Transmembrane Protein 63A Is a Partner Protein of Haemonchus
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Yuan et al. Parasites & Vectors (2015) 8:211 DOI 10.1186/s13071-015-0816-3 RESEARCH Open Access Transmembrane protein 63A is a partner protein of Haemonchus contortus galectin in the regulation of goat peripheral blood mononuclear cells Cheng Yuan, Hui Zhang, Wang Wang, Yan Li, RuoFeng Yan, LiXin Xu, XiaoKai Song and XiangRui Li* Abstract Background: Hco-gal-m and -f were two isoforms of galectin cloned from male and female Haemonchus contortus, respectively, and it was demonstrated that recombinant Hco-gal-m and -f could act as immune suppressors. However, little is known about the receptors or binding partners of these galectins in the host. The research of the molecular mechanisms that govern the interactions between these galectins and host molecules will fill a gap in our understanding how parasite galectins interact with host cells. Methods: A yeast two-hybrid system was used to identify the binding partners of Hco-gal-m and -f in this research. The interaction between rHco-gal-m and candidate binding protein was validated by co-immunoprecipitation. The localization of transmembrane protein 63A (TMEM63A) in peripheral blood mononuclear cells (PBMCs) was detected by immunofluorescence. The distribution of TMEM63A in T cells, B cells and monocytes in PBMCs was detected by flow cytometry. The immunomodulatory effects of Hco-gal-m and TMEM63A on cell proliferation, migration, apoptosis, nitric oxide production and cytokine secretion were observed by co-incubation of rHco-gal-m and TMEM63A-siRNA with goat PBMCs and monocytes. Results: We found that TMEM63A, a functionally unknown protein, from goat PBMCs could bind to Hco-gal-m and -f. Immunofluorescence showed that TMEM63A was localized to the cell membrane. Flow cytometric analysis revealed that TMEM63A was expressed in the majority of goat PBMCs. After using RNA interference to knockdown expression of TMEM63A, the PBMC proliferation and migration were significantly increased, while the influence of rHco-gal-m on monocyte phagocytosis, PBMC nitric oxide production and migration were potently blocked. In addition, the production of IL-10, IFN-γ and TGF-β induced by rHco-gal-m were also altered. Conclusions: Our results show that TMEM63A is a binding partner of Hco-gal-m/f, and involved in the immune responses of host PBMCs induced by Hco-gal-m for the first time. Keywords: Galectin, Haemonchus contortus,Partnerprotein,TMEM63A Background been demonstrated that the interaction of galectin with Galectins are β-galactoside-binding lectins that are its receptor on the target cell governs the function of present not only in the cytosol and nucleus but also in the galectin. The carbohydrate-dependent binding of the extracellular space [1-3]. Secreted galectins of mam- galectin-4 secreted by intestinal epithelial cells to the malian can bind to the cell surface and exert a range of CD3 epitope is fully functional and inhibited T cell acti- functions, including leading to cell adhesion, activating vation, cycling and expansion [8]. Galectin-8 interacts signaling pathways of functional relevance in the control with several members of the integrin family and modu- of receptor endocytosis, host-pathogen interactions, and lates the interactions of integrin with the extracellular activation and homeostasis of immune cells [4-7]. It has matrix and thus regulates cell adhesion and cell survival [9]. Thurston et al. suggest that galectin-8 serves as a * Correspondence: [email protected] versatile receptor for vesicle-damaging pathogens in the College of Veterinary Medicine, Nanjing Agricultural University, Nanjing cytosol [10]. Galectin-9 and galectin-1 both kill thymocytes, 210095, People’s Republic of China © 2015 Yuan et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yuan et al. Parasites & Vectors (2015) 8:211 Page 2 of 13 peripheral T cells, and T cell lines. However, Bi et al. still unknown. In the present research, we identified that have found that galectin-9 and galectin-1 require differ- the effects of Hco-gal-m/f on the proliferation, migration ent glycan ligands and glycoprotein receptors to trigger phagocytosis, nitric oxide and some cytokine productions T cell death [11]. Galectin-3 and galectin-9 can both of the goat PBMC were all altered after the TMEM63A recognize Leishmania major (L. major)bybindingto (NCBI accession number KF850508) gene was knocked the L. major-specific polygalactosyl epitope, but only down by specific small interference RNA (siRNA). Our re- galectin-9 can promote the interaction between L. major sults firstly show that TMEM63A is a binding partner of and macrophages [12,13]. Hco-gal-m/f, and involved in the immune responses of Galectin-like proteins have also been identified in para- host PBMCs induced by Hco-gal-m. sitic nematodes, cestodes and