RESUMEN
Plant apyrases are nucleoside triphosphate (NTP) diphosphohydrolases (NTPDases) and have been implicated in an array of functions within the plant including the regulation of extracellular ATP. Arabidopsis encodes a family of seven membrane bound apyrases (AtAPY1-7) that comprise three distinct clades, all of which contain the five conserved apyrase domains. With the exception of AtAPY1 and AtAPY2, the biochemical and the sub-cellular characterization of the other members are currently unavailable. In this research, we have shown all seven Arabidopsis apyrases localize to internal membranes comprising the cis-Golgi, endoplasmic reticulum (ER) and endosome, indicating an endo-apyrase classification for the entire family. In addition, all members, with the exception of AtAPY7, can function as endo-apyrases by complementing a yeast double mutant (Δynd1Δgda1) which lacks apyrase activity. Interestingly, complementation of the mutant yeast using well characterized human apyrases could only be accomplished by using a functional ER endo-apyrase (NTPDase6), but not the ecto-apyrase (NTPDase1). Furthermore, the substrate specificity analysis for the Arabidopsis apyrases AtAPY1-6 indicated that each member has a distinct set of preferred substrates covering various NDPs (nucleoside diphosphates) and NTPs. Combining the biochemical analysis and sub-cellular localization of the Arabidopsis apyrases family, the data suggest their possible roles in regulating endomembrane NDP/NMP (nucleoside monophosphate) homoeostasis.
Asunto(s)
Apirasa/metabolismo , Proteínas de Arabidopsis/metabolismo , Homeostasis , Membranas Intracelulares/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Apirasa/clasificación , Apirasa/genética , Arabidopsis , Proteínas de Arabidopsis/clasificación , Proteínas de Arabidopsis/genética , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Aparato de Golgi/metabolismo , Immunoblotting , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Familia de Multigenes , Filogenia , Plantas Modificadas Genéticamente , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Apyrases (ATP-diphosphohydrolase) comprise a ubiquitous class of glycosylated nucleotidases that hydrolyse extracellular ATP and ADP to orthophosphate and AMP. One class of newly-described, Ca2+-dependent, salivary apyrases known to counteract blood-clotting, has been identified in haematophagous arthropods. Herein, we have identified a gene (Oos-apy-1) encoding a protein that structurally conforms to the Ca2+-activated apyrase from the bed bug, Cimex lectularius, by immunologically screening an Ostertagia L4 cDNA expression library. The expressed protein (rOos-APY-1) was biochemically functional in the presence of Ca2+ only, with greatest activity on ATP, ADP, UTP and UDP. Host antibodies to the fusion protein appeared as early as 14 days post-infection (p.i.) and increased through 30 days p.i. Immunohistochemical and Western blot analyses demonstrated that the native Oos-APY-1 protein is present in the glandular bulb of the oesophagus and is confined to the L4. A putative signal sequence at the N-terminus and near 100% identity with a Teladorsagia circumcincta L4 secreted protein is consistent with the native protein being secreted at the cellular level. Predicated upon substrate specificity, the native protein may be used by the parasite to control the levels of host extracellular nucleotides released by locally-damaged tissues in an effort to modulate immune intervention and inflammation.
Asunto(s)
Apirasa/clasificación , Calcio/farmacología , Nucleotidasas/metabolismo , Ostertagia/enzimología , Ostertagia/crecimiento & desarrollo , Animales , Chinches/enzimología , Western Blotting , Esófago/enzimología , Biblioteca de Genes , Proteínas del Helminto/clasificación , Proteínas del Helminto/metabolismo , Inmunohistoquímica , Larva/enzimología , Nucleotidasas/clasificación , Glándulas Salivales/enzimologíaRESUMEN
Salivary apyrases are nucleotide-metabolising enzymes that blood-feeding parasites utilise for modulation of extracellular nucleotides to prevent platelet activation and aggregation. In this study a 5'-nucleotidase specific degenerate primer was used to identify homologous transcripts from Ornithodoros savignyi salivary gland cDNA. Two 5'-nucleotidase isoforms that share significant sequence identity to putative apyrases from Rhipicephalus appendiculatus and Ixodes scapularis were identified. Structure prediction showed a tertiary structure similar to periplasmic ecto-5'-nucleotidase from Escherichia coli, with high conservation of functional residues. The O. savignyi 5'-nucleotidase isoform I was recombinantly expressed in Pichia pastoris. Cross-reactivity was demonstrated with polyclonal anti-apyrase antisera produced against O. savignyi apyrase. Subsequent Edman sequencing and MS/MS analysis of purified O. savignyi apyrase identified peptide sequence fragments that shared sequence identity with both newly identified 5'-nucleotidase isoforms. It was concluded that wild-type apyrase is a mixture of the isoforms identified from the salivary glands of O. savignyi. These results represent the first confirmation of a soft (argasid) tick apyrase that belongs to the 5'-nucleotidase family of enzymes.
