Eur. J. Entomol. 114: 430-438, 2017 | DOI: 10.14411/eje.2017.055

Characterization and functional analysis of the serpin-10 gene from oak silkworm, Antheraea pernyi (Lepidoptera: Saturniidae)

Saima KAUSAR, Cen QIAN, Muhammad Nadeem ABBAS, Bao-Jian ZHU, Ya LIU, Lei WANG, Guo-Qing WEI, Yu SUN, Chao-Liang LIU*
College of Life Sciences, Anhui Agricultural University, 130 Changjiang West Road, Hefei, 230036, China; e-mails: drkausarsn@hotmail.com, qiancenqiancen@163.com, abbasmndr@gmail.com, zhubaojian@ahau.edu.cn, 243685181@qq.com, wanglei20041225@163.com, weiguoqing@tom.com, nagato0621@163.com, anhuicansang@163.com

Serpin is a broadly distributed superfamily of proteins that have a crucial role in regulating various immune reactions. Herein we identified a serpin-10 gene from Antheraea pernyi that encodes a 1557 amino acid residue protein with a predicted molecular weight of 58.76 kDa. Recombinant Apserpin-10 protein was expressed in a prokaryotic expression system (Escherichia coli) and the purified protein was used to prepare rabbit anti-Apserpin-10 polyclonal antibodies. Quantitative real-time polymerase chain reaction and western blot analysis indicate that Apserpin-10 was transcribed in all the tissues examined, including haemolymph, malpighian tubules, fat body, silk gland, integument and mid gut; the greatest expression level of Apserpin-10 was recorded in the fat body and haemocytes. The comparison of different developmental stages showed that Apserpin-10 transcript level was highest in 5th instar larvae, while the lowest expression was recorded at the egg stage. We also investigated the expression patterns of Apserpin-10 in fat body and haemocyte samples, following administration of heat-inactivated gram-positive bacteria (Micrococcus luteus), gram negative bacteria (Escherichia coli), a fungus (Beauveria bassiana) and virus (nuclear polyhedrosis virus, NPV). A substantial up-regulation of Apserpin-10 expression was recorded following pathogen challenge in both the tissues tested. Further the knock down of Apserpin-10 led to down regulation of antimicrobial peptide genes. Altogether, our results indicate that Apserpin-10 is involved in the innate immunity of A. pernyi.

Keywords: Lepidoptera, Saturniidae, Antheraea pernyi, serpin, innate immunity, haemolymph protein

Received: July 10, 2017; Accepted: September 25, 2017; Published online: October 23, 2017Show citation

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KAUSAR, S., QIAN, C., ABBAS, M.N., ZHU, B., LIU, Y., WANG, L., ... LIU, C. (2017). Characterization and functional analysis of the serpin-10 gene from oak silkworm, Antheraea pernyi (Lepidoptera: Saturniidae). Eur. J. Entomol.114(1), 2017.000. doi: 10.14411/eje.2017.055.
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References

