RESUMEN
A cDNA representing the gene Teladorsagia circumcincta apyrase-1 (Tci-apy-1) was isolated, by PCR, from a T. circumcincta fourth-stage larval (L4) cDNA library. The closest orthologue of this gene is a Ca(2+)-dependent apyrase from Ostertagia ostertagi, with 92% amino acid identity across all 339 residues. Tci-apy-1 is transcribed in a stage-specific manner, the transcript being predominant in L4, detectable in the adult cDNA, but absent from eggs and infective third-stage larvae (L3). The protein, Tci-APY-1, was detected by immunoblotting in extracts of L4 nematodes and was present in excretory/secretory products from the same developmental stage. A recombinant version of Tci-APY-1 was expressed in bacteria as an active enzyme that hydrolysed nucleoside triphosphate substrates with a preference of ATP over other nucleoside triphosphates. Recombinant Tci-APY-1 hydrolysed ATP and ADP but not AMP. Apyrase activity was divalent cation-dependent, with no hydrolysis in the presence of Mg(2+), but activation in the presence of Ca(2+). Recombinant Tci-APY-1 was bound by IgG present in serum and both IgG and IgA present in abomasal mucus from trickle-infected, immune sheep but not in material derived from lambs exposed to a single infection. The potential immunomodulatory roles of this Tci-APY-1 are discussed in relation to purinergic signalling.
Asunto(s)
Apirasa/inmunología , Apirasa/metabolismo , Calcio/metabolismo , Trichostrongyloidea/enzimología , Trichostrongyloidea/inmunología , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos Helmínticos/genética , Apirasa/genética , Cationes Bivalentes/metabolismo , ADN Complementario/genética , ADN de Helmintos/genética , Activadores de Enzimas/metabolismo , Perfilación de la Expresión Génica , Proteínas del Helminto/genética , Datos de Secuencia Molecular , Ostertagia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Ovinos , Enfermedades de las Ovejas/inmunología , Trichostrongyloidea/genética , Tricostrongiloidiasis/inmunología , Tricostrongiloidiasis/veterinariaRESUMEN
The bovine interleukin-15 (IL-15) sequence was cloned from abomasal lymph node mRNA by enzymatic amplification of cDNA using human primers proximal to and including the translation start and stop sites. The open reading frame is 486 base pairs in length, and the proposed protein sequence shows 78.4% and 73.5% similarity with that predicted for the human and mouse sequences, respectively. Expressed and purified recombinant bovine IL-15 in the absence of the 48-amino acid leader sequence stimulated the proliferation of bovine lymphoblast cells at least 12-fold over background at maximum concentration levels. Competitive reverse transcriptase-polymerase chain reaction analysis showed constitutive levels of IL-15 mRNA within a broad range of tissues and cell types. Lipopolysaccharide addition to adherent lymph node populations caused moderate increases in IL-15 transcription, whereas the addition of phorbol 12-myristate 13-acetate and calcium ionophore failed to induce gene expression for this cytokine. Transcription of IL-15 was also downregulated in the presence of low concentrations of human recombinant interleukin-2.