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SDS-induced Serine Protease Activity of an Antiviral Red Fluorescent Protein

Authors: Kalyankumar M. Matti, Chandrashekhar J. Savanurmath, Shivayogeppa B. Hinchigeri


A rare phenomenon of SDS-induced activation of a latent protease activity associated with the purified silkworm excretory red fluorescent protein (SE-RFP) was noticed. SE-RFP aliquots incubated with SDS for different time intervals indicated that the protein undergoes an obligatory breakdown into a number of subunits which exhibit autoproteolytic (acting upon themselves) and/or heteroproteolytic (acting on other proteins) activities. A strong serine protease activity of SE-RFP subunits on Bombyx mori nucleopolyhedrovirus (BmNPV) polyhedral protein was detected by zymography technique. A complete inhibition of BmNPV infection to silkworms was observed by the oral administration assay of the SE-RFP. Here, it is proposed that the SE-RFP prevents the initial infection of BmNPV to silkworms by obliterating the polyhedral protein. This is the first report on a silkworm red fluorescent protein that exhibits a protease activity on exposure to SDS. The present studies would help in understanding the antiviral mechanism of silkworm red fluorescent proteins.

Keywords: BmNPV, polyhedra, SE-RFP, SDS-induced protease activity, zymography

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[1] T. Yamada, H. Ohta, T. Masuda, M. Ikeda, N. Tomita, A. Ozawa1, Y. Shioi and K. Takamiya "Purification of a novel type of SDS-dependent protease in maize using a monoclonal antibody", Plant Cell Physiol, vol. 39. pp. 106-114, 1998.
[2] T. Yamada, A. Kondo, H. Ohta, T. Masuda, H. Shimada, K. Takamiya "Isolation of the protease component of maize cysteine protease-cystatin complex: release of cystatin is not crucial for the activation of the cysteine protease", Plant Cell Physiol, vol. 42. pp. 710-716, 2001.
[3] C. Wittenberg and E. Triplett "A detergent-activated tyrosinase from Xenopus laeuis I. Purification and partial characterization", J Biol Chem,vol. 260. pp.12535-12541, 1985a.
[4] C. Wittenberg and E. Triplett "A detergent-activated tyrosinase from Xenopus laeuis I. Purification and partial characterization", J Biol Chem,vol. 260, pp. 12542-12546, 1985b.
[5] J. H Golbeck and K. V. Cammarata "Spinach thylakoid polyphenoloxidase. Isolation, activation and properties of native chloroplast enzyme", Plant Physiol,vol. 67. pp. 977-984, 1981.
[6] B. M. Moore and W. H. Flurkey "Sodium dodecyl sulphate activation of a plant polyphenoloxidase. Effect of sodium dodecylsulfate on enzymatic and physical characteristics of purified broad bean polyphenoloxidase", J Biol Chem vol. 265, pp. 4982-4990, 1990.
[7] H. Decker, M. Ryan, E. Jaenicke and N. Terwilligeri "SDS-induced Phenoloxidase Activity of Hemocyanins from Limulus polyphemus, Eurypelma californicum, and Cancer magister", J Biol Chem,vol. 276. pp.17796-17799, 2001.
[8] Y. M. Rustum, E. J. Massaro, E. A. Barnard "Yeast hexokinase. V. Subunit structure and hybridization of isoenzymes", Biochemistry, vol. 10, pp. 3509-3516, 1971.
[9] W. Diezel, K. Nissler, W. Heilmann, G. Kopperschlager and E. Hofmann "Evidence for adsorbed proteolytic activity on isolated yeast phosphofructokinase", FEBS Lett. vol. 27, pp. 195-197, 1972.
[10] H. K. Mitchell, N. S. Peterson and C. H. Buzin "Self-degradation of heat shock proteins", Proc Natl Acad Sci USA, vol. 82, pp. 4969-4973, 1985.
[11] H. K. Mitchell and N. S. Peterson "Spontaneous fragmentation of several proteins in Drosophila Pupae", J Biol Chem,vol. 262, pp. 1429- 14304, 1987.
[12] J. Fibla, S. Atrian, and R. Gonzàlez-Duarte "Evidence of serine-protease activity closely associated with Drosophila alcohol dehydrogenase" Eur J Biochem, vol. 211, pp. 357-365, 1993.
[13] J. Mukai, J. Inoue, and S. Akune "A red fluorescent protein from silkworm", Agric. Biol. Chem, vol. 33, pp. 125-127. 1969.
[14] K. Hayashiya, J. Nishida and Y Uchida "The mechanism of formation of the red fluorescent protein in the digestive juice of silkworm larvae-the formation of chlorophyllide-a", J. Appl. Entomol. Zool, vol. 20, pp. 37- 43. (1976)
[15] Y. Uchida, F. Kawamoto, M. Himano and K. Hayashiya "A virus inactivating protein isolated from the digestive juice of the silkworm, B. mori" J. Invertebr. Pathol, vol. 43, pp. 182-189, 1984.
[16] B. Nagaraja-Sethuraman, N. Nagaraju and R. K. Datta "Purification and partial characterization of antiviral protein in silkworm, Bombyx mori", Indian J. Sericult,vol. 32, pp. 63-67, 1993.
