Although SW-AT-1, a serpin-type trypsin inhibitor from silkworm (Bombyx mori), was identified in previous study, its structure-function relationship has not been studied. In this study, SW-AT-1 was cloned from the body wall of silkworm and expressed in E. coli. rSW-AT-1 inhibited both trypsin and chymotrypsin in a concentration-dependent manner. The association rate constant for rSW-AT-1 and trypsin is 1.31×10？5 M？1s？1, for rSW-AT-1 and chymotrpsin is 2.85×10？6 M？1s？1. Circular dichroism (CD) assay showed 33% α-helices, 16% β-sheets, 17% turns, and 31% random coils in the secondary structure of the protein. Enzymatic and CD analysis indicated that rSW-AT-1 was stable at wide pH range between 4–10, and exhibited the highest activity at weakly acidic or alkaline condition. The predicted three-dimensional structure of SW-AT-1 by PyMOL (v1.4) revealed a deductive reactive centre loop (RCL) near the C-terminus, which was extended from the body of the molecule. In addition to trypsin cleavage site in RCL, matrix-assisted laser desorption ionization time of flight mass spectrometry indicated that the chymotrypsin cleavage site of SW-AT-1 was between F336 and T337 in RCL. Directed mutagenesis indicated that both the N- and C-terminal sides of RCL have effects on the activity, and G327 and E329 played an important role in the proper folding of RCL. The physiological role of SW-AT-1 in the defense responses of silkworm were also discussed.
References
[1]
Carrell RW, Pemberton PA, Boswell DR (1987) The Serpins: Evolution and adaptation in a family of protease Inhibitors. Cold Spring Harb Symp Quant Biol 52: 527–535. doi: 10.1101/sqb.1987.052.01.060
[2]
Rau JC, Beaulieu LM, Huntington JA, Church FC (2007) Serpins in thrombosis, hemostasis and fibrinolysis. J Thromb Haemost 5 Suppl 1: 102–15. doi: 10.1111/j.1538-7836.2007.02516.x
[3]
Grigoryev SA, Woodcock CL (1998) Chromatin structure in granulocytes. A link between tight compaction and accumulation of a heterochromatin-associated protein (MENT). J Biol Chem 273: 3082–3089. doi: 10.1074/jbc.273.5.3082
[4]
Irving JA, Shushanov SS, Pike RN, Popova EY, Bromme D, et al. (2002) Inhibitory activity of a heterochromatin-associated serpin (MENT) against papain-like cysteine proteinases affects chromatin structure and blocks cell proliferation. J Biol Chem 277: 13192–13201. doi: 10.1074/jbc.m108460200
[5]
Ligoxygakis P, Roth S, Reichhart JM (2003) A serpin regulates dorsalventral axis formation in the Drosophila embryo. Curr Biol 13: 2097–2102. doi: 10.1016/j.cub.2003.10.062
[6]
Levashina EA, Langley E, Green C, Gubb D, Ashburner M, et al. (1999) Constitutive activation of toll-mediated antifungal defense in serpin-deficient Drosophila. Science 285: 1917–1919. doi: 10.1126/science.285.5435.1917
[7]
Pak SC, Kumar V, Tsu C, Luke CJ, Askew YS, et al. (2004) SRP-2 is a cross-class inhibitor that participates in postembryonic development of the nematode Caenorhabditis elegans: initial characterization of the clade L serpins. J Biol Chem 279: 15448–15459. doi: 10.1074/jbc.m400261200
[8]
Ray CA, Black RA, Kronheim SR, Greenstreet TA, Sleath PR, et al. (1992) Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1 beta converting enzyme. Cell 69: 597–604. doi: 10.1016/0092-8674(92)90223-y
[9]
Huntington JA (2011) Serpin structure, function and dysfunction. J Thromb Haemost 9 Suppl 1: 26–34. doi: 10.1111/j.1538-7836.2011.04360.x
[10]
Michael RK (1990) Isolation and characterization of four serine proteinase inhibitors (serpins) from hemolymph of Manduca sexta. Insect Biochem Mol Biol 20: 141–147. doi: 10.1016/0020-1790(90)90006-g
[11]
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. doi: 10.1016/j.ygeno.2008.12.010
[12]
Zhou Y, Gao L, Shi HF, Xia HC, Gao L, et al. (2009) Microarray analysis of gene expression profile in resistant and susceptible Bombyx mori strains reveals resistance-related genes to nucleopolyhedrovirus. Genomics 101: 256–262. doi: 10.1016/j.ygeno.2013.02.004
Takuji S (1985) The reactive site of silkworm hemolymph antichymotrypsin is located at the COOH-terminal region of the molecule. Biochem Biophys Res Commun 132: 320–326. doi: 10.1016/0006-291x(85)91025-3
[15]
Narumi H, Tomoaki H, Kenzo N, Takuji S (1990) Amino acid sequence of silkworm (Bombyx mori) hemolymph antitrypsin deduced from its cDNA nucleotide sequence: Confirmation of its homology with serpins. J Biol Chem 108: 372–378.
[16]
Takuji S, Kazuto K, Tomochika O (1987) Interaction of Silkworm Larval Hemolymph Antitrypsin and Bovine Trypsin. J Biol Chem 102: 433–441.
