receptor domain class A domains involved in formation of the constitutively active form. The results validate LRP5/6 as physiological targets of ADAMs. Furthermore, they indicate that SVMP-induced cleavage of LRP5/6 causes disruption of cell-cell adhesion and haemorrhage, potentially opening new avenues for the treatment of snake bites.
Asunto(s)
Proteínas ADAM/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Venenos de Crotálidos/metabolismo , Hemorragia/inducido químicamente , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/fisiología , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/fisiología , Metaloendopeptidasas/metabolismo , Proteínas ADAM/farmacología , Proteína ADAM12/metabolismo , Proteína ADAM12/farmacología , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Resistencia a Medicamentos , Fibrinógeno/metabolismo , Fibronectinas/metabolismo , Células HeLa , Células Endoteliales de la Vena Umbilical Humana , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/química , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/química , Masculino , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/farmacología , Ratones , Modelos Moleculares , Simulación del Acoplamiento Molecular , Dominios Proteicos , Estructura Secundaria de Proteína/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Vertebrados/metabolismo , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/fisiologíaRESUMEN
Although ascidians are hermaphroditic, many species including Halocynthia roretzi are self-sterile. We previously reported that a vitelline coat polymorphic protein HrVC70, consisting of 12 EGF (epidermal growth factor)-like repeats, is a candidate allorecognition protein in H. roretzi, because the isolated HrVC70 shows higher affinity to nonself-sperm than to self-sperm. Here, we show that a sperm 35-kDa glycosylphosphatidylinositol-anchored CRISP (cysteine-rich secretory protein)-like protein HrUrabin in a low density detergent-insoluble membrane fraction is a physiological binding partner for HrVC70. We found that HrVC70 specifically interacts with HrUrabin, which had been separated by SDS-PAGE and transferred onto a nitrocellulose membrane. HrUrabin has an N-linked sugar chain, essential for binding to HrVC70. HrUrabin mRNA is expressed in the testis but not in the ovary, and the protein appears to be localized on the surface of sperm head and tail. Anti-HrUrabin antibody, which neutralizes the interaction between HrUrabin and HrVC70, potently inhibited fertilization and allorecognizable sperm-binding to HrVC70-agarose. However, no significant difference in the binding ability of HrUrabin to HrVC70 was observed in autologous and allogeneic combinations by Far Western analyses. These results indicate that sperm-egg binding in H. roretzi is mediated by the molecular interaction between HrUrabin on the sperm surface and HrVC70 on the vitelline coat, but that HrUrabin per se is unlikely to be a direct allorecognition protein.