  1. Abraham E.G., Pinto S.B., Ghosh A., Vanlandingham D.L., Budd A., Higgs S., Kafatos F.C., Jacobs L.M. & Michel K. 2005: An immune-responsive serpin, SRPN6, mediates mosquito defence against malaria parasites. - Proc. Natn. Acad. Sci. U.S.A. 102: 16327-6332. Go to original source...
  2. An C., Ishibashi J., Ragan E.J., Jiang H. & Kanost M.R. 2009: Functions of Manduca sexta hemolymph proteinases HP6 and HP8 in two innate immune pathways. - J. Biol. Chem. 284: 19716-19726. Go to original source...
  3. Aymeric J.L., Alain G. & Bernard D. 2010: Imd pathway is involved in the interaction of Drosophila melanogaster with the entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus luminescens. - Mol. Immunol. 47: 2342-2348. Go to original source...
  4. Bulet P., Hetru C., Dimarcq J.L. & Hoffmann D. 1999: Antimicrobial peptides in insects: structure and function. - Dev. Comp. Immunol. 23: 329-344. Go to original source...
  5. Danielli A., Kafatos F.C. & Loukeris T.G. 2003: Cloning and characterization of four Anopheles gambiae serpin isoforms, differentially induced in the midgut by Plasmodium berghei invasion. - J. Biol. Chem. 278: 4184-4193. Go to original source...
  6. De Gregorio E., Han S.J., Lee W.J., Baek M.J., Osaki T., Kawabata S., Lee B.L., Iwanaga S., Lemaitre B. & Brey P.T. 2002: An immune-responsive serpin regulates the melanization cascade in Drosophila. - Dev. Cell 3: 581-592. Go to original source...
  7. Feng C., Huang J., Song Q., Stanley D., Lu W., Zhang Y. & Huang Y. 2011: Parasitization by Macrocentrus cingulum (Hymenoptera: Braconidae) influences expression of prophenoloxidase in Asian corn borer Ostrinia furnacalis. - Arch. Insect Biochem. Physiol. 77: 99-117. Go to original source...
  8. Fullaondo A., Garcia-Sanchez S., Sanz-Parra A., Recio E., Lee S.Y. & Gubb D. 2011: Spn1 regulates the GNBP3-dependent Toll signaling pathway in Drosophila melanogaster. - Mol. Cell Biol. 31: 2960-2972. Go to original source...
  9. Gooptu B. & Lomas D.A. 2009: Conformational pathology of the serpins: themes, variations, and therapeutic strategies. - Annu. Rev. Biochem. 78: 147-176. Go to original source...
  10. Green C., Levashina E., McKimmie C., Dafforn T., Reichhart J.M. & Gubb D. 2000: The necrotic gene in Drosophila corresponds to one of a cluster of three serpin transcripts mapping at 43A1.2. - Genetics 156: 1117-1127.
  11. Gubb D., Robertson A., Dafforn T., Troxler L. & Reichhart J.M. 2007: Drosophila serpins: Regulatory cascades in innate immunity and morphogenesis. In Silverman G.A. & Lomas D.A (eds): Molecular and Cellular Aspects of the Serpinopathies and Disorders in Serpin Activity. World Scientific Publishing, Singapore, pp. 207-227.
  12. Gulley M.M., Zhang X. & Michel K. 2013: The roles of serpins in mosquito immunology and physiology. - J. Insect Physiol. 59: 138-147. Go to original source...
  13. Han P., Fan J., Liu Y., Cuthbertson A.G.S., Yan S., Qiu B.L. & Ren S. 2014: RNAi-mediated knockdown of serine protease inhibitor genes increases the mortality of Plutella xylostella challenged by destruxin A. - PLoS One 9: e97863, 16 pp. Go to original source...
  14. Harlow E. & Lane D. 1999: Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York, 726 pp.
  15. Hegedus D.D., Erlandson M., Baldwin D., Hou X. & Chamankhah M. 2008: Differential expansion and evolution of the exon family encoding the serpin-1 reactive centre loop has resulted in divergent serpin repertoires among the Lepidoptera. - Gene 418: 15-21. Go to original source...
  16. Irving J.A., Pike R.N., Lesk A.M. & Whisstock J.C. 2000: Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function. - Genome Res. 10: 1845-1864. Go to original source...
  17. Jiang H. 2008: The biochemical basis of antimicrobial responses in Manduca sexta. - Insect Sci. 15: 53-66. Go to original source...
  18. Jiang H. & Kanost M.R. 1997: Characterization and functional analysis of 12 naturally occurring reactive site variants of serpin-1 from Manduca sexta. - J. Biol. Chem. 272: 1082-1087. Go to original source...
  19. Kanost M.R. 1999: Serine proteinase inhibitors in arthropod immunity. - Dev. Comp. Immunol. 23: 291-301. Go to original source...
  20. Kausar S., Abbas M.N., Qian C., Zhu B.J., Sun Y., Sun Y.X., Wang L., Wei G., Maqsood I. & Liu C.L. 2017: Serpin-14 negatively regulates prophenoloxidase activation and expression of antimicrobial peptides in Chinese oak silkworm Antheraea pernyi. - Dev. Comp. Immunol. 76: 45-55. Go to original source...
  21. Kim C.H., Kim S.J., Kan H., Kwon H.M., Roh K.B., Jiang R., Yang Y., Park J.W., Lee H.H., Ha N.C., Kang H.J., Nonaka M., Soderha K. & Lee B.L. 2008: A three step proteolytic cascade mediates the activation of the peptidoglycan-induced Toll pathway in an insect. - J. Biol. Chem. 283: 7599-7607. Go to original source...
  22. Law R.H., Zhang Q., McGowan S., Buckle A.M., Silverman G.A., Wong W., Rosado C.J., Langendorf C.G., Pike R.N., Bird P.I. & Whisstock J.C. 2006: An overview of the serpin superfamily. - Genome Biol. 7: 216, 11 pp. Go to original source...
  23. Levashina E.A. 1999: Constitutive activation of toll-mediated antifungal defense in serpin-deficient Drosophila. - Science 285: 1917-1919. Go to original source...
