Cloning and Expression of Scavenger Receptor Class B BmSCRB8 in Silkworm Bombyx mori

Yuzu Zhao1 Kui Zhang1 Mei Tang1 Man Xu1 Chongyang Li1 Guangzhao Pan1 Li Shen1 Hongjuan Cui1 Liqun Yang1

(1.State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China 400716)

【Abstract】Scavenger receptor class B is involved in various indispensable physiological processes, like the formation and inhibition of atherosclerosis or other cardiovascular diseases, innate immune defense and the removal of apoptotic cells. Here, we cloned BmSCRB8, a member of scavenger receptor class B in silkworm. We obtained the full-length cDNA sequence of BmSCRB8 by rapid amplification of cDNA ends (RACE), including 2 668 bp. The ORF of BmSCRB8 is 1 704 bp, encoding 567 amino acids. Online software prediction indicated that the molecular weight of BmSCRB8 was 63.87 kDa and the isoelectric point (pI) was 6.06. The space-time expression profile of BmSCRB8 was detected by reverse transcription PCR (RT-PCR), which implicated that BmSCRB8 was extensively expressed in each tissue and at each stage of blood. In addition, BmSCRB8 was highest expressed in fat body of silkworm, and was highly expressed in metamorphosis periods. Anti-BmSCRB8 polyclonal antibody was generated through prokaryotic expression, protein purification and mice immunization. Simultaneously, we constructed BmSCRB8 eukaryotic vector and then transfected embryonic cell line of silkworm. Immunofluorescence and overexpression showed that BmSCRB8 expressed specifically in membrane. Western blotting demonstrated that BmSCRB8 protein can be specifically recognized by anti-serum generated after mice immunization.

【Keywords】 Bombyx mori; BmSCRB8; expression profile; polyclonal antibody; subcellular localization;

【DOI】

【Funds】 National Basic Research Program of China (973 Program) (No. 2012CB114603) National Basic Research Program of China(973 Program)(No.2012CB114603) National Natural Science Foundation of China (No. 81201551) National Natural Science Foundation of China(No.81201551) Fundamental Research Funds for the Central Universities (No. XDJK2013B020) the Fundamental Research Funds for the Central Universities(No.XDJK2013B020) Disciplinary Team Program of the Fundamental Research Funds for the Central Universities (No. 2362015XK09) Disciplinary Team Program of the Fundamental Research Funds for the Central Universities(No.2362015XK09) Postgraduate Technology Innovation Program of Chongqing (No. CYB2015067) Postgraduate Technology Innovation Program of Chongqing(No.CYB2015067) Basic Research Specialized Funds of Southwest University (No. XDJK2015D021) Basic Research Specialized Funds of Southwest University(No.XDJK2015D021)

Download this article

(Translated by FANG Huaming)

    References

    [1]Murphy JE, Tedbury PR, Homer-Vanniasinkam S, et al. Biochemistry and cell biology of mammalian scavenger receptors. Atherosclerosis, 2005, 182(1): 1–15.

    [2]González-Lázaro M, Dinglasan RR, de la Cruz Hernández-Hernández F, et al. Anopheles gambiae Croquemort SCRBQ2, expression profile in the mosquito and its potential interaction with the malaria parasite Plasmodium berghei. Insect Biochem Mol Biol, 2009, 39(5/6): 395–402.

    [3]Jones RS, Minogue AM, Connor TJ, et al. Amyloid-β-induced astrocytic phagocytosis is mediated by CD36, CD47 and RAGE. J Neuroimmune Pharmacol, 2013, 8(1): 301–311.

    [4]Plüddemann A, Mukhopadhyay S, Gordon S. The interaction of macrophage receptors with bacterial ligands. Expert Rev Mol Med, 2006, 8(28): 1–25.

    [5]Prabhu Das M, Bowdish D, Drickamer K, et al. Standardizing scavenger receptor nomenclature. J Immunol, 2014, 192(5): 1997–2006.

    [6]Neculai D, Schwake M, Ravichandran M, et al. Structure of LIMP-2 provides functional insights with implications for SR-BI and CD36. Nature, 2013, 504(7478): 172–176.

    [7]Baranova IN, Vishnyakova TG, Bocharov AV, et al. scavenger receptor class B types I and II and CD36 mediate bacterial recognition and proinflammatory signaling induced by Escherichia coli, lipopolysaccharide, and cytosolic chaperonin60. J Immunol, 2012, 188(3): 1371–1380.

    [8]Erdman LK, Cosio G, Helmers AJ, et al. CD36 and TLR interactions in inflammation and phagocytosis: implications for malaria. J Immunol, 2009, 183(10): 6452–6459.

    [9]Kim E, Tolhurst AT, Qin LY, et al. CD36/fatty acid translocase, an inflammatory mediator, is involved in hyperlipidemia-induced exacerbation in ischemic brain injury. J Neurosci, 2008, 28(18): 4661–4670.

