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Expression and characterization of honeybee, Apis mellifera, larva chymotrypsin-like protease

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Previously, we found three enzyme fractions containing activities for the hydrolysis of royal jelly proteins from honeybee queen larvae. In this study, we identified a honeybee chymotrypsin-like protease (HCLPase) by LC-MS/MS and expressed it as a recombinant protein in Escherichia coli. The protease had an estimated molecular weight of around 26 kDa and showed high specificity for succinyl-Ala-Ala-Pro-Phe p-nitroanilide as a proteolytic substrate. Furthermore, the protease had an optimal pH of 9, and the activity was markedly inhibited by phenylmethylsulfonyl fluoride but not tosyl phenylalanyl chloromethyl ketone, both of which are irreversible inhibitors of chymotrypsin-like serine proteases. These results suggested that this recombinant protease, HCLPase, was a chymotrypsin-like serine protease with different characteristics from mammalian chymotrypsin.

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  • Botos, I., Meyer, E., Nguyen, M., Swanson, S.M., Koomen, J.M., Russell, D.H., Meyer, E.F. (2000) The structure of an insect chymotrypsin. J. Mol. Biol. 298, 895–901

    Article  CAS  PubMed  Google Scholar 

  • Brødsgaard, H.F., Brødsgaard, C.J., Hansen, H., Lövei, G.L. (2003) Environmental risk assessment of transgene products using honey bee (Apis mellifera) larvae. Apidologie 34, 139–145

    Article  Google Scholar 

  • Burgess, E.P.J., Malone, L.A., Christeller, J.T. (1996) Effect of two proteinase inhibitors on the digestive enzymes and survival of honey bees (Apis mellifera). J. Insect Physiol. 42, 823–828

    Article  CAS  Google Scholar 

  • Dahlmann, B., Jany, K.D., Pfleiderer, G. (1978) The midgut endopeptidase of the honey bee (Apis mellifica): comparison of the enzymes in different ontogenetic stages. Insect Biochem. 8, 203–211

    Article  CAS  Google Scholar 

  • Elpidina, E.N., Tsybina, T.A., Dunaevsky, Y.E., Belozersky, M.A., Zhuzhikov, D.P., Oppert, B. (2005) A chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae. Biochimie 87, 771–779

    Article  CAS  PubMed  Google Scholar 

  • Giebel, W., Zwilling, R., Pfleiderer, G. (1971) The evolution of endopeptidases-XII. The proteolytic enzymes of the honeybee (Apis mellifica L.). Comp. Biochem. Physiol. 38B, 197–210

    Google Scholar 

  • Guo, H., Ekusa, A., Iwai, K., Yonekura, M., Takahata, Y., Morimatsu, F. (2008) Royal jelly peptides inhibit lipid peroxidation in vitro and in vivo. J. Nutr. Sci. Vitaminol. 54, 191–195

    Article  CAS  PubMed  Google Scholar 

  • Guo, H., Kouzuma, Y., Yonekura, M. (2009) Structures and properties of antioxidative peptides derived from royal jelly protein. Food Chem. 113, 238–245

    Article  CAS  Google Scholar 

  • Herbert Jr., E.W., Shimanuki, H. (1983) Effect of diet pH on the consumption, brood rearing, and pH of worker jelly produced by caged honey bees. Apidologie 14, 191–196

    Article  Google Scholar 

  • Herrero, S., Combes, E., Van Oers, M.M., Vlak, J.M., de Maagd, R.A., Beekwilder, J. (2005) Identification and recombinant expression of a novel chymotrypsin from Spodoptera exigua. Insect Biochem. Mol. Biol. 35, 1073–1082

    Article  CAS  PubMed  Google Scholar 

  • Hidaka, S., Okamoto, Y., Uchiyama, S., Nakatsuoma, A., Hashimoto, K., Ohnishi, S.T., Yamaguchi, M. (2006) Royal jelly prevents osteoporosis in rats: beneficial effects in ovariectomy model and in bone tissue culture model. eCAM 3, 339–348

    PubMed Central  PubMed  Google Scholar 

  • Honeybee Genome Sequencing Consortium (2006) Insights into social insects from the genome of the honeybee Apis mellifera. Nature 443, 931–949

    Article  Google Scholar 

  • Jimenez, D.R., Gilliam, M. (1989) Age-related changes in midgut ultrastructure and trypsin activity in the honey bee, Apis mellifera. Apidologie 20, 287–303

    Article  Google Scholar 

  • Jones, J.C., Myerscough, M.R., Graham, S., Oldroyd, B.P. (2004) Honey bee nest thermoregulation: diversity promotes stability. Science 305, 402–404

    Article  CAS  PubMed  Google Scholar 

  • Kamakura, M. (2011) Royalactin induces queen differentiation in honeybees. Nature 473, 478–483

    Article  CAS  PubMed  Google Scholar 

  • Louati, H., Zouari, N., Miled, N., Gargouri, Y. (2011) A new chymotrypsin-like serine protease involved in dietary protein digestion in a primitive animal, Scorpio maurus: purification and biochemical characterization. Lipids Health Dis. 10, 121

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Matsui, T., Yukiyoshi, A., Doi, S., Sugimoto, H., Yamada, H., Matsumoto, K. (2002) Gastrointestinal enzyme production of bioactive peptides from royal jelly protein and their antihypertensive ability in SHR. J. Nutr. Biochem. 13, 80–86

