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Abstract
Lysophospholipase I (LYPLA1) is an important protein with multiple functions. In this study, the full-length cDNA of the LYPLA1 gene from Ovis aries (OaLypla1) was cloned using primers and rapid amplification of cDNA ends (RACE) technology. The full-length OaLypla1 was 2457 bp with a 5′-untranslated region (UTR) of 24 bp, a 3′-UTR of 1740 bp with a poly (A) tail, and an open reading frame (ORF) of 693 bp encoding a protein of 230 amino acid residues with a predicted molecular weight of 24,625.78 Da. Phylogenetic analysis showed that the OaLypla1 protein shared a high amino acid identity with LYPLA1 of Bos taurus. The recombinant OaLypla1 protein was expressed and purified, and its phospholipase activity was identified. Monoclonal antibodies (mAb) against OaLypla1 that bound native OaLypla1 were generated. Real-time PCR analysis revealed that OaLypla1 was constitutively expressed in the liver, spleen, lung, kidney, and white blood cells of sheep, with the highest level in the kidney. Additionally, the mRNA levels of OaLypla1 in the buffy coats of sheep challenged with virulent or avirulent Brucella strains were down-regulated compared to untreated sheep. The results suggest that OaLypla1 may have an important physiological role in the host response to bacteria. The function of OaLypla1 in the host response to bacterial infection requires further study in the future.
Highlights
Lysophospholipase I (LYPLA1), known as acyl-protein thioesterase 1 (APT1), is a widely distributed enzyme with phospholipase A2 [1], lysophospholipase [1,2] and acyl-protein thioesterase [3,4,5] activity
The expression profiles of OaLypla1 were analyzed using quantitative real-time PCR (qPCR), and the results confirmed that the transcriptional levels of OaLypla1 in white blood cells (WBCs) were down-regulated by infection of Brucella compared with the uninfected sheep
Tissue distribution of OaLypla1 in the liver, spleen, lung, kidney and white blood cells (WBCs) of sheep was analyzed by quantitative real-time PCR and Western blotting
Summary
Lysophospholipase I (LYPLA1), known as acyl-protein thioesterase 1 (APT1), is a widely distributed enzyme with phospholipase A2 [1], lysophospholipase [1,2] and acyl-protein thioesterase [3,4,5] activity. In contrast to other lipid modifications, such as isoprenylation and myristoylation, palmitoylation is a unique reversible post-translational modification [17] that allows proteins to rapidly shuttle between intracellular membrane compartments [18,19] and can be dynamically regulated by specific extracellular stimuli [18] This modification is important for regulating protein subcellular localization, stability, trafficking, translocation to lipid rafts, aggregation, and interaction with effectors and other protein functions [17]. A time course suppression subtractive hybridization (SSH) cDNA library of buffy coats from sheep (Ovis aries) infected with different virulent Brucella strains was constructed to analyze the modulation of transcriptional profiles of hosts exposed to Brucella infection, and the differentially transcribed genes were screened. 3 of 16 3 of 15 waassseesxspedre.sTshede taisnsduepudirsitfireibdu, taionndoitfsOpahLoyspplah1owlipasasdeetaecrtmiviintyedw, ansdasdsieffsesreedn.tiTahl extpisrseusesiodnisptrriobfuilteisonofof OOaLayLpylpal1a1winasthdeetbeurmffyinceoda,tsanofdsdhiefefeprefonltlioawl einxgprcehsasliloengperowfiiltehsdoiffOeraeLnytpvlair1uilnentthBerbuuceffllya csotraatisnosfwshereeep foolbloswerivnegdc. hFaullrethnegremwoirteh, dwifefegrentervairteudlenatmBrouncoelcllaosntarlaiannstwibeordeyob(mseArvbe)dt.hFaut rrtehaecrtms owreit,hwtehegennaetriavteed aOmaoLnyopcllao1n. aTlhaenrteibsuoldtsyf(rmomAbth)itshsatturdeyacmtsaywfiathciltihtaetenafutirvteheOrasLtuydpylao1f. tThheefurenscutilotsnsfroofmOathLiyspslat1udinytmheay fahcoilsittarteespfuorntsheetrositnufdeyctoiofnthweitfhunBcrtuiocenllsa.of OaLypla in the host response to infection with Brucella
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