基于不同纯化策略的4种对虾血蓝蛋白的凝集活性及分子基础对比分析
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摘要
既往研究表明,对虾血蓝蛋白(hemocyanin, HMC)是一种具有抗病毒、抗菌等多种免疫学活性的免疫球蛋白超家族(immunoglobulin superfamily, IgSF)分子,但迄今为止,其功能多样性的分子基础尚不是很清楚。本研究以凡纳滨对虾HMC为研究对象,采用亲和层析、凝集素层析技术获得4种HMC成分:A-HMCs、A-HMCl、AL-HMCs和AL-HMCl,发现其对不同细菌的凝集活性存在较大差异。其中,AL-HMCs和AL-HMCl对大肠杆菌和副溶血弧菌的凝集活性明显强于A-HMCs和A-HMCl,前者约为后者的2～32倍。继而,通过双向凝胶电泳(two-dimensional gel electrophoresis, 2-DE)和凝集素印迹技术对不同HMC的蛋白质组成和糖基化修饰水平进行对比分析。结果显示,4种HMC在2-DE图谱上表现为6～7个差异明显的蛋白质点,且与伴刀豆凝集素(concanavalin A, ConA)、花生凝集素(peanut agglutinin, PNA)、荆豆凝集素(ulex europaeus agglutinin, UEA)和双花扁豆凝集素(dolichos bifows agglutinin, DBA)等4种凝集素的识别存在显著性差异。其中,A-HMCl 6个蛋白点均可识别4种凝集素,而A-HMCs 6个蛋白点中仅有4、3个点分别与UEA、DBA反应呈阳性,AL-HMCs和AL-HMCl可以与UEA、PNA特异性显色的点分别为其总蛋白点的3/7、2/6。由此推测,对虾血蓝蛋白功能多样性的分子基础可能与其蛋白质组成和糖基化修饰水平的多样性密切相关。
It has been confirmed that hemocyanin(HMC) is a kind of immunoglobulin superfamily(IgSF) protein with antiviral and antibacterial activity. But until now, the molecular basis of its functional diversity is not well understood. In this study, four kinds of HMC fractions from Litopenaeus vannamei, i.e. A-HMCs, A-HMCl, ALHMCs and AL-HMCl, were purified by affinity chromatography and lectin chromatography, which possessed distinct agglutinative activities against different pathogens. Among them, the agglutination activities of AL-HMCs and AL-HMCl were significantly(about 2–32 times) stronger than those of A-HMCs and A-HMCl against Escherichia coli K12 and Vibrio parahaemolyticus. Furthermore, the protein component and glycosylation modification of the 4 HMCs were analyzed by 2-DE and 2-D lectin blotting. The results showed that they had 6-7 spots in the 2-DE map with significant difference in isoelectric point and molecular weight. In particular, these protein spots could bind with 4 lectins, including concanavalin A(ConA), peanut agglutinin(PNA), ulex europaeus agglutinin(UEA) and dolichos bifows agglutinin(DBA), to different degrees. Of these, all of the 6 protein spots from A-HMCl could bind to 4 kinds of lectins, while only 4 and 3 protein spots from A-HMCs showed positive to UEA and DBA, respectively. AL-HMCs and AL-HMCl could respectively react with PNA and UEA, to produce 3 spots out of 7 total protein spots and 2 spots out of 6 total protein spots, respectively. Collectively, our data demonstrated that the molecular basis of functional diversity of shrimp hemocyanin may be closely related to the diversity of protein composition and glycosylation.
It has been confirmed that hemocyanin(HMC) is a kind of immunoglobulin superfamily(IgSF) protein with antiviral and antibacterial activity. But until now, the molecular basis of its functional diversity is not well understood. In this study, four kinds of HMC fractions from Litopenaeus vannamei, i.e. A-HMCs, A-HMCl, ALHMCs and AL-HMCl, were purified by affinity chromatography and lectin chromatography, which possessed distinct agglutinative activities against different pathogens. Among them, the agglutination activities of AL-HMCs and AL-HMCl were significantly(about 2–32 times) stronger than those of A-HMCs and A-HMCl against Escherichia coli K12 and Vibrio parahaemolyticus. Furthermore, the protein component and glycosylation modification of the 4 HMCs were analyzed by 2-DE and 2-D lectin blotting. The results showed that they had 6-7 spots in the 2-DE map with significant difference in isoelectric point and molecular weight. In particular, these protein spots could bind with 4 lectins, including concanavalin A(ConA), peanut agglutinin(PNA), ulex europaeus agglutinin(UEA) and dolichos bifows agglutinin(DBA), to different degrees. Of these, all of the 6 protein spots from A-HMCl could bind to 4 kinds of lectins, while only 4 and 3 protein spots from A-HMCs showed positive to UEA and DBA, respectively. AL-HMCs and AL-HMCl could respectively react with PNA and UEA, to produce 3 spots out of 7 total protein spots and 2 spots out of 6 total protein spots, respectively. Collectively, our data demonstrated that the molecular basis of functional diversity of shrimp hemocyanin may be closely related to the diversity of protein composition and glycosylation.
