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Characterization and functional analysis of serpin-1 like gene from oak silkworm Antheraea pernyi

Published online by Cambridge University Press:  23 February 2017

H.M. Yu ,
B.J. Zhu ,
Y. Sun ,
G.Q. Wei ,
L. Wang ,
C. Qian ,
M. Nadeem Abbas  and
C.L. Liu
H.M. Yu
Affiliation:
College of Life Sciences, Anhui Agricultural University, Hefei, 230036, China
B.J. Zhu
Affiliation:
College of Life Sciences, Anhui Agricultural University, Hefei, 230036, China
Y. Sun
Affiliation:
College of Life Sciences, Anhui Agricultural University, Hefei, 230036, China
G.Q. Wei
Affiliation:
College of Life Sciences, Anhui Agricultural University, Hefei, 230036, China
L. Wang
Affiliation:
College of Life Sciences, Anhui Agricultural University, Hefei, 230036, China
C. Qian
Affiliation:
College of Life Sciences, Anhui Agricultural University, Hefei, 230036, China
M. Nadeem Abbas
Affiliation:
College of Life Sciences, Anhui Agricultural University, Hefei, 230036, China
C.L. Liu*
Affiliation:
College of Life Sciences, Anhui Agricultural University, Hefei, 230036, China
*
*Author for correspondence Phone: +86 551 6578 -6325 Fax: +86 551 6578-6325 E-mail: clliu@ahau.edu.cn
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Abstract

Serpins are a broadly distributed family of proteases found in various organisms that play an important role in regulating the immune response. Here, we identified a serpin-1 gene from Antheraea pernyi that encodes a 279 amino acid protein with a molecular weight of 30.8 kDa. We expressed the recombinant Ap-serpin-1 protein in Escherichia coli and used the purified protein to prepare rabbit anti-Ap-serpin-1 polyclonal antibodies. We calculated the enzyme-linked immunosorbent assay titer of the antibody as 1:128000. Quantitative real-time polymerase chain reaction analysis revealed that Ap-serpin-1 was expressed in all examined tissues, including hemolymph, malpighian tubules, midgut, silk gland, integument and the fat body; the highest Ap-serpin-1 expression levels was detected in the fat body. We next investigated the expression patterns of Ap-serpin-1 in both fat body and hemolymph samples, following treatment with E. coli, Beauveria bassiana, Micrococcus luteus and nuclear polyhedrosis virus (NPV). We reported that NPV and M. luteus significantly enhanced Ap-serpin-1 expression in the fat body. While, in the hemolymph samples, treatment with B. bassiana and M. luteus was shown to upregulate Ap-serpin-1 expression at 24 h induction. Altogether, our results suggest that Ap-serpin-1 is involved in the innate immunity of A. pernyi.

Keywords

Antheraea pernyiserpin-1innate immunityexpression
Type
Research Papers
Information
Bulletin of Entomological Research , Volume 107 , Issue 5 , October 2017 , pp. 620 - 626
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Copyright
Copyright © Cambridge University Press 2017 

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Footnotes

†

These authors contributed equally to this work.