    [10]Seizer P, Schiemann S, Merz T, et al. CD36 and macrophage scavenger receptor a modulate foam cell formation via inhibition of lipid-laden platelet phagocytosis. Semin Thromb Hemost, 2010, 36(2): 157–162.

    [11]Stewart CR, Stuart LM, Wilkinson K, et al. CD36ligands promote sterile inflammation through assembly of a toll-like receptor 4 and 6 heterodimer. Nat Immunol, 2010, 11(2): 155–161.

    [12]Cai L, Ji AL, de Beer FC, et al. SR-BI protects against endotoxemia in mice through its roles in glucocorticoid production and hepatic clearance. J Clin Invest, 2008, 118(1): 364–375.

    [13]Okumura T, Jamieson GA. Platelet glycocalicin. I. Orientation of glycoproteins of the human platelet surface. J Biol Chem, 1976, 251(19): 5944–5949.

    [14]Okumura I, Lombart C, Jamieson GA. Platelet glycocalicin. II. Purification and characterization. J Biol Chem, 1976, 251(19): 5950–5955.

    [15]Franc NC, Dimarcq JL, Lagueux M, et al. Croquemort, a novel drosophila hemocyte/macrophage receptor that recognizes apoptotic cells. Immunity, 1996, 4(5): 431–443.

    [16]Voolstra O, Kiefer C, Hoehne M, et al. The Drosophila scavenger receptor class B Nina D-I is a cell surface receptor mediating carotenoid transport for visual chromophore synthesis. Biochemistry, 2006, 45(45): 13429–13437.

    [17]Nichols Z, Vogt RG. The SNMP/CD36 gene family in Diptera, Hymenoptera and Coleoptera: Drosophila melanogaster, D. pseudoobscura, Anopheles gambiae, Aedes aegypti, Apis mellifera, and Tribolium castaneum. Ins Bio Mol Biol, 2008, 38(4): 398–415.

    [18]Benton R, Vannice KS, Vosshall LB. An essential role for a CD36-related receptor in pheromone detection in Drosophila. Nature, 2007, 450(7167): 289–293.

    [19]Tanaka H, Ishibashi J, Fujita K, et al. A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori. Insect Biochem Mol Biol, 2008, 38(12): 1087–1110.

    [20]Sakudoh T, Iizuka T, Narukawa J, et al. A CD36-related transmembrane protein is coordinated with an intracellular lipid-binding protein in selective carotenoid transport for cocoon coloration. J Biol Chem, 2010, 285(10): 7739–7751.

    [21]Sakudoh T, Kuwazaki S, Iizuka T, et al. CD36homolog divergence is responsible for the selectivity of carotenoid species migration to the silk gland of the silkworm Bombyx mori. J Lipid Res, 2013, 54(2): 482–495.

    [22]Dong ZP. Master's thesis. Clone, Identification and study on the function of scavenger receptor class B genes in silkworm, Bombyx mori[D]. Chongqing: Southwest University, 2011(in Chinese).

    [23]Dong ZP, Chai CL, Dai FY, et al. Expression pattern and tissue localization of the scavenger receptor class B Bm SCRBQ4 in Bombyx mori. Insect Sci, 2015, 22(6): 739–747.

    [24]Benard EL, Roobol SJ, Spaink HP, et al. Phagocytosis of mycobacteria by zebrafish macrophages is dependent on the scavenger receptor Marco, a key control factor of pro-inflammatory signalling. Dev Comp Immunol, 2014, 47(2): 223–233.

    [25]Kiefer C, Sumser E, Wernet MF, et al. A scavenger receptor class B mediates the cellular uptake of carotenoids in Drosophila. Proc Natl Acad Sci USA, 2002, 99(16): 10581–10586.

    [26]Price GM. Protein and nucleic acid metabolism in insect fat body. Biol Rev, 1973, 48(3): 333–372.

    [27]Aggrawal K, Silverman N. Peptidoglycan recognition in Drosophila. Biochem Soc Trans, 2007, 35(Pt6): 1496–1500.

    [28]Zhang WB, Zhou QM, Zhang K, et al. Integrin-mediated signaling pathway and its research advances in insects. Sci Sericult, 2015, 41(1): 159–165(in Chinese).

    [29]Su JJ, Chen SY, Zhang K, et al. Identification and expression analysis of cathepsin O gene in silkworm(Bombyx mori). Sci Agr Sin, 2015, 48(22): 4564–4573(in Chinese).

    [30]Arrese EL, Soulages JL. Insect fat body: energy, metabolism, and regulation. Annu Rev Entomol, 2010, 55: 207–225.

    [31]Tan J, Zhang K, Xu M, et al. Identification and subcellular localization of BmintegrinαPS3 from silkworm(Bombyx mori). Sci Agr Sin, 2013, 46(22): 4808–015(in Chinese).

This Article

ISSN:1000-3061

CN: 11-1998/Q

Vol 32, No. 10, Pages 1408-1421

October 2016

Downloads:1

Share
Article Outline

Knowledge

Abstract

  • 1 Materials and methods
  • 2 Results and analysis
  • 3 Discussion
  • References