CLIP-domain serine proteases in Drosophila innate immunity

  • Jang, In-Hwan ;
  • Nam, Hyuck-Jin ;
  • Lee, Won-Jae
  • Accepted : 2008.02.14
  • Published : 2008.02.29


Extracellular proteases play an important role in a wide range of host physiological events, such as food digestion, extracellular matrix degradation, coagulation and immunity. Among the large extracellular protease family, serine proteases that contain a "paper clip"-like domain and are therefore referred to as CLIP-domain serine protease (clip-SP), have been found to be involved in unique biological processes, such as immunity and development. Despite the increasing amount of biochemical information available regarding the structure and function of clip-SPs, their in vivo physiological significance is not well known due to a lack of genetic studies. Recently, Drosophila has been shown to be a powerful genetic model system for the dissection of biological functions of the clip-SPs at the organism level. Here, the current knowledge regarding Drosophila clip-SPs has been summarized and future research directions to evaluate the role that clip-SPs play in Drosophila immunity are discussed.


CLIP domain;Drosophila;Pro-phenoloxidase;Serine protease;Toll


  1. Muta, T., Hashimoto, R., Miyata, T., Nishimura, H., Toh, Y. and Iwanaga, S. (1990) Proclotting enzyme from horseshoe crab hemocytes. cDNA cloning, disulfide locations, and subcellular localization. The Journal of Biological Chemistry 265, 22426-22433
  2. Jiang, H. and Kanost, M. R. (2000) The clip-domain family of serine proteinases in arthropods. Insect Biochemistry and Molecular Biology 30, 95-105
  3. Ross, J., Jiang, H., Kanost, M. R. and Wang, Y. (2003) Serine proteases and their homologs in the Drosophila melanogaster genome: an initial analysis of sequence conservation and phylogenetic relationships. Gene 304, 117-131
  4. Piao, S., Song, Y. L., Kim, J. H., Park, S. Y., Park, J. W., Lee, B. L., Oh, B. H. and Ha, N. C. (2005) Crystal structure of a clip-domain serine protease and functional roles of the clip domains. The EMBO Journal 24, 4404-4414
  5. Chasan, R. and Anderson, K. V. (1989) The role of easter, an apparent serine protease, in organizing the dorsal-ventral pattern of the Drosophila embryo. Cell 56, 391-400
  6. Chasan, R., Jin, Y. and Anderson, K. V. (1992) Activation of the easter zymogen is regulated by five other genes to define dorsal-ventral polarity in the Drosophila embryo. Development (Cambridge, England) 115, 607-616
  7. Stein, D. and Nusslein-Volhard, C. (1992) Multiple extracellular activities in Drosophila egg perivitelline fluid are required for establishment of embryonic dorsal-ventral polarity. Cell 68, 429-440
  8. DeLotto, Y. and DeLotto, R. (1998) Proteolytic processing of the Drosophila Spatzle protein by easter generates a dimeric NGF-like molecule with ventralising activity. Mech. Dev. 72, 141-148
  9. Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J. M. and Hoffmann, J. A. (1996) The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86, 973-983
  10. Levashina, E. A., Langley, E., Green, C., Gubb, D., Ashburner, M., Hoffmann, J. A. and Reichhart, J. M. (1999) Constitutive activation of toll-mediated antifungal defense in serpin-deficient Drosophila. Science (New York, N.Y) 285, 1917-1919
  11. Gobert, V., Gottar, M., Matskevich, A. A., Rutschmann, S., Royet, J., Belvin, M., Hoffmann, J. A. and Ferrandon, D. (2003) Dual activation of the Drosophila toll pathway by two pattern recognition receptors. Science (New York, N.Y) 302, 2126-2130
  12. Gottar, M., Gobert, V., Matskevich, A. A., Reichhart, J. M., Wang, C., Butt, T. M., Belvin, M., Hoffmann, J. A. and Ferrandon, D. (2006) Dual detection of fungal infections in Drosophila via recognition of glucans and sensing of virulence factors. Cell 127, 1425-1437