引文
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[2] Nesterova N V, Zagorodnya S D, Moshtanska V, et al.Antiviral activity of hemocyanin isolated from marine snail Rapana venosa[J]. Antiviral Research, 2011, 90(2):38.
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[13] Pan J Y, Zhang Y L, Wang S Y, et al. Dodecamer is required for agglutination of Litopenaeus vannamei hemocyanin with bacterial cells and red blood cells[J]. Mar-ine Biotechnology, 2008, 10(6):645-652.
[14]章跃陵,邢立刚,严芳,等.5种凡纳滨对虾血蓝蛋白的糖基化修饰及功能对比分析[J].中国生物化学与分子生物学报,2009, 25(7):655-661.Zhang Y L, Xing L G, Yan F, et al. Comparative analyses of five hemocyanin isomers from shrimp Litopenaeus vannamei[J]. Chinese Journal of Biochemistry and Molecular Biology, 2009, 25(7):655-661(in Chinese).
[15] Cao J S, Wang Z H, Zhang Y L, et al. Identification and characterization of the related immune-enhancing proteins in crab Scylla paramamosain stimulated with rhubarb polysaccharides[J]. Molecular Immunology, 2014,57(2):263-273.
[16] Figueroa-Soto C G, De La Barca A M C, VazquezMoreno L, et al. Purification of hemocyanin from white shrimp(Penaeus vannamei Boone)by immobilized metal affinity chromatography[J]. Comparative Biochemistry and Physiology-Part B:Biochemistry and Molecular Biology,1997, 117(2):203-208.
[17] Zhao X L, Guo L L, Lu X, et al. Evidences of abundant hemocyanin variants in shrimp Litopenaeus vannamei[J].Molecular Immunology,2016, 77:103-112.
[18] Lu X, Lu H, Guo L L, et al. Cloning and characterization of a novel hemocyanin variant LvHMCV4 from shrimp Litopenaeus vannamei[J]. Fish&Shellfish Immunology, 2015, 46(2):398-405.
[19] Zhao S, Lu X, Zhang Y L, et al. Identification of a novel alternative splicing variant of hemocyanin from shrimp Litopenaeus vannamei[J]. Immunology Letters, 2013,154(1-2):1-6.
[20] Zhao X L, Guo L L, Zhang Y L, et al. SNPs of hemocyanin C-terminal fragment in shrimp Litopenaeus vannamei[J]. FEBS Letters, 2012, 586(4):403-410.
[21] Guo L L, Zhao X L, Zhang Y L, et al. Evidences of SNPs in the variable region of hemocyanin Ig-like domain in shrimp Litopenaeus vannamei[J]. Fish&Shellfish Immunology, 2013,35(5):1532-1538.
[22] Brudzynski K, Sjaarda C. Honey glycoproteins containing antimicrobial peptides, Jelleins of the Major Royal Jelly Protein 1, are responsible for the cell wall lytic and bactericidal activities of honey[J]. PLoS ONE, 2015,10(4):e0120238.
[23]代智,刘银坤,郭坤,等.CK8寡糖链结构及翻译水平改变与人肝癌细胞转移相关性研究[J].生物化学与生物物理进展,2007, 34(2):196-202.Dai Z, Liu Y K, Guo K, et al. Alteration of CK8 both in its expression level and its glycan parts related to HCC metastatic ability[J]. Progress in Biochemistry and Biophysics, 2007, 34(2):196-202(in Chinese).
[24] Munoz-Antoli C, Cortes A, Sotillo J, et al. Differential expression and glycosylation of proteins in the rat ileal epithelium in response to Echinostoma caproni infection[J]. Journal of Proteomics, 2014, 101:169-178.
[25] Dolashka P, Velkova L, Shishkov S, et al. Glycan structures and antiviral effect of the structural subunit RvH2of Rapana hemocyanin[J]. Carbohydrate Research,2010, 345(16):2361-2367.
[2] Nesterova N V, Zagorodnya S D, Moshtanska V, et al.Antiviral activity of hemocyanin isolated from marine snail Rapana venosa[J]. Antiviral Research, 2011, 90(2):38.
[3] Zhang Y L, Wang S Y, Xu A L, et al. Affinity proteomic approach for identification of an IgA-like protein in Litopenaeus vannamei and study on its agglutination characterization[J]. Journal of Proteome Research, 2006,5(4):815-821.
[4] Jaenicke E, Fraune S, May S, et al. Is activated hemocyanin instead of phenoloxidase involved in immune response in woodlice?[J]. Developmental and Comparative Immunology, 2009, 33(10):1055-1063.
[5] Zhang Y L, Yan F, Hu Z, et al. Hemocyanin from shrimp Litopenaeus vannamei shows hemolytic activity[J]. Fish&Shellfish Immunology, 2009, 27(2):330-335.
[6] Zheng L Y, Zhao X L, Zhang P, et al. Hemocyanin from shrimp Litopenaeus vannamei has antiproliferative effect against HeLa cell in vitro[J]. PLoS ONE, 2016,11(3):e0151801.