  24. Li W., Terenius O., Hirai M., Nilsson A.S. & Faye I. 2005: Cloning, expression and phylogenetic analysis of hemolin, from the Chinese oak silkmoth, Antheraea pernyi. - Dev. Comp. Immunol. 29: 853-864. Go to original source...
  25. Liu D.R., Wang L., Yang L., Qian C., Wei G.Q., Dai L.S., Li J., Zhu B.J. & Liu C.L. 2015: Serpin-15 from Bombyx mori inhibits prophenoloxidase activation and expression of antimicrobial peptides. - Dev. Comp. Immunol. 51: 22-28. Go to original source...
  26. Livak K.J. & Schmittgen T.D. 2001: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. - Methods 25: 402-408. Go to original source...
  27. Lu W.X., Yue D., Hai Z.J., Daihua W., Yi Z.M., Fu W.C. & Rong Z. 2015: Cloning, expression, and characterization of prophenoloxidase from Antheraea pernyi. - Arch. Insect Biochem. Physiol. 88: 45-63. Go to original source...
  28. Pan Y., Xia H., Lü P., Chen K., Yao Q., Chen H., Gao L., He Y. & Wang L. 2009: Molecular cloning, expression and characterization of Bmserpin-2 gene from Bombyx mori. - Acta Biochim. Pol. 56: 671-677.
  29. Potempa J., Korzus E. & Travis J. 1994: The serpin superfamily of proteinase inhibitors: structure, function, and regulation. - J. Biol. Chem. 269: 15957-15960.
  30. Rawlings N.D., Tolle D.P. & Barrett A.J. 2004: MEROPS: the peptidase database. - Nucl. Acids Res. 32: 160-164. Go to original source...
  31. Reichhart J.M. 2005: Tip of another iceberg: Drosophila serpins. - Trends Cell Biol. 15: 659-665. Go to original source...
  32. Roh K.B., Kim C.H., Lee H., Kwon H.M., Park J.W., Ryu J.H., Kurokawa K., Ha N.C., Lee W.J., Lemaitre B., Soderhall K. & Lee B.L. 2009: Proteolytic cascade for the activation of the insect toll pathway induced by the fungal cell wall component. - J. Biol. Chem. 284: 19474-19481. Go to original source...
  33. Schleef R.R. & Chuang T.L. 2000: Protease inhibitor 10 inhibits tumor necrosis factor alpha-induced cell death. Evidence for the formation of intracellular high M(r) protease inhibitor 10-containing complexes. - J. Biol. Chem. 275: 26385-26389. Go to original source...
  34. Silverman G.A., Bird P.I., Carrell R.W., Church F.C., Coughlin P.B., Gettins P.G., Irving J.A., Lomas D.A., Luke C.J., Moyer R.W., Pemberton P.A., Remold-O'Donnell E., Salvesen G.S., Travis J. & Whisstock J.C. 2001: The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. - J. Biol. Chem. 276: 33293-33296. Go to original source...
  35. Suwanchaichinda C. & Kanost M.R. 2009: The serpin gene family in Anopheles gambiae. - Gene 442: 47-54. Go to original source...
  36. Tamura K., Peterson D., Peterson N., Stecher G., Nei M. & Kumar S. 2011: MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. - Mol. Biol. Evol. 28: 2731-2739. Go to original source...
  37. Tanaka H. & Yamakawa M. 2011: Regulation of the innate immune responses in the silkworm, Bombyx mori. - Invert. Surviv. J. 8: 59-69.
  38. Wang X., Zhang J., Chen Y., Ma Y., Zou W., Ding G., Li W., Zhao M., Wu C. & Zhang R. 2013: A novel pattern recognition protein of the Chinese oak silkmoth, Antheraea pernyi, is involved in the pro-PO activating system. - BMB Rep. 46: 358-363. Go to original source...
  39. Wang X., Wang K., He Y., Lu X., Wen D., Wu C., Zhang J. & Zhang R. 2017: The functions of serpin-3, a negative-regulator involved in prophenoloxidase activation and antimicrobial peptides expression of Chinese oak silkworm, Antheraea pernyi. - Dev. Comp. Immunol. 69: 1-11. Go to original source...
  40. Yang H.X., Zhu X.R. & Lu H.S. 2002: Research progress on application of silkworm pupa in medical science. - Bull. Sci. Tech. 18: 318-322.
  41. Youlei M., Jinghai Z., Yuntao Z., Jiaoshu L., Tianyi W., Chunfu W. & Rong Z. 2013: Purification and characterization of a 1,3-beta-D-glucan recognition protein from Antheraea pernyi larve that is regulated after a specific immune challenge. - BMB Rep. 46: 264-269. Go to original source...
  42. Yu H., Zhu B.J., Yu S., Wei G.Q., Wang L., Qian C., Abbas M.N. & Liu C.L. 2017: Characterization and functional analysis of serpin-1 like gene from oak silkworm Antheraea pernyi. - Bull. Entomol. Res. 107: 620-626. Go to original source...
  43. Zhang B., Wu T.Y., Tang X.W., Zhang S.Y., Xu Q.W., Zhao Y., Wang Y.J. & Feng C.J. 2016: Cloning, expression and characterization of Ostrinia furnacalis serpin1, a regulator of the prophenoloxidase activation system. - Comp. Biochem. Physiol. (B) 192: 9-20. Go to original source...
  44. Zheng Y.P., He W.Y., Beliveau C., Nisole A., Stewart D., Zheng S.C., Doucet D., Cusson M. & Feng Q.L. 2009: Cloning, expression and characterization of four serpin-1 cDNA variants from the spruce budworm, Choristoneura fumiferana. - Comp. Biochem. Physiol. (B) 154: 165-173. Go to original source...
  45. Zhou J. & Han D. 2006: Safety evaluation of protein of silkworm (Antheraea pernyi) pupae. - Food Chem. Toxicol. 44: 1123-1130. Go to original source...
  46. Zou Z., Picheng Z., Weng H., Mita K. & Jiang H. 2009: A comparative analysis of serpin genes in the silkworm genome. - Genomics 93: 367-375. Go to original source...