Identification of Schistosoma japonicum schistosomula proteins interacted with Microtus fortis resistance-related ILKAP by co-immunoprecipitation

doi:10.12140/j.issn.1000-7423.2019.02.002

Abstract

Abstract:

Objective To understand the mechanism underlying the natural resistance to Schistosoma infection in Microtus fortis, the schistosomula proteins interacted with ILKAP(integrin-linked kinase-associated serine/threonine phosphatase), one of the resistant-related proteins in M. fortis, were screened and identified. Methods The crude proteins were extracted from S. japonicum schistosomula and those proteins specifically bound with M. fortis ILKAP(MfILKAP) were isolated and identified by immunoprecipitation using magnetic beads conjugated with anti-ILKAP IgG. Briefly, the beads conjugated with anti-ILKAP IgG were co-incubated with bone marrow proteins of M. fortis and S. japonicum schistosomula extracts, the proteins bound to the beads were extracted in SDS-loading buffer and separated by SDS-PAGE. The separated bands were cut from the gel and analyzed by MALDI-TOF-MS. The schistosomula proteins bound to ILKAP were identified by searching through genome database of S. japonicum. In order to further determine the binding activity, genes for one of the ILKAP-bound S. japonicum proteins, tegument antigen （TA), and MfILKAP were amplified and cloned into eukaryotic expression plasmids Flag-pCMV-2b and Myc-pcDNA3.1/(-)b, respectively. The recombinant plasmid DNAs were co-transfected into HEK293T cells. The expression of recombinant tegument antigen of S. japonicum (SjTA) and MfILKAP and their interaction were confirmed by co-immunoprecipitation with anti-ILKAP. Results A total of 8 S. japonicum schistosomula proteins bound to MfILKAP were identified by co-immunoprecipitation with anti-ILKAP IgG and MALDI-TOF-MS analysis. One of the major ILKAP-binding proteins was SjTA. After RT-PCR amplification and enzymatic digestion, fragments of 1 147 bp, and 4 300 bp from M. fortis, and fragments of 561 bp and 5 500 bp from Schistosomula were obtained, respectively. In order to further determine their interacting activity, MfILKAP and SjTA were successfully cloned from their corresponding cDNA and the recombinant proteins were effectively expressed in HEK293T cells determined by Western blotting with specific antibodies. The binding activity of recombinant MfILKAP to recombinant SjTA was further confirmed by co-immunoprecipitation with anti-ILKAP IgG from the co-expressed cell lysates and recognition by the specific antibodies. Conclusion Total 8 S. japonicum schistosomula proteins have been identified to bind to MfILKAP. One of the ILKAP-binding proteins, SjTA, was cloned and co-expressed in HEK293T cells. The interaction between SjTA and MfILKAP was further confirmed by the binding of both recombinant proteins.

Key words: Microtus fortis, Schistosomula, Integrin-linked kinase-associated serine/threonine phosphatase, Co-immunoprecipitation

CLC Number:

R383.24

Rong LI, De-hui XIONG, Wei-xin HU. Identification of Schistosoma japonicum schistosomula proteins interacted with Microtus fortis resistance-related ILKAP by co-immunoprecipitation[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2019, 37(2): 121-126.