References

Abraham, E.G., Pinto, S.B., Ghosh, A., Vanlandingham, D.L., Budd, A., Higgs, S., Kafatos, F.C., Jacobs-Lorena, M. & Michel, K. (2005) An immune-responsive serpin, SRPN6, mediates mosquito defense against malaria parasites. Proceedings of the National Academy of Sciences of the United States of America 102, 16327–16332.Google Scholar
An, C., Ragan, E.J. & Kanost, M.R. (2011) Serpin-1 splicing isoform J inhibits the proSpatzle-activating proteinase HP8 to regulate expression of antimicrobial hemolymph proteins in Manduca sexta . Developmental & Comparitive Immunology 35, 135–141.Google Scholar
Bilang Liu, X.Q., Han, Q., Jia, L., Xiang, Z. & He, N. (2012) A silkworm hemolymph protein is a prophenoloxidase activation blocker. American Journal of Molecular Biology 2, 332–340.Google Scholar
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.Google Scholar
Buss, H., Chan, T.P., Sluis, K.B., Domigan, N.M. & Winterbourn, C.C. (1997) Protein carbonyl measurement by a sensitive ELISA method. Free Radical Biology & Medicine 23, 361–366.Google Scholar
Chamankhah, M., Braun, L., Visal-Shah, S., O'Grady, M., Baldwin, D., Shi, X., Hemmingsen, S.M., Alting-Mees, M. & Hegedus, D.D. (2003) Mamestra configurata serpin-1 homologues: cloning, localization and developmental regulation. Insect Biochemistry and Molecular Biology 33, 355–369.Google Scholar
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. Developmental Cell 3, 581–592.Google Scholar
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 . Archieves of Insect Biochemistry and Physiology 77, 99–117.Google Scholar
Gettins, P.G.W. (2002) Serpin structure, mechanism, and function. Chemical Reviews 102, 4751–4803.Google Scholar
Harlow, E. & Lane, D. (1999) Using Antibodies: a Laboratory Manual: Portable Protocol no. i., Cold Spring Harbor Laboratory Press.Google Scholar
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 Research 10, 1845–1864.Google Scholar
Jiang, H.B. (2008) The biochemical basis of antimicrobial responses in Manduca sexta . Insect Science 15, 53–66.Google Scholar
Jiang, H. & Kanost, M.R. (1997) Characterization and functional analysis of 12 naturally occurring reactive site variants of serpin-1 from Manduca sexta. Journal of Biological Chemistry 272, 1082–1087.Google Scholar
Kanost, M.R. (1999) Serine proteinase inhibitors in arthropod immunity. Developmental & Comparitive Immunology 23, 291–301.Google Scholar
Kanost, M.R., Prasad, S.V., Huang, Y. & Willott, E. (1995) Regulation of serpin gene-1 in Manduca sexta . Insect Biochemistry and Molecular Biology 25, 285–291.Google Scholar
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 Biology 7, 216.Google Scholar
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. Developmental & Comparitive Immunology 51, 22–28.Google Scholar
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.Google Scholar
Sasaki, T. & Kobayashi, K. (1984) Isolation of two novel proteinase inhibitors from hemolymph of silkworm larva, Bombyx mori. Comparison with human serum proteinase inhibitors. Journal of Biochemistry 95, 1009–1017.Google Scholar
Sun, Y., Wang, L., Qian, C., Dai, L.S., Li, J., Zhang, C.F., Zhu, B.J. & Liu, C.L. (2015) Molecular cloning and expression analysis of a hemolin-like molecule from Antheraea pernyi . International Immunopharmacology 26, 65–71.Google Scholar
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. Molecular Biology and Evolution 28, 2731–2739.Google Scholar
Tang, H., Kambris, Z., Lemaitre, B. & Hashimoto, C. (2008) A serpin that regulates immune melanization in the respiratory system of Drosophila. Developmental Cell 15, 617–626.Google Scholar
Tong, Y., Jiang, H. & Kanost, M.R. (2005) Identification of plasma proteases inhibited by Manduca sexta serpin-4 and serpin-5 and their association with components of the prophenol oxidase activation pathway. Journal of Biological Chemistry 280, 14932–14942.Google Scholar
Walker, J.M. (1994) The bicinchoninic acid (BCA) assay for protein quantitation. Methods in Molecular Biology 32, 5–8.Google Scholar
Yamasaki, M., Sendall, T.J., Harris, L.E., Lewis, G.M. & Huntington, J.A. (2010) Loop-sheet mechanism of serpin polymerization tested by reactive center loop mutations. Journal of Biological Chemistry 285, 30752–30758.Google Scholar
Zhang, C.F., Dai, L.S., Wang, L., Qian, C., Wei, G.Q., Li, J., Zhu, B.J. & Liu, C.L. (2015) Eicosanoids mediate sHSP 20.8 gene response to biotic stress in larvae of the Chinese oak silkworm Antheraea pernyi . Gene 562, 32–39.Google Scholar
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. Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology 192, 9–20.Google Scholar
Zhao, P., Dong, Z., Duan, J., Wang, G., Wang, L., Li, Y., Xiang, Z. & Xia, Q. (2012) Genome-wide identification and immune response analysis of serine protease inhibitor genes in the silkworm, Bombyx mori . PLoS ONE 7, e31168.Google Scholar
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 . Comparitive Biochemistry and Physiology B Biochemical and Molecular Biology 154, 165–173.Google Scholar
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.Google Scholar