[7] Coates C J, Decker H. Immunological properties of oxygen-transport proteins:hemoglobin, hemocyanin and hemerythrin[J]. Cellular and Molecular Life Sciences,2017, 74(2):293-317.
[8] Coates C J, Nairn J. Diverse immune functions of hemocyanins[J]. Developmental and Comparative Immunology, 2014,45(1):43-55.
[9] Velayutham M, Munusamy A. Humoral immune responses of antibacterial hemocyanin(Ab-Hcy)in mud crab, Scylla serrata[J]. Aquaculture, 2016, 464:428-433.
[10] Cong Y, Zhang Q F, Woolford D, et al. Structural mechanism of SDS-induced enzyme activity of scorpion hemocyanin revealed by electron cryomicroscopy[J].Structure,2009, 17(5):749-758.
[11] Jiang N X, Tan N S, Ho B, et al. Respiratory proteingenerated reactive oxygen species as an antimicrobial strategy[J]. Nature Immunology,2007, 8(10):1114-1122.
[12] Dolashka-Angelova P, Lieb B, Velkova L, et al. Identification of glycosylated sites in Rapana hemocyanin by mass spectrometry and gene sequence, and their antiviral effect[J]. Bioconjugate Chemistry, 2009, 20(7):1315-1322.
[13] Pan J Y, Zhang Y L, Wang S Y, et al. Dodecamer is required for agglutination of Litopenaeus vannamei hemocyanin with bacterial cells and red blood cells[J]. Mar-ine Biotechnology, 2008, 10(6):645-652.
[14]章跃陵,邢立刚,严芳,等.5种凡纳滨对虾血蓝蛋白的糖基化修饰及功能对比分析[J].中国生物化学与分子生物学报,2009, 25(7):655-661.Zhang Y L, Xing L G, Yan F, et al. Comparative analyses of five hemocyanin isomers from shrimp Litopenaeus vannamei[J]. Chinese Journal of Biochemistry and Molecular Biology, 2009, 25(7):655-661(in Chinese).
[15] Cao J S, Wang Z H, Zhang Y L, et al. Identification and characterization of the related immune-enhancing proteins in crab Scylla paramamosain stimulated with rhubarb polysaccharides[J]. Molecular Immunology, 2014,57(2):263-273.
[16] Figueroa-Soto C G, De La Barca A M C, VazquezMoreno L, et al. Purification of hemocyanin from white shrimp(Penaeus vannamei Boone)by immobilized metal affinity chromatography[J]. Comparative Biochemistry and Physiology-Part B:Biochemistry and Molecular Biology,1997, 117(2):203-208.
[17] Zhao X L, Guo L L, Lu X, et al. Evidences of abundant hemocyanin variants in shrimp Litopenaeus vannamei[J].Molecular Immunology,2016, 77:103-112.
[18] Lu X, Lu H, Guo L L, et al. Cloning and characterization of a novel hemocyanin variant LvHMCV4 from shrimp Litopenaeus vannamei[J]. Fish&Shellfish Immunology, 2015, 46(2):398-405.
[19] Zhao S, Lu X, Zhang Y L, et al. Identification of a novel alternative splicing variant of hemocyanin from shrimp Litopenaeus vannamei[J]. Immunology Letters, 2013,154(1-2):1-6.
[20] Zhao X L, Guo L L, Zhang Y L, et al. SNPs of hemocyanin C-terminal fragment in shrimp Litopenaeus vannamei[J]. FEBS Letters, 2012, 586(4):403-410.
[21] Guo L L, Zhao X L, Zhang Y L, et al. Evidences of SNPs in the variable region of hemocyanin Ig-like domain in shrimp Litopenaeus vannamei[J]. Fish&Shellfish Immunology, 2013,35(5):1532-1538.
[22] Brudzynski K, Sjaarda C. Honey glycoproteins containing antimicrobial peptides, Jelleins of the Major Royal Jelly Protein 1, are responsible for the cell wall lytic and bactericidal activities of honey[J]. PLoS ONE, 2015,10(4):e0120238.
[23]代智,刘银坤,郭坤,等.CK8寡糖链结构及翻译水平改变与人肝癌细胞转移相关性研究[J].生物化学与生物物理进展,2007, 34(2):196-202.Dai Z, Liu Y K, Guo K, et al. Alteration of CK8 both in its expression level and its glycan parts related to HCC metastatic ability[J]. Progress in Biochemistry and Biophysics, 2007, 34(2):196-202(in Chinese).
[24] Munoz-Antoli C, Cortes A, Sotillo J, et al. Differential expression and glycosylation of proteins in the rat ileal epithelium in response to Echinostoma caproni infection[J]. Journal of Proteomics, 2014, 101:169-178.
[25] Dolashka P, Velkova L, Shishkov S, et al. Glycan structures and antiviral effect of the structural subunit RvH2of Rapana hemocyanin[J]. Carbohydrate Research,2010, 345(16):2361-2367.