[17] V. J. Lakkappan, S. S. Ingalhalli, S. B. Hinchigeri and Savanurmath, C. J. Oral administration of red fluorescent proteins in the silkworm Bombyx mori against nuclear polyhedrosis virus. J. Entomol. Res, vol. 31, pp. 251-254, 2007.
[18] B. Mauchamp, C. Royer, A. Garel, A. Jalabert, M. Da Rocha, A. M. Grenier, V. Labas, J. Vinh, K. Mita, K. Kadono and G. Chavancy "Polycalin chlorophyllid A binding protein: A novel, very large fluorescent lipocalin from the midgut of the domestic silkworm Bombyx mori L." Insect. Biochem. Molec, vol. 36, pp. 623-633, 2006.
[19] G. S. Suresh, L. N. Srikar, P. Anitha, L. Shanthala and M.A. Shankar "Purification and partial characterization of red fluorescent protein from silkworm, Bombyx mori", Indian J. Agric. Biochem. Vol. 20, 2007.
[20] S. G. Sunagar, V. J. Lakkappan, S. S. Ingalhalli , C. J. Savanurmath and Hinchigeri, S. B "Characterization of the photochromic pigments in red fluorescent proteins purified from the gut juice of the silkworm Bombyx mori L." Photochem. Photobiol, vol. 84, pp. 1440-1444, 2008.
[21] G. N. Pandian, T. Ishikawa, M. Togashi, Y. Shitomi, K. Haginoya, S. Yamamoto, T. Nishiumi and H. Hori. "Bombyx mori midgut membrane protein P252, which binds to Bacillus thuringiensis Cry1A, is a chlorophyllide-binding protein, and the resulting complex has antimicrobial activity", Appl. Environ. Microbiol, vol. 74, pp. 1324- 1331, 2008.
[22] R. Selot , V. Kumar , S. Shukla , K. Chandrakuntal , M. Brahmaraju , S. B. Dandin , M. Laloraya and P. G Kumar "Identification of a soluble NADPH oxidoreductase (BmNOX) with antiviral activities in the gut juice of Bombyx mori" Biosci Biotechnol Biochem, vol. 71. pp. 200-205, 2007.
[23] K. M. Ponnuvel, H. Nakazawa, S. Furukawa, A. Asaoka, J. Ishibashi H. Tanaka and M. Yamakawa "A lipase isolated from the silkworm Bombyx mori shows antiviral activity against nucleopolyhedrovirus", J. Virol, vol. 77, pp. 10725-10729, 2003.
[24] H. Nakazawa, E. Tsuneishi, K. M. Ponnuvel, S. Furukawa, A. Asaoka, H. Tanaka, J. Ishibashi, and M. Yamakawa "Antiviral activity of a serine protease from the digestive juice of Bombyx mori larvae against nucleopolyhedrovirus", Virology, vol. 321, pp. 154-162, 2004.
[25] M. Eguchi and A. Iwamoto "Comparison of three alkaline proteases from digestive fluid of the silkworm, Bombyx mori L.", Comp Biochem Physiol B, vol. 71, pp. 663-8, 1982.
[26] K. M. Matti, S. S. Singh, C. J. Savanurmath and Hinchigeri, S. B "A unique red fluorescent protein of silkworm, bearing two photochromic moieties", Photochem. Photobiol. Sci, vol 8, pp. 1364-1372, 2009.
[27] K. M. Matti, C. J. Savanurmath and S. B. Hinchigeri "A promising broad spectrum antimicrobial red fluorescent protein present in silkworm excreta", Biol. Pharm. Bull, (in press), 33 (7), 2010.
[28] U. K. Laemmli, "Cleavage of structural proteins during the assembly of the head of bacteriophage T4", Nature, vol. 227, pp. 680-685, 1970.
[29] P. Cuervo , C. Mesquita-Rodrigues , C. M. d'Avila Levy , C. Britto , F. A. Pires , R. Gredilha , C. R. Alves and J. B. Jesus "Serine protease activities in Oxysarcodexia thornax (Walker) (Diptera: Sarcophagidae) first instar larva", Mem Inst Oswaldo Cruz, vol. 103, pp. 504-506.
[30] K. M. Ponnuvel, V. Kumar, A. M. Babu and R. K. Datta "Effect of alkalinity and protease in the digestive juice of silkworm, Bombyx mori on BmNPV infection", Italian Journal of Zoology, 66: 2, 121 ÔÇö 124, 1999.
[31] S. S. Hiremath, L. N. Srikar, T. K. Narayanaswamy and K. R. Shashidhar "Lipid Profile of Silkworm Bombyx Mori L. as Influenced By Mulberry Grown Through Organic Based Nutrient Management" e Journal of Biological Sciences, vol. 1, pp. 101-104, 2009.
[32] B. Dahlmann, M. Rutschmann, L. Kuehn and H. Reinaur "Activation of the multicatalytic proteinase from rat skeletal muscle by fatty acids or sodium dodecyl sulphate", Biochem. J, vol. 228, pp. 171-177, 1985.
[33] N. Watanabe and S. Yamada "Activation of 20S proteasomes from spinach leaves by fatty acids" Plant Cell Physiol,vol. 37, pp. 147-151, 1996.
[34] E.A. Kozlov, N. Nijazova, M.T. Bobrovskaja, T.V. Shirina "Investigation of structure-function interaction of Bombyx mori nuclear polyhedrosis virus polyhedrin with polyhedra protease and RNA. Effect of protease on polyhedrin dissociation during Sepharose 6B gelfiltration" Biopolymers and cell, vol. 22, pp. 190-200, 2006.