Asunto(s)
5'-Nucleotidasa/clasificación , Apirasa/clasificación , Ornithodoros/enzimología , 5'-Nucleotidasa/química , 5'-Nucleotidasa/genética , Secuencia de Aminoácidos , Animales , Animales Domésticos/parasitología , Apirasa/química , Apirasa/genética , Secuencia de Bases , Western Blotting , Clonación Molecular , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Regulación Enzimológica de la Expresión Génica , Isoenzimas/química , Isoenzimas/clasificación , Isoenzimas/genética , Datos de Secuencia Molecular , Ornithodoros/clasificación , Ornithodoros/genética , Filogenia , Pichia/enzimología , Glándulas Salivales/enzimología , Análisis de Secuencia , Dióxido de Silicio , Sudáfrica , Espectrometría de Masas en Tándem , Infestaciones por Garrapatas/parasitología , Infestaciones por Garrapatas/veterinariaRESUMEN
Extracellular nucleotides are hydrolysed by enzymes of the plasma membrane with an extracellularly oriented catalytic site (ectonucleotidases). Members of several families of ectonucleotidases can contribute to extracellular nucleotide hydrolysis. They have been characterized in molecular and functional terms. A major role of these enzymes is in the modulation of ligand availability at nucleotide and nucleoside receptors. The enzymes reveal a wide and partially overlapping tissue distribution. The diversity of the individual family members is considerable and it is still difficult to assign identified enzymes to the modulation of purinergic signalling pathways. In the brain, members of all ectonucleotidase-families are expressed. Proposed physiological functions include modulation of synaptic transmission, of the ATP-mediated propagation of glial Ca2+ waves, of microglial function, adult neurogenesis or the control of vascular tone, haemostasis and thromboregulation.
Asunto(s)
Antígenos CD/metabolismo , Apirasa/metabolismo , Isoenzimas/metabolismo , Proteínas de la Membrana/metabolismo , Sistema Nervioso/enzimología , Nucleótidos/metabolismo , Animales , Antígenos CD/química , Antígenos CD/clasificación , Antígenos CD/genética , Apirasa/química , Apirasa/clasificación , Apirasa/genética , Vasos Sanguíneos/enzimología , Humanos , Isoenzimas/química , Isoenzimas/clasificación , Isoenzimas/genética , Morfogénesis , Neuroglía/enzimología , Neuroglía/fisiología , Neuronas/enzimología , Neuronas/fisiología , Filogenia , Conformación Proteica , Agonistas del Receptor Purinérgico P1 , Antagonistas de Receptores Purinérgicos P1 , Agonistas del Receptor Purinérgico P2 , Antagonistas del Receptor Purinérgico P2 , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2/metabolismo , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Immune responses to sandfly saliva have been shown to protect animals against Leishmania infection. Yet very little is known about the molecular characteristics of salivary proteins from different sandflies, particularly from vectors transmitting visceral leishmaniasis, the fatal form of the disease. Further knowledge of the repertoire of these salivary proteins will give us insights into the molecular evolution of these proteins and will help us select relevant antigens for the development of a vector based anti-Leishmania vaccine. RESULTS: Two salivary gland cDNA libraries from female sandflies Phlebotomus argentipes and P. perniciosus were constructed, sequenced and proteomic analysis of the salivary proteins was performed. The majority of the sequenced transcripts from the two cDNA libraries coded for secreted proteins. In this analysis we identified transcripts coding for protein families not previously described in sandflies. A comparative sandfly salivary transcriptome analysis was performed by using these two cDNA libraries and two other sandfly salivary gland cDNA libraries from P. ariasi and Lutzomyia longipalpis, also vectors of visceral leishmaniasis. Full-length secreted proteins from each sandfly library were compared using a stand-alone version of BLAST, creating formatted protein databases of each sandfly library. Related groups of proteins from each sandfly species were combined into defined families of proteins. With this comparison, we identified families of salivary proteins common among all of the sandflies studied, proteins to be genus specific and proteins that appear to be species specific. The common proteins included apyrase, yellow-related protein, antigen-5, PpSP15 and PpSP32-related protein, a 33-kDa protein, D7-related protein, a 39- and a 16.1- kDa protein and an endonuclease-like protein. Some of these families contained multiple members, including PPSP15-like, yellow proteins and D7-related proteins suggesting gene expansion in these proteins. CONCLUSION: This comprehensive analysis allows us the identification of genus- specific proteins, species-specific proteins and, more importantly, proteins common among these different sandflies. These results give us insights into the repertoire of salivary proteins that are potential candidates for a vector-based vaccine.