Figures/Tables 7

Table 1

Fig. 1

Table 2

Fig. 2

Fig. 3

Fig. 4

Fig. 5

References 21

[1]	Utzinger J, Becker SL, Knopp S, et al. Neglected tropical diseases: diagnosis, clinical management, treatment and control[J]. Swiss Med Wkly, 2012, 142(47): w13727.
[2]	张利娟, 徐志敏, 钱颖骏, 等. 2016年全国血吸虫病疫情通报[J]. 中国血吸虫病防治杂志, 2017, 29(6): 669-677.
[3]	周智君, 苏志杰, 俞远京. 东方田鼠地方标准的编制以及相关研究进展[J]. 中国实验动物学报, 2014, 22(6): 103-105.
[4]	邬国军, 秦志强, 罗赛群, 等. 东方田鼠血清组分对日本血吸虫童虫的体外杀伤力[J]. 中国寄生虫学与寄生虫病杂志, 2005, 23(6): 404-407.
[5]	蒋守富, 魏梅雄, 林矫矫, 等. 东方田鼠血清被动转移抗日本血吸虫的保护力研究[J]. 中国病原生物学杂志, 2004, 17(5): 298-300.
[6]	程红兵, 周云飞, 张树菊, 等. pVAX1/IL-18对日本血吸虫DNA疫苗效果影响的初步研究[J]. 中国人兽共患病学报, 2018, 34(9): 774-777.
[7]	姚利晓, 林矫矫, 傅志强, 等. 日本血吸虫新基因SjPP的筛选、克隆及对小鼠的免疫效果[J]. 中国农业科学, 2006, 39(8): 1659-1666.
[8]	阎玉涛, 刘述先, 宋光承, 等. 东方田鼠天然抗体相关的日本血吸虫抗原基因筛选和克隆[J]. 中国寄生虫学与寄生虫病杂志, 2001, 19(3): 153-156.
[9]	Zarowlecki MZ, Huyse T, Littlewood DT.Making the most of mitochondrial genomes-markers for phylogeny, molecular ecology and barcodes in Schistosoma (Platyhelminthes:Digenea)[J]. Int J Parasitol, 2007, 37(12): 1401-1418.
[10]	熊德慧. 东方田鼠抗日本血吸虫相关基因E77.43全长cDNA的克隆及生物信息学分析 [D]. 长沙: 中南大学, 2009.
[11]	Gong Q, Cheng G, Qin ZQ, et al. Identification of the resistance of a novel molecule heat shock protein 90αlpha (HSP90αlpha) in Microtus fortis to Schistosoma japonicum infection[J]. Acta Trop, 2010, 115(3): 220-226.
[12]	Cheng G, Gong Q, Gai N, et al. Karyopherin alpha 2 (KPNA2) is associated with the natural resistance to Schistosoma japanicum infection in Microtus fortis[J]. Biomed Pharmacother, 2011, 65(3): 230-237.
[13]	Li R, Wu GJ, Xiong DH, et al. A Microtus fortis protein, is a novel inhibitor of Schistosoma japonicum schistosomula[J]. Mem Inst Oswarld Cruz, 2013, 180(7): 865-872.
[14]	Hu Y, Xu YX, Lu WY, et al. De novo assembly and transcriptome characterization: novel insights into the natural resistance mechanisms of Microtus fortis against Schistosoma japonicum[J]. BMC Genomics, 2014, 15: 417-430.
[15]	You H, McManus DP, Hu W, et al. Transcriptional responses of in vivo praziquantel exposure in schistosomes identifies a functional role for calcium signalling pathway member camKII[J]. PLoS One, 2013, 9(3): e1003254.
[16]	Hehlgans S, Haase M, Cordes N.Signalling via integrins: implications for cell survival and anticancer strategies[J]. Biochim Biophys Acta, 2007, 1775(1): 163-180.
[17]	钱门宝,胡薇. 血吸虫体被的结构功能及其蛋白质组研究[J]. 中国寄生虫学与寄生虫病杂志, 2008, 26(6): 466-471.
[18]	Hu S, Law Pk, Lv Z, et al. Molecular characterization of a calcium-binding protein SjCa8 from Schistosoma japonicum[J]. Parasitol Res, 2008, 103(5): 1047-1053.
[19]	张旻. 日本血吸虫体被表膜蛋白分析及表膜蛋白Sj NPP-5的初步研究[D]. 上海: 上海兽医研究所, 2011.
[20]	Liu Y, Brindley PJ. Zeng Q, et al. Identification of phage display peptides with affinity for the tegument of Schistosoma japonicum schistosomula[J]. Mol Biochem Parasitol, 2011, 180(2): 86-98.
[21]	Zhang Z, Xu H, Gan W, et al. Schistosoma japonicum calcium-binding tegumental protein SjTP22.4 immunization confers praziquantel schistosomulumicide and antifecundity effect in mice[J]. Vaccine, 2012 30(34): 5140-5150.

基因 Gene	引物（3′→5′） Primer （3′→5′）
MfILKAP	F: ATTACCCGGGATGGACCTGTTCGGGGATCTG
	R: ATATGCGGCCGCCAAGTCAAGAGTGCATGTGCTG
SjTA	F: GGCAAGCTTATGGCGAGTACAATGGAACAAT
	R: GCTGCTAGCGTGTACGCCAAGCAAGAATGA

蛋白名称 Protein description	蛋白GI号 Accession no.	蛋白相对分子质量 M_r	得分 Score
表膜抗原 Tegument antigen	gi-189503050	21 793	84
SjCHGC0600 protein	gi-56759178	20 016	50
先天性胰腺脂肪过多症Shwachman-bodian-diamond syndrome protein	gi-2266487060	18 124	48
Hypothetical protein, conserved	gi-CMU-008010	34 695	37
谷胱甘肽转移酶同工酶Glutathione S-transferase class-mu 26 kDa isozyme	gi-121697	25 710	49
Hypothetical protein CLF_105248	gi-3582543661	40 586	29
3-磷酸甘油醛脱氢酶 Glyceraldehyde-3-phosphate dehydrogenase	gi-1660996	36 788	26
Retrovirus-related pol polymrotein from type-1 retrotransposable elemeng R2	gi-358255808	36 908	266