微小隐孢子虫Ⅰ型跨膜蛋白Cgd7_1430的亚细胞定位及其HCT-8细胞黏附特性研究
|
摘要
目的对微小隐孢子虫Ⅰ型跨膜蛋白Cgd7_1430进行亚细胞定位,研究其对HCT-8细胞的黏附特性及肝素结合特性,为深入研究微小隐孢子虫的侵入机制提供依据。方法对Cgd7_1430序列进行生物信息学分析,合成特异性多肽并制备抗多肽抗体,通过间接免疫荧光对该蛋白质在子孢子进行亚细胞定位;通过PCR扩增Cgd7_1430全长基因并克隆至PGEX-4T-1载体,用大肠埃希菌BL21(DE3)菌株表达重组蛋白;通过细胞亲和ELISA和流式细胞术确定重组蛋白与HCT-8细胞的黏附特性;通过Pull-down确定重组蛋白的肝素结合特性。结果成功获得抗Cgd7_1430多肽抗体及其重组蛋白质,间接免疫荧光表明该蛋白定位于子孢子表膜,ELISA、流式细胞术表明该重组蛋白能够与HCT-8细胞结合,并呈现剂量依赖性和可饱和性,Pull-down实验表明重组蛋白能够与肝素结合。结论 Cgd7_1430蛋白质是一种子孢子表膜蛋白,能够与HCT-8细胞特异性结合,该蛋白为肝素结合蛋白,可能与宿主细胞表面的硫化肝素受体结合介导虫体的侵入过程。本研究为深入研究隐孢子虫的侵入机制提供依据。
Objective Type I transmembrane proteins play an important role during invasion by parasitic alveolates. This study examined the subcellular localization and preliminary adhesion characteristics of Cryptosporidium parvum type I transmembrane protein Cgd7_1430 in order to provide evidence for the study of the parasite's mechanism of invasion. Methods Cgd7_1430 was bioinformatically analyzed, and a specific peptide was designed and synthesized to prepare an anti-peptide antibody. Subcellular localization of the protein in sporozoites was identified using indirect immunofluorescence. The full-length Cgd7_1430 gene was cloned into a PGEX-4 T-1 vector and expressed in E. coli BL21(DE3). The binding of the recombinant protein to HCT-8 cells was identified using cell-binding ELISA and flow cytometry. The binding of the recombinant protein to heparin was investigated using a pull-down assay. Results The anti-peptide antibody and recombinant protein were successfully prepared. An indirect immunofluorescence assay indicated that the protein was localized to the membrane of sporozoites. ELISA and flow cytometry indicated that the recombinant protein bound to HCT-8 cells in a dose-dependent and saturable manner. A pull-down assay indicated that the recombinant protein bound to heparin. Conclusion Cgd7_1430 protein is located on the membrane of sporozoites and can specifically bind to HCT_8 cells and heparin, which may mediate the invasion process by adhesion to heparin sulfate on host cells. This study may provide some evidence for further study of the parasite's mechanism of invasion.
Objective Type I transmembrane proteins play an important role during invasion by parasitic alveolates. This study examined the subcellular localization and preliminary adhesion characteristics of Cryptosporidium parvum type I transmembrane protein Cgd7_1430 in order to provide evidence for the study of the parasite's mechanism of invasion. Methods Cgd7_1430 was bioinformatically analyzed, and a specific peptide was designed and synthesized to prepare an anti-peptide antibody. Subcellular localization of the protein in sporozoites was identified using indirect immunofluorescence. The full-length Cgd7_1430 gene was cloned into a PGEX-4 T-1 vector and expressed in E. coli BL21(DE3). The binding of the recombinant protein to HCT-8 cells was identified using cell-binding ELISA and flow cytometry. The binding of the recombinant protein to heparin was investigated using a pull-down assay. Results The anti-peptide antibody and recombinant protein were successfully prepared. An indirect immunofluorescence assay indicated that the protein was localized to the membrane of sporozoites. ELISA and flow cytometry indicated that the recombinant protein bound to HCT-8 cells in a dose-dependent and saturable manner. A pull-down assay indicated that the recombinant protein bound to heparin. Conclusion Cgd7_1430 protein is located on the membrane of sporozoites and can specifically bind to HCT_8 cells and heparin, which may mediate the invasion process by adhesion to heparin sulfate on host cells. This study may provide some evidence for further study of the parasite's mechanism of invasion.
引文
[1] Checkley W, White AC, Jaganath D, et al. A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for Cryptosporidium[J]. Lancet Infect Dis, 2015, 15(1): 85-94.
[2] Chen XM, Keithly JS, Paya CV, et al. Current concepts: Cryptosporidiosis [J]. New Engl J Med, 2002, 346(22):1723-31.
[3] Kotloff KL, Nataro JP, Blackwelder WC, et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study [J]. Lancet, 2013, 382(9888): 209-22.