Asunto(s)
Proteínas de Insectos/clasificación , Insectos Vectores/genética , Phlebotomus/genética , Proteínas y Péptidos Salivales/clasificación , Secuencia de Aminoácidos , Animales , Apirasa/clasificación , Evolución Molecular , Femenino , Biblioteca de Genes , Proteínas de Insectos/genética , Proteínas de Insectos/inmunología , Insectos Vectores/inmunología , Leishmaniasis Visceral/prevención & control , Leishmaniasis Visceral/transmisión , Datos de Secuencia Molecular , Phlebotomus/inmunología , Filogenia , Proteómica , Vacunas Antiprotozoos/inmunología , Glándulas Salivales/metabolismo , Proteínas y Péptidos Salivales/genética , Proteínas y Péptidos Salivales/inmunología , Alineación de Secuencia , Transcripción GenéticaAsunto(s)
Apirasa/genética , Hidrolasas Diéster Fosfóricas/genética , Animales , Apirasa/clasificación , Apirasa/fisiología , Bovinos , Pollos , Humanos , Isoenzimas/genética , Isoenzimas/fisiología , Ratones , Fosfodiesterasa I , Hidrolasas Diéster Fosfóricas/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Terminología como Asunto , Distribución TisularRESUMEN
An extracellular ATPase (E-type ATPase) clone was isolated from a human brain cDNA library and sequenced. The transcript shows similarity to the previously published chicken smooth muscle and rat brain ecto-ATPase cDNAs, human CD39L1 cDNA (putative human ecto-ATPase), and mammalian CD39 (lymphoid cell activation antigen, ecto-apyrase, ATPDase, ATP-diphosphohydrolase) cDNAs. The full-length human brain cDNA encodes a 529 amino acid glycoprotein with a putative membrane spanning region near each terminus, with the majority of the protein found extracellularly. Expression of this clone in mammalian COS-1 cells yielded NaN3-sensitive ATPase and ADPase activity detectable both on intact cells and cell membrane preparations. The nucleotide hydrolysis ratio of the expressed protein is approx. 2.75:1 (ATPase:ADPase activity), classifying it as an ecto-apyrase. However, this hydrolysis ratio is intermediate between that observed for the ecto-ATPases and the CD39 ecto-apyrases (L. Plesner, Int. Rev. Cytol. 158 (1995) 141-214). Quantitative analyses of amino acid identities and similarities between this ecto-apyrase and other vertebrate E-type ATPases suggest that this human brain enzyme is nearly equally related to the ecto-ATPases and the CD39s, and phylogenetic analysis suggests that it could be an ancestral enzyme from which both ecto-ATPases and CD39 ecto-apyrases are derived.
Asunto(s)
Apirasa/genética , Encéfalo/enzimología , Adenosina Trifosfatasas/genética , Secuencia de Aminoácidos , Antígenos CD/genética , Apirasa/biosíntesis , Apirasa/clasificación , Secuencia de Bases , ADN Complementario/genética , Evolución Molecular , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
We have recently described different isoforms of mammalian ATP diphosphohydrolase (ATPDase; EC 3.6.1.5). In the present study, we purified the lung ATPDase by column chromatographies followed by polyacrylamide gel electrophoresis under nondenaturing conditions. The active polypeptide that has a molecular mass of 78 kDa was identified by affinity labeling to the ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA), followed by detection on Western blot with an antibody specific for FSBA. N-glycosidase F treatment shifted the molecular mass of the 78-kDa polypeptide down to 54 kDa, indicating that the enzyme bears approximately 6-12 NH2-linked oligosaccharide chains. A polyclonal antibody raised against the pancreas ATPDase, which specifically recognized the 78-kDa glycoprotein on Western blot, was used to carry out an immunological survey of the enzyme distribution in bovine lungs. Immunoreactivity was detected on airway epithelia from the trachea down to alveolar cells, airway and vascular smooth muscle cells, submucous glands, chondrocytes, leucocytes, as well as endothelial and mesothelial cells. Such a wide distribution suggests that the ATPDase may affect a variety of physiological effects mediated by extracellular nucleotides, such as airway smooth muscle tone, surfactant secretion, platelet aggregation, and inflammation.