RESUMEN
The Gamma interferon inducible lysosomal thiol reductase (GILT) plays a key biological role in the immune responses and involves in the processing of class II MHC-restricted antigen by stimulating disulfide bond reduction in mammals. To determine the biological function of GILT in the innate immune system of crustaceans, we sequenced and cloned GILT gene from red swamp crayfish, Procambarus clarkii (Pc-GILT). The deduced amino acid sequence of Pc-GILT contained the putative conserved structures of the GILT family proteins: the GILT signature (CQHGX2ECX2NX4C) sequence and the active site (CXXS) motif. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis suggested that a recombinant Pc-GILT protein was successfully expressed in Escherichia coli (E. coli). Quantitative real-time PCR analysis showed that Pc-GILT transcript level was highest in the hepatopancreas followed by the gut, heart and muscles. Additionally, we analyzed the transcription level of Pc-GILT gene in hepatopancreas of red swamp crayfish under biotic stress conditions. The expression of Pc-GILT gene upregulated after viral (poly I:C) and bacterial (peptidoglycan, lipopolysaccharide) infection. The suppression of Pc-GILT by double stranded RNA influenced the transcript levels of various immune-related genes. These observations indicate that the Pc-GILT probably plays a key biological role in the innate immune responses of red swamp crayfish, since it modulates the expression of genes associated with immune pathways.
Asunto(s)
Proteínas de Artrópodos/inmunología , Astacoidea/inmunología , Inmunidad Innata/genética , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/inmunología , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/antagonistas & inhibidores , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Astacoidea/clasificación , Astacoidea/genética , Secuencia de Bases , Expresión Génica , Regulación de la Expresión Génica/inmunología , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/antagonistas & inhibidores , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/química , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Filogenia , Interferencia de ARN , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Alineación de Secuencia , Estrés Fisiológico/genética , Estrés Fisiológico/inmunología , Distribución TisularRESUMEN
Peroxiredoxin 6 (Prx6) is an important member of the peroxiredoxin family that plays critical roles in protecting host against the toxicity of oxidative stress and participates in cell signaling. Herein, we report Prx6 gene from red swamp crayfish, Procambarus clarkii. The cDNA fragment of PcPrx6 was 660 bp, encoding a 219 amino acid residues protein. The quantitative real time PCR analysis showed ubiquitous expression of PcPrx6 mRNA in the tested tissues. The challenge with peptidoglycan and Poly I:C remarkably suppressed the mRNA level of PcPrx6 in hepatopancreas at 3, 12, 48â¯h compared with the PBS control. However, the expression level significantly increased after 36â¯h of their treatment. The knockdown of PcPrx6 by small interference RNA significantly enhanced the transcript levels of Toll pathway-responsive genes at 24â¯h. Recombinant PcPrx6 protein was purified using affinity chromatography and analyzed for its biological role. The results revealed that the recombinant PcPrx6 protein manifested the ability to protect supercoiled DNA damage from oxidative stress elicited by mixed function oxidative assay. Altogether, PcPrx6 may have multiple functional roles in the physiology of P. clarkii, since it negatively regulates the Toll signaling transduction and protects supercoiled DNA damage from oxidative stress.
Asunto(s)
Astacoidea/genética , Astacoidea/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Peroxiredoxina VI/genética , Peroxiredoxina VI/inmunología , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/inmunología , Secuencia de Bases , Cromatografía de Afinidad , Daño del ADN , ADN Superhelicoidal/fisiología , Perfilación de la Expresión Génica , Estrés Oxidativo , Peptidoglicano/farmacología , Peroxiredoxina VI/química , Filogenia , Poli I-C/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Alineación de SecuenciaRESUMEN
Anti-lipopolysaccharide factors are a group of small proteins with broad spectrum antiviral property and antibacterial activity. Herein, we obtained the genomic sequence of the Procambarus clarkii anti-lipopolysaccharide factor (PcALF) gene by using polymerase chain reaction to investigate its expression pattern in various tissues and in the immune tissues (Hepatopancreas) following exposure to pathogens. The deduced protein of PcALF was conserved; it displayed the signal peptides and putative lipo-polysaccharide binding domain, particularly the two conserved cysteine amino acid residues at both ends of the domain. The recombinant protein of PcALF was successfully expressed in Escherichia coli and rabbit anti-PcALF polyclonal antibodies were prepared. The qRT-PCR analysis showed unequal distribution of PcALF transcript in the examined tissues, however the transcript level was greatest in hepatopancreas. The challenge with peptidoglycan (PGN), lipo-polysaccharide (LPS) and Poly I:C significantly enhanced expression level of PcALF in hepatopancreas when compared with the PBS control. RNA interference of PcALF affected the mRNA expression levels of immune-related genes. Taken together, our data suggested that PcALF is an inducible protein and could play a key biological role in the innate immune defense of P. clarkii.