Bilang Liu, Xiwu Qi, Qi Han, Ling Jia, Zhonghuai Xiang, Ningjia He^*

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State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China.
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Abstract: Melanization in insect hemolymph is triggered by the recognition of pathogen-associated molecular patterns via pattern recognition receptors. The signal transduction leads to the activation of the prophenoloxidase and hence the generation of melanin. The proPO activation process must be tightly controlled to minimize the host damage caused by reactive intermediates during melanin synthesis. The full-length cDNA sequence of a 20 kDa hemolymph protein from Bombyx (Bmhp20) was determined. Bmhp20 gene was expressed in larval fat body, integument, trachea, and ovary and was induced by the challenge of B. bombyseptieus. Binding of recombinant Bmhp20 to microbial cell wall components as well as gram-positive bacteria and fungi was confirmed. Phenoloxidase activity assay indicated that recombinant Bmhp20 blocked the proPO activation in hemolymph that was triggered by peptidoglycan or beta-1, 3-glucan. Our data suggest that Bmhp20 plays bifunctional roles in silkworm humoral responses: to participate in pattern recognition and to block the activation of proPO.

Keywords: Bombyx Mori; Humoral Responses; Pattern Recognition Receptors; Phenoloxidase Activity

Cite this paper: Liu, B. , Qi, X. , Han, Q. , Jia, L. , Xiang, Z. and He, N. (2012) A silkworm hemolymph protein is a prophenoloxidase activation blocker. American Journal of Molecular Biology, 2, 332-340. doi: 10.4236/ajmb.2012.24034.

References

[1]   Hoffmann, J. A. and Reichhart, J. M. (2002) Drosophila innate immunity: an evolutionary perspective. Nat Immunology 3(2): 121-126.

[2]   Gillespie, J. P., Kanost, M. R. and Trenczek, T. (1997) Biological mediators of insect immunity. Annu. Rev. Entomol 42: 611–643.

[3]   Yoshida, H., Ochiai, M. and Ashida, M. (1986) Beta-1, 3-glucan receptor and peptidoglycan receptor are present as separate entities within insect prophenoloxidase activating system. Biochem Biophys Res Commun 141(3):1177-84.

[4]   Ochiai, M. and Ashida, M. (1988) Purification of a beta-1, 3-glucan recognition protein in the prophenoloxidase activating system from hemolymph of the silkworm, Bombyx mori. J Biol Chem. 263(24):12056-62.

[5]   Janeway, C. A. and Medzhitov, R. (2002) Innate immune recognition. Annu. Rev. Immunol 20:197-216.

[6]   Mogensen, T. H. (2009) Signaling in Innate Immune Defenses Pathogen Recognition and Inflammatory. Clin. Microbiol. Rev 22(2):240. DOI: 10.1128/CMR.00046-08.

[7]   Yoshida, H., Kinoshita, K. and Ashida, M. (1996) Purification of a peptidoglycan recognition protein from hemolymph of the silkworm, Bombyx mori. J Biol Chem. 271(23):13854-60.

[8]   S?derh?ll, K. and Cerenius, L. (1998) Role of the prophenoloxidase activating system in invertebrate immunity. Current Opinion in Immunology 10: 23–28.

[9]   González-Santoyo, I. and Córdoba-Aguilar, A. (2011) Phenoloxidase: a key component of the insect immune system. Entomologia Experimentalis et Applicata DOI:10.1111/j.1570-7458.2011.01187.x

[10]   Jiang, R., Kim, E. H, Gong, J. H., et al. (2009) Three Pairs of Protease-Serpin Complexes Cooperatively Regulate the Insect Innate Immune Responses. J Biol Chem 284: 35652-35658.

[11]   Ligoxygakis, P., Pelte, N., Leclerc, V., et al. (2002) A serpin mutant links Toll activation to melanization in the host defence of Drosophila. EMBO J 1: 6330-6337.

[12]   Lu, Z. and Jiang, H. (2007) Regulation of phenoloxidase activity by high-and low-molecular-weight inhibitors from the larval hemolymph of Manduca sexta. Insect biochemistry and molecular biology 37: 478-485.

[13]   Tsukamoto, T., Ichimaru, Y., Kanegae, N., et al. (1992) Identification and isolation of endogenous insect phenoloxidase inhibitors. Biochem Biophys Res Commun 184: 86-92.

[14]   Cheng, T. C., Xia, Q. Y., Xu, P. Z., et al. (2009) Identification and comparative analysis of immune-related genes and signaling pathways in the silkworm, Bombyx mori. Acta Entomologica Sinica 52 (3): 235-245.

[15]   Takase, H., Watanabe, A., Yoshizawa, Y., et al. (2009) Identification and comparative analysis of three novel C-type lectins from the silkworm with functional implications in pathogen recognition, Devel and Comp Immunology 33 (6): 789-800.

[16]   Huang, L., Cheng, T., Xu, P., et al. (2009) A genome-wide survey for host response of silkworm, Bombyx mori during pathogen Bacillus bombyseptieus infection. PLoS One 4: e8098.

[17]   Li, J.Y., Li, J. S. and Zhong B. X. (2011) Proteomic profiling of the hemolymph at the fifth instar of the silkworm Bombyx mori. Insect Science 00: 1-14.

[18]   Hou, Y., Zou, Y, Wang, F., et al. (2010) Comparative analysis of proteome maps of silkworm hemolymph during different developmental stages. Proteome Science 8: 45-54.

[19]   He, N., Fujii, H., Kusakabe, T., et al. (2004) Overexpression in Escherichia coli and purification of recombinant CI-b1, a Kunitz-type chymotrypsin inhibitor of silkworm. Protein expression and purification 38: 9-16.

[20]   Gould, C. M., Diella, F., Via, A., et al. (2012) ELM: the status of the 2010 eukaryotic linear motif resource. Nucleic Acids Research 38: 167-180.

[21]   Futahashi, R., Okamoto, S., Kawasaki, H., et al. (2008) Genome-wide identification of cuticular protein genes in the silkworm, Bombyx mori. Insect Biochem Mol Biol 38: 1138-1146.

[22]   Tsuchiya, M., Asahi, N., Suzuoki, F., et al. (1996) Detection of peptidoglycan and β-glucan with silkworm larvae plasma test. FEMS Immunology and Medical Microbiology 15: 129-134.

[23]   Kobayashi, T., Tani, T., Yokota, T., et al. (2000) Detection of peptidoglycan in human plasma using the silkworm larvae plasma test. FEMS Immunology and Medical Microbiology 28: 49-53.

[24]   Aigaki, T., Kasuga, H. and Osanai, M. (1987) A specific endopeptidase, BAEE esterase, in the glandula prostatica of the male reproductive system of the silkworm, Bombyx mori. Insect Biochem 17 (2): 323-328.

[25]   Satoh, D., Horii, A., Ochiai, M., et al. (1999) Prophenoloxidase-activating enzyme of the silkworm, Bombyx mori. Purification, characterization, and cDNA cloning. J Biol Chem. 274(11): 7441-53.

[26]   Ashida, M. and Sasaki, T. (1994) A target protease activity of serpins in insect hemolymph. Insect Biochem and Mol Bio 24(10): 1037-1041.