SDS-induced Serine Protease Activity of an Antiviral Red Fluorescent Protein
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.
[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.
[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.
@article{"International Journal of Biological, Life and Agricultural Sciences:60094", author = "Kalyankumar M. Matti and Chandrashekhar J. Savanurmath and Shivayogeppa B. Hinchigeri", title = "SDS-induced Serine Protease Activity of an Antiviral Red Fluorescent Protein", abstract = "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.", volume = "4", number = "8", pages = "621-6", }