[4] Gargala G, Delaunay A, Li XD, et al. Efficacy of nitazoxanide, tizoxanide and tizoxanide glucuronide against Cryptosporidium parvum development in sporozoite-infected HCT-8 enterocytic cells [J]. J Antimicrob Chemother, 2000, 46(1): 57-60.
[5] Dreyfuss JL, Regatieri CV, Jarrouge TR, et al. Heparan sulfate proteoglycans: structure, protein interactions and cell signaling [J]. An Acad Bras Cienc, 2009, 81(3): 409-29.
[6] Kobayashi K, Kato K, Sugi T, et al. Plasmodium falciparum BAEBL binds to heparan sulfate proteoglycans on the human erythrocyte surface [J]. J Biol Chem, 2010, 285(3): 1716-25.
[7] Azzouz N, Kamena F, Laurino P, et al. Toxoplasma gondii secretory proteins bind to sulfated heparin structures [J]. Glycobiology, 2013, 23(1): 106-20.
[8] Love DC, Esko JD, Mosser DM. A heparin-binding activity on Leishmania amastigotes which mediates adhesion to cellular proteoglycans [J]. J Cell Biol, 1993, 123(3): 759-66.
[9] Copeland R, Balasubramaniam A, Tiwari V, et al. Using a 3-O-sulfated heparin octasaccharide to inhibit the entry of herpes simplex virus type 1 [J]. Biochemistry-Us, 2008, 47(21): 5774-83.
[10] Liu J, Thorp SC. Cell surface heparan sulfate and its roles in assisting viral infectious [J]. Med Res Rev, 2002, 22(1): 1-25.
[11] Schulze A, Gripon P, Urban S. Hepatitis B virus infection initiates with a large surface protein-dependent binding to heparan sulfate proteoglycans [J]. Hepatology, 2007, 46(6): 1759-68.
[12] Wuppermann FN, Hegemann JH, Jantos CA. Heparan sulfate-like glycosaminoglycan is a cellular receptor for Chlamydia pneumoniae [J]. J Infect Dis, 2001, 184(2): 181-7.
[13] Liang OD, Ascencio F, Fransson LA, et al. Binding of heparan sulfate to Staphylococcus aureus [J]. Infect Immun, 1992, 60(3): 899-906.
[14] Ludington JG, Ward HD. The Cryptosporidium parvum C-Type lectin CpClec mediates infection of intestinal epithelial cells via interactions with sulfated proteoglycans [J]. Infect Immun, 2016, 84(5): 1593-602.
[15] Inomata A, Murakoshi F, Ishiwa A, et al. Heparin interacts with elongation factor 1alpha of Cryptosporidium parvum and inhibits invasion [J]. Sci Rep, 2015(5): 11599.
[16] Putignani L, Possenti A, Cherchi S, et al. The thrombospondin-related protein CpMIC1 (CpTSP8) belongs to the repertoire of micronemal proteins of Cryptosporidium parvum [J]. Mol Biochem Parasitol, 2008, 157(1): 98-101.
[17] Spano F, Putignani L, Naitza S, et al. Molecular cloning and expression analysis of a Cryptosporidium parvum gene encoding a new member of the thrombospondin family [J]. Mol Biochem Parasitol, 1998, 92(1): 147-62.
[18] Zarei O, Irajian GR, Zarnani AH, et al. Peptide-based Polyclonal Antibody Production against P110 Protein of Mycoplasma genitalium[J]. Avi J Med Biotechnol, 2011, 3(2): 79-85.
[19] Hadavi R, Zarnani AH, Ahmadvand N, et al. Production of monoclonal antibody against human nestin[J]. Avicenna J Med Biotechnol, 2010, 2(2): 69-77.
[20] Bhat N, Joe A, PereiraPerrin M, et al. Cryptosporidium p30, a galactose/N-acetylgalactosamine-specific lectin, mediates infection in vitro [J]. J Biol Chem, 2007, 282(48): 34877-87
[21] Inomata A, Murakoshi F, Ishiwa A, et al. Heparin interacts with elongation factor 1alpha of Cryptosporidium parvum and inhibits invasion [J]. Sci Rep, 2015(5): 11599.
[22] McGuckin MA, Linden SK, Sutton P, et al. Mucin dynamics and enteric pathogens [J]. Nat Rev Microbiol, 2011, 9(4): 265-278.
[23] Kato K, Ishiwa A: The role of carbohydrates in infection strategies of enteric pathogens [J]. Trop Med Health, 2015, 43(1): 41-52.
[24] Dreyfuss JL, Regatieri CV, Jarrouge TR, et al. Heparan sulfate proteoglycans: structure, protein interactions and cell signaling [J]. An Acad Bras Cienc, 2009, 81(3): 409-29.
[25] Cardin AD, Weintraub HJR. Molecular modeling of protein-Glycosaminogly-can interactions[J]. Arteriosclerosis, 1989, 9(1): 21-32.
[2] Chen XM, Keithly JS, Paya CV, et al. Current concepts: Cryptosporidiosis [J]. New Engl J Med, 2002, 346(22):1723-31.