trematodes, such as Oncho- cerca volvulus, Teladorsagia circumcincta, Haemonchus Methods contortus, Trichostrongylus colubriformis, Taenia serialis Ethics statement and Fasciola hepatica [4]. Galectins secreted or excreted The animals were handled according to the guideline of the by parasitic nematodes or other helminths can signifi- Animal Ethics Committee, Nanjing Agricultural University, cantly modulate the host immune response [14-16]. How- China.Allanimalexperimentscomplied with the guidelines ever, little is known about the receptors or binding of the Animal Welfare Council of China. All experimental partners of these galectins in the host. In this case, the re- protocols were approved by the Science and Technology search of the molecular mechanisms that govern the inter- Agency of Jiangsu Province. The approval ID is SYXK (SU) actions between these galectins and host molecules will 2010–0005. The least hardship was certified. fill a gap in our understanding how parasite galectins interact with host cells. Haemonchus contortus (H. contortus) is one of the Animal and cell most economically important parasites of small rumi- Local crossbred goats (3–6-month-old) were fed with hay nants worldwide. Infection can lead to anaemia, loss of and whole shelled corn and watered with libitum and condition and death of the host, especially lambs [17,18]. housed indoor in pens healthily at Nanjing Agricultural Simultaneously, it can cause immunosuppression and re- University. All goats were dewormed twice at 2 week in- sults in a low level immunity in the host [19-21]. This tervals with levamisole (8 mg/kg bodyweight) orally at worm is also the most widely used model parasitic the time of housing to remove naturally acquired strongy- nematode in drug discovery [22], vaccine development lid infection [32]. After 2 weeks, a fecal sample from [23-25] and anthelmintic resistance research [26-29]. each goat was examined by microscopy for helminth eggs, Hco-gal-m (Acc. No. AY253330) and Hco-gal-f (Acc.No. according to standard parasitological techniques. Goats AY253331) are two isoforms of galectin which were cloned exhibiting no eggs were used in the subsequent study from male and female H. contortus respectively. Our previ- and daily health observations were performed throughout ous studies showed that recombinant Hco-gal-m/f (rHco- the experiment. gal-m/f) could bind to the surface of goat PBMCs, and Goat peripheral venous blood samples were collected function as immune suppressors by inducing cell apoptosis from healthy goats consistently. The goat PBMCs were and inhibiting the transcription of IL-1β,IL-4,IFN-γ and separated from blood of six healthy adult goats with the TNF-α mRNA in goat PBMCs in vitro [30,31]. Our further standard Ficoll-hypaque (GE Healthcare, USA) gradient research indicated that rHco-gal-m/f could influence cell centrifugation method [33] and were adjusted to a migration, T cell proliferation and differentiation, the ex- density of 1 × 106 cells/mL in RPMI 1640 or DMEM pression of MHC-II on monocytes and that of CD25 on (GIBCO,UK) containing 10% heat inactivated fetal calf T cells [30,32]. Meanwhile, a combined proteomic and tran- serum (GIBCO, UK), 100 IU/mL penicillin and 100 mg/mL scriptomic analysis revealed that the activations of vascular streptomycin (GIBCO, UK) at 37°C in a humidified atmos- endothelial growth factor pathway, free radical producing phere with 5% CO2. pathway, NFκB pathway and ubiquitin–proteasome path- Monocytes were isolated by their adherence to plastic wayingoatPBMCweredown-regulatedbyrHco-gal-m/f surface [34]. The goat PBMCs were seeded in a 6 wells [30]. These findings suggested that Hco-gal-m/f were multi- flat-bottom tissue culture plates (Corning, USA) in cell functional molecules that can influence many biological culture medium RPMI 1640 (GIBCO,UK) containing processes, especially those relevant to immune responses 10% heat inactivated fetal calfserum (GIBCO, UK), 100 or evasion. The discovery of the binding partner of Hco- U/mL penicillin and 100 mg/mL streptomycin (GIBCO, gal-m/f in goat PBMCs would challenge the current under- UK). Plates were incubated at 37°C in a humidified at- standing of the H. contortus parasite-host interactions. mosphere with 5% CO2 for 1 h [35]. Non-adherent cells Transmembrane protein 63A (TMEM63A) is a member were removed by washing twice with phosphate buffered of the transmembrane protein family. But its function is saline (PBS). The adherent cells were collected and Yuan et al. Parasites & Vectors (2015) 8:211 Page 3 of 13 adjusted to a density of 1 × 106 cells/mL in cell medium pH 7.4) [37], containing a protease inhibitor cocktail at 37°C in a humidified atmosphere with 5% CO2. (Merck, Darmstadt, Germany). Cell lysates were pre- Cells used for the experiments were freshly isolated from cleared by adding 1 μg rat normal IgG and 20 μL Protein goat peripheral blood.