[17]
Takuji S (1991) Patchwork-structure serpins from silkworm (Bombyx morz) larval hemolymph. Eur J Biochem 202: 255–261. doi: 10.1111/j.1432-1033.1991.tb16370.x
[18]
Liu HF, Yi N, Li RJ, Cui WZ, Mu ZM, et al. (2011) Alternative splicing of the antitrypsin gene in the silkworm, Bombyx mori. Mol Biol Rep 38: 2793–2799. doi: 10.1007/s11033-010-0424-4
[19]
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685. doi: 10.1038/227680a0
[20]
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72: 248–254. doi: 10.1006/abio.1976.9999
[21]
Masaaki A, Takuji S (1994) A target protease activity of serpins in insect hemolymph. Insect Biochem Mol Biol 24: 1037–1041. doi: 10.1016/0965-1748(94)90141-4
[22]
Koji S, Hiroshi F, Hiroshi D, Hisao I (2000) Synthesis and resorption of a humoral chymotrypsin inhibitor, CI-8, by fat body of the silkworm, Bombyx mori.. Insect Biochem Mol Biol 30: 363–368. doi: 10.1016/s0965-1748(00)00003-5
[23]
Patston PA, Gettins PG (1996) Significance of secondary structure predictions on the reactive center loop region of serpins: a model for the folding of serpins into a metastable state. FEBS Lett 383: 87–92. doi: 10.1016/0014-5793(96)00231-1
[24]
Peter GW (2002) Serpin Structure, Mechanism, and Function. Chem Rev 102: 4751–4804. doi: 10.1021/cr010170+
[25]
Huntington JA (2006) Shape-shifting serpins-advantages of a mobile mechanism. Trends Biochem Sci 31: 427–435. doi: 10.1016/j.tibs.2006.06.005
[26]
Alfonso C, Gabriella S, Claire D (2011) Bowman-Birk inhibitors from legumes and human gastrointestinal health: Current status and perspectives. Curr Protein Pept Sci 12: 358–373. doi: 10.2174/138920311796391133
[27]
Bryant J, Green TR, Gurusaddaiah T, Ryan CA (1976) Proteinase inhibitor II from potatoes: lsolation and characterization of its protomer components. Biochemistry 15: 3418–3424. doi: 10.1021/bi00661a004
[28]
Plunkett G, Senear DF, Zuroske G, Ryan CA (1982) Proteinase inhibitors l and II from leaves of wounded tomato plants: Purification and properties. Arch Biochem Biophys 213: 463–472. doi: 10.1016/0003-9861(82)90572-0
[29]
Atkinson AH, Heath RL, Simpson RJ, Clarke AE, Anderson MA (1993) Proteinase inhibitors in Nicotiana alata stigmas are derived from a precursor protein which is processed into five homologous inhibitors. The Plant Cell 5: 203–213. doi: 10.2307/3869586
[30]
Huang L, Cheng T, Xu P, Cheng D, Fang T, et al. (2009) A genome-wide survey for host response of silkworm, Bombyx mori during pathogen Bacillus bombyseptieus infection. PloS one 4: e8098. doi: 10.1371/journal.pone.0008098
[31]
De Gregorio E, Han S-J, Lee W-J, Baek M-J, Osaki T, et al. (2002) An Immune-Responsive Serpin Regulates the Melanization Cascade in Drosophila. Developmental cell 3: 581–592. doi: 10.1016/s1534-5807(02)00267-8
[32]
Ferrandon D, Imler J-L, Hetru C, Hoffmann JA (2007) The Drosophila systemic immune response: sensing and signalling during bacterial and fungal infections. Nature Reviews Immunology 7: 862–874. doi: 10.1038/nri2194
[33]
Charnley AK (2003) Fungal pathogens of insects: cuticle degrading enzymes and toxins. Advances in Botanical Research 40: 241–321. doi: 10.1016/s0065-2296(05)40006-3
[34]
Steinert S, Levashina EA (2011) Intracellular immune responses of dipteran insects. Immunological reviews 240: 129–140. doi: 10.1111/j.1600-065x.2010.00985.x
[35]
Huntington JA, Read RJ, Carrell RW (2000) Structure of a serpin-protease complex shows inhibition by deformation. Nature 407: 923–926.
[36]
Whisstock JC, Silverman GA, Bird PI, Bottomley SP, Kaiserman D, et al. (2010) Serpins flex their muscle II. Structural insights into target peptidase recognition, polymerization, and transport functions. J Biol Chem 285: 24307–24312. doi: 10.1074/jbc.r110.141408
[37]
Olson ST, Swanson R, Day D, Verhamme I, Kvassman J, et al. http://pubs.acs.org/doi/abs/10.1021/bi01？07290-bi0107290AF4 (2001) Resolution of michaelis complex, acylation, and conformational change steps in the reactions of the serpin, plasminogen activator inhibitor-1, with tissue plasminogen activator and trypsin. Biochemistry 40: 11742–11756.
[38]
Dementiev A, Simonovic M, Volz K, Gettins PG (2003) Canonical Inhibitor-like Interactions Explain Reactivity of α1-Proteinase Inhibitor Pittsburgh and Antithrombin with Proteinases. J Biol Chem 278: 37881–37887. doi: 10.1074/jbc.m305195200
[39]
Irving JA, Cabrita LD, Rossjohn J, Pike RN, Bottomley SP, et al. (2003) The 1.5 ？ crystal structure of a prokaryote serpin: controlling conformational change in a heated environment. Structure 11: 387–397. doi: 10.1016/s0969-2126(03)00057-1
[40]
Dunstone MA, Dai WW, Whisstock JC, Rossjohn J, Pike RN, et al. (2000) Cleaved antitrypsin polymers at atomic resolution. Protein Science 9: 417–420. doi: 10.1110/ps.9.2.417
[41]
Ricagno S, Caccia S, Sorrentino G, Antonini G, Bolognesi M (2009) Human Neuroserpin: Structure and Time-Dependent Inhibition. J Biol Chem 388: 109–121. doi: 10.1016/j.jmb.2009.02.056