[3] Kotloff KL, Nataro JP, Blackwelder WC, et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study [J]. Lancet, 2013, 382(9888): 209-22.
[4] Gargala G, Delaunay A, Li XD, et al. Efficacy of nitazoxanide, tizoxanide and tizoxanide glucuronide against Cryptosporidium parvum development in sporozoite-infected HCT-8 enterocytic cells [J]. J Antimicrob Chemother, 2000, 46(1): 57-60.
[5] Dreyfuss JL, Regatieri CV, Jarrouge TR, et al. Heparan sulfate proteoglycans: structure, protein interactions and cell signaling [J]. An Acad Bras Cienc, 2009, 81(3): 409-29.
[6] Kobayashi K, Kato K, Sugi T, et al. Plasmodium falciparum BAEBL binds to heparan sulfate proteoglycans on the human erythrocyte surface [J]. J Biol Chem, 2010, 285(3): 1716-25.
[7] Azzouz N, Kamena F, Laurino P, et al. Toxoplasma gondii secretory proteins bind to sulfated heparin structures [J]. Glycobiology, 2013, 23(1): 106-20.
[8] Love DC, Esko JD, Mosser DM. A heparin-binding activity on Leishmania amastigotes which mediates adhesion to cellular proteoglycans [J]. J Cell Biol, 1993, 123(3): 759-66.
[9] Copeland R, Balasubramaniam A, Tiwari V, et al. Using a 3-O-sulfated heparin octasaccharide to inhibit the entry of herpes simplex virus type 1 [J]. Biochemistry-Us, 2008, 47(21): 5774-83.
[10] Liu J, Thorp SC. Cell surface heparan sulfate and its roles in assisting viral infectious [J]. Med Res Rev, 2002, 22(1): 1-25.
[11] Schulze A, Gripon P, Urban S. Hepatitis B virus infection initiates with a large surface protein-dependent binding to heparan sulfate proteoglycans [J]. Hepatology, 2007, 46(6): 1759-68.
[12] Wuppermann FN, Hegemann JH, Jantos CA. Heparan sulfate-like glycosaminoglycan is a cellular receptor for Chlamydia pneumoniae [J]. J Infect Dis, 2001, 184(2): 181-7.
[13] Liang OD, Ascencio F, Fransson LA, et al. Binding of heparan sulfate to Staphylococcus aureus [J]. Infect Immun, 1992, 60(3): 899-906.
[14] Ludington JG, Ward HD. The Cryptosporidium parvum C-Type lectin CpClec mediates infection of intestinal epithelial cells via interactions with sulfated proteoglycans [J]. Infect Immun, 2016, 84(5): 1593-602.
[15] Inomata A, Murakoshi F, Ishiwa A, et al. Heparin interacts with elongation factor 1alpha of Cryptosporidium parvum and inhibits invasion [J]. Sci Rep, 2015(5): 11599.
[16] Putignani L, Possenti A, Cherchi S, et al. The thrombospondin-related protein CpMIC1 (CpTSP8) belongs to the repertoire of micronemal proteins of Cryptosporidium parvum [J]. Mol Biochem Parasitol, 2008, 157(1): 98-101.
[17] Spano F, Putignani L, Naitza S, et al. Molecular cloning and expression analysis of a Cryptosporidium parvum gene encoding a new member of the thrombospondin family [J]. Mol Biochem Parasitol, 1998, 92(1): 147-62.
[18] Zarei O, Irajian GR, Zarnani AH, et al. Peptide-based Polyclonal Antibody Production against P110 Protein of Mycoplasma genitalium[J]. Avi J Med Biotechnol, 2011, 3(2): 79-85.
[19] Hadavi R, Zarnani AH, Ahmadvand N, et al. Production of monoclonal antibody against human nestin[J]. Avicenna J Med Biotechnol, 2010, 2(2): 69-77.
[20] Bhat N, Joe A, PereiraPerrin M, et al. Cryptosporidium p30, a galactose/N-acetylgalactosamine-specific lectin, mediates infection in vitro [J]. J Biol Chem, 2007, 282(48): 34877-87
[21] Inomata A, Murakoshi F, Ishiwa A, et al. Heparin interacts with elongation factor 1alpha of Cryptosporidium parvum and inhibits invasion [J]. Sci Rep, 2015(5): 11599.
[22] McGuckin MA, Linden SK, Sutton P, et al. Mucin dynamics and enteric pathogens [J]. Nat Rev Microbiol, 2011, 9(4): 265-278.
[23] Kato K, Ishiwa A: The role of carbohydrates in infection strategies of enteric pathogens [J]. Trop Med Health, 2015, 43(1): 41-52.
[24] Dreyfuss JL, Regatieri CV, Jarrouge TR, et al. Heparan sulfate proteoglycans: structure, protein interactions and cell signaling [J]. An Acad Bras Cienc, 2009, 81(3): 409-29.
[25] Cardin AD, Weintraub HJR. Molecular modeling of protein-Glycosaminogly-can interactions[J]. Arteriosclerosis, 1989, 9(1): 21-32.