    Article  CAS  PubMed  Google Scholar 

  • Matsuoka, T., Kawashima, T., Nakamura, T., Kanamaru, Y., Yabe, T. (2012) Isolation and characterization of proteases that hydrolyze royal jelly proteins from queen bee larvae of the honeybee, Apis mellifera. Apidologie 43, 685–697

    Article  CAS  Google Scholar 

  • Moore, S. (1968) Amino acid analysis: aqueous dimethyl sulfoxide as solvent for the ninhydrin reaction. J. Biol. Chem. 243, 6281–6283

    CAS  PubMed  Google Scholar 

  • Moritz, B., Crailsheim, K. (1987) Physiology of protein digestion in the midgut of honeybee (Apis mellifera L.). J. Insect Physiol. 12, 923–931

    Article  Google Scholar 

  • Nagai, T., Inoue, R., Suzuki, N., Nagashima, T. (2006) Antioxidant properties of enzymatic hydrolysates from royal jelly. J. Med. Food 9, 363–367

    Article  CAS  PubMed  Google Scholar 

  • Nakasa, T., Ueda, S., Nakatsuka, M., Okinaka, O. (2003) Effect of protease-treated royal jelly on plasma and liver lipids in rats fed on a high fat plus high cholesterol diet. Nippon Shokuhin Kagaku Kogaku Kaishi 10, 463–467

    Article  Google Scholar 

  • Niu, K., Guo, H., Guo, Y., Ebihara, S., Asada, M., Ohrui, T., Furukawa, K., Ichinose, M., Yanai, K., Kubo, Y., Arai, H., Okazaki, T., Nagatomi, R. (2013) Royal jelly prevents the progression of sarcopenia in aged mice in vivo and in vitro. J. Gerontol. A Biol. Sci. Med. Sci. 68, 1482–1492

    Article  PubMed  Google Scholar 

  • Shevchenko, A., Wilm, M., Vorm, O., Mann, M. (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858

    Article  CAS  PubMed  Google Scholar 

  • Terra, W.R., Ferreira, C. (1994) Insect digestive enzymes: properties, compartmentalization and function. Comp. Biochem. Physiol. 109B, 1–62

    CAS  Google Scholar 

  • Tokunaga, K., Yoshida, C., Suzuki, K., Maruyama, H., Futamura, Y., Araki, Y., Mishima, S. (2004) Antihypertensive effect of peptides from royal jelly in spontaneously hypertensive rats. Biol. Pharm. Bull. 27, 189–192

    Article  CAS  PubMed  Google Scholar 

  • Tsybina, T.A., Dunaevsky, Y.E., Belozersky, M.A., Zhuzhikov, D.P., Oppert, B., Elpidina, E.N. (2005) Digestive proteinases of yellow mealworm (Tenebrio molitor) larvae: purification and characterization of a trypsin-like proteinase. Biochemistry (Moscow) 70, 370–377

    Article  Google Scholar 

  • Vinokurov, K.D., Elpidina, E.N., Oppert, B., Prabhakar, S., Zhuzhikov, D.P., Dunaevsky, Y.E., Belozersky, M.A. (2006) Diversity of digestive proteinases in Tenebrio molitor (Coleoptera: Tenebrionidae) larvae. Comp. Biochem. Physiol. B 145, 126–137

    Article  CAS  PubMed  Google Scholar 

  • Wagner, W., Möhrlen, F., Schnetter, W. (2002) Characterization of the proteolytic enzymes in the midgut of the European cockchafer, Melolontha melolontha (Coleoptera: Scarabaeidae). Insect Biochem. Mol. Biol. 32, 803–814

    Article  CAS  PubMed  Google Scholar 

  • Whitworth, S.T., Blum, M.S., Travis, J. (1998) Proteolytic enzymes from larvae of the fire ant, Solenopsis invicta: isolation and characterization of four serine endopeptidases. J. Biol. Chem. 273, 14430–14434

    Article  CAS  PubMed  Google Scholar 

  • Zou, Z., Lopez, D.L., Kanost, M.R., Evans, J.D., Jiang, H. (2006) Comparative analysis of serine protease-related genes in the honey bee genome: possible involvement in embryotic development and innate immunity. Insect Biochem. Mol. Biol. 15, 603–614

    Article  CAS  Google Scholar 

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We wish to thank Dr. Yokichi Hayashi of the Department of Life Science, Asahikawa Medical Collage, for helpful suggestions. We also thank Kenji Ota, Suzuyo Watanabe, Yuri Kashima, and Satoshi Kanematsu of Akitaya Honten Co., Ltd. for their help in the procurement and preparation of larval samples.

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Correspondence to Tomio Yabe.

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Handling Editor: Klaus Hartfelder

Expression et caractérisation d’une protéase analogue à la chymotrypsine chez les larves de l’abeille, Apis mellifera

Larve / reine / protéase analogue à la chymotrypsine

Expression und Charakterisierung einer Chymotrypsin-ähnlichen Protease in Larven der Honigbiene, Apis mellifera

Apis mellifera / Königinnenlarven / Chymotrypsin-ähnliche Protease

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Matsuoka, T., Takasaki, A., Mishima, T. et al. Expression and characterization of honeybee, Apis mellifera, larva chymotrypsin-like protease. Apidologie 46, 167–176 (2015).

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