Arginine kinase from Haemonchus contortus decreased the proliferation and increased the apoptosis of goat PBMCs in vitro

Muhammad Ehsan,Yujian Wang,Lixin Xu,Mingmin Lu,Xiaokai Song,Wenxiang Gao,Ruofeng Yan,Xiangrui Li,Javaid Ali Gadahi,Xinchao Liu

doi:10.1186/s13071-017-2244-z

Muhammad Ehsan, Yujian Wang + Show 8 more

Open Access

https://doi.org/10.1186/s13071-017-2244-z

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Abstract

BackgroundArginine kinase (AK), an important member of phosphagen kinase family has been extensively studied in various vertebrates and invertebrates. Immunologically, AKs are important constituents of different body parts, involved in various biological and cellular functions, and considered as immune-modulator and effector for pro-inflammatory cytokines. However, immunoregulatory changes of host cells triggered by AK protein of Haemonchus contortus, a parasitic nematode of ruminants, are still unknown. The current study was focused on cloning and characterisation of Hc-AK, and its regulatory effects on cytokines level, cell migration, cell proliferation, nitric oxide production and apoptosis of goat peripheral blood mononuclear cells (PBMCs) were observed.MethodsThe full-length sequence of the Hc-AK gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sub-cloned into the prokaryotic expression vector pET-32a. The biochemical characteristics of recombinant protein Hc-AK, which was purified by affinity chromatography, were performed based on the enzymatic assay. Binding of rHc-AK with PBMCs was confirmed by immunofluorescence assay (IFA). Immunohistochemical analysis was used to detect localisation of Hc-AK within adult worms sections. The immunoregulatory effects of rHc-AK on cytokine secretions, cell proliferation, cell migration, nitric oxide production and apoptosis were determined by co-incubation of rHc-AK with goat PBMCs.ResultsThe full-length ORF (1080 bp) of the Hc-AK gene was successfully cloned, and His-tagged AK protein was expressed in the Escherichia coli strain BL21. The recombinant protein of Hc-AK (rHc-AK) was about 58.5 kDa together with the fused vector protein of 18 kDa. The biochemical assay showed that the protein encoded by the Hc-ak exhibited enzymatic activity. Western blot analysis confirmed that the rHc-AK was recognised by the sera from rat (rat-antiHc-AK). The IFA results showed that rHc-AK could bind on the surface of goat PBMCs. Immunohistochemically, Hc-AK was localised at the inner and outer membrane as well as in the gut region of adult worms. The binding of rHc-AK to host cells increased the levels of IL-4, IL-10, IL-17, IFN-γ, nitric oxide (NO) production and cell apoptosis of goat PBMCs, whereas, TGF-β1 levels, cell proliferation and PBMCs migration were significantly decreased in a dose dependent manner.ConclusionsOur findings suggested that rHc-AK is an important excretory and secretory (ES) protein involved in host immune responses and exhibit distinct immunomodulatory properties during interaction with goat PBMCs.

Highlights

Arginine kinase (AK), an important member of phosphagen kinase family has been extensively studied in various vertebrates and invertebrates
Molecular cloning and sequence analysis of H. contortus AK gene (Hc-AK) gene The amplified PCR products of Hc-AK gene were obtained from H. contortus cDNA using a specific pair of primers, and a correct fragment size of 1080 bp was detected
These results indicated that Hc-AK had been successfully inserted into the frame of pET32a vector

Summary

Introduction

Arginine kinase (AK), an important member of phosphagen kinase family has been extensively studied in various vertebrates and invertebrates. Arginine kinase (AK), being a highly conserved member of the phosphagen kinase (PKs) family, has been studied extensively with a high degree of sequence similarity among various invertebrate species including H. contortus [5], Trypanosoma cruzi [6], Caenorhabditis elegans [7], Heterodera glycines [8], Toxocara canis and Ascaris lumbricoides [9] and proteobacteria [10]. Co-substrate of AKs and nitric oxide synthase (NOS), L-arginine has been considered to cause immunomodulation through nitric oxide (NO) synthesis via different biochemical pathways and decreased the level of pro-inflammatory cytokines [12]. The induced expression levels of Chlamys farreri AK and NO concentration after LPS stimulation indicated that AK played an important role in immunomodulation during invading pathogens [17]

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