  13. Kim, Y. S., Ryu, J. H., Han, S. J., Choi, K. H., Nam, K. B., Jang, I. H., Lemaitre, B., Brey, P. T. and Lee, W. J. (2000) Gram-negative bacteria-binding protein, a pattern recognition receptor for lipopolysaccharide and beta-1,3-glucan that mediates the signaling for the induction of innate immune genes in Drosophila melanogaster cells. The Journal of Biological Chemistry 275, 32721-32727
  14. Lee, W. J., Lee, J. D., Kravchenko, V. V., Ulevitch, R. J. and Brey, P. T. (1996) Purification and molecular cloning of an inducible gram-negative bacteria-binding protein from the silkworm, Bombyx mori. Proceedings of the National Academy of Sciences of the United States of America 93, 7888-7893
  15. Michel, T., Reichhart, J. M., Hoffmann, J. A. and Royet, J. (2001) Drosophila Toll is activated by Gram-positive bacteria through a circulating peptidoglycan recognition protein. Nature 414, 756-759
  16. Ligoxygakis, P., Pelte, N., Hoffmann, J. A. and Reichhart, J. M. (2002) Activation of Drosophila Toll during fungal infection by a blood serine protease. Science (New York, N.Y) 297, 114-116
  17. Ashida, M. (1971) Purification and characterization of pre-phenoloxidase from hemolymph of the silkworm Bombyx mori. Arch. Biochem. Biophys. 144, 749-762
  18. Ashida, M. (1990) The prophenoloxidase cascade in insect immunity. Res. Immunol. 141, 908-910
  19. Katsumi, Y., Kihara, H., Ochiai, M. and Ashida, M. (1995) A serine protease zymogen in insect plasma. Purification and activation by microbial cell wall components. Eur. J. Biochem. 228, 870-877
  20. Jang, I. H., Chosa, N., Kim, S. H., Nam, H. J., Lemaitre, B., Ochiai, M., Kambris, Z., Brun, S., Hashimoto, C., Ashida, M., Brey, P. T. and Lee, W. J. (2006) A Spatzleprocessing enzyme required for toll signaling activation in Drosophila innate immunity. Dev. Cell. 10, 45-55
  21. Kambris, Z., Brun, S., Jang, I. H., Nam, H. J., Romeo, Y., Takahashi, K., Lee, W. J., Ueda, R. and Lemaitre, B. (2006) Drosophila immunity: a large-scale in vivo RNAi screen identifies five serine proteases required for Toll activation. Curr. Biol. 16, 808-813
  22. Jang, I. H. (2007) Les mecanismes regulatoires de l'immunite systemique et locale epitheliale dans Drosophila melanogaster. PhD. thesis, University of Paris VI, France
  23. Ashida, M. and Brey, P. T. (1998) Molecular mechanisms of immune responses; in Insects, Brey, P. T. and Hultmark, D. (eds.) Chapman and Hall, London
  24. Ashida, M. and Yamazaki, H. I. (1990) Biochemistry of the prophenoloxidase; in Insects, with special reference to its activation., Ohnishi, E. and Ishizaki, I. (eds.) Japan Sci. Society Press., Tokyo/Springer Verlag, Berlin
  25. Ashida, M. and Ohnishi, E. (1967) Activation of pre-phenol oxidase in hemolymph of the silkworm, Bombyx mori. Arch. Biochem. Biophys. 122, 411-416
  26. Ashida, M. and Brey, P. T. (1995) Role of the integument in insect defense: pro-phenol oxidase cascade in the cuticular matrix. Proceedings of the National Academy of Sciences of the United States of America 92, 10698-10702
  27. Gorman, M. J., Wang, Y., Jiang, H. and Kanost, M. R. (2007) Manduca sexta hemolymph proteinase 21 activates prophenoloxidase-activating proteinase 3 in an insect innate immune response proteinase cascade. The Journal of Biological Chemistry 282, 11742-11749
  28. Lee, H. S., Cho, M. Y., Lee, K. M., Kwon, T. H., Homma, K., Natori, S. and Lee, B. L. (1999) The pro-phenoloxidase of coleopteran insect, Tenebrio molitor, larvae was activated during cell clump/cell adhesion of insect cellular defense reactions. FEBS Letters 444, 255-259
  29. Park, J. W., Kim, C. H., Kim, J. H., Je, B. R., Roh, K. B., Kim, S. J., Lee, H. H., Ryu, J. H., Lim, J. H., Oh, B. H., Lee, W. J., Ha, N. C. and Lee, B. L. (2007) Clustering of peptidoglycan recognition protein-SA is required for sensing lysine-type peptidoglycan in insects. Proceedings of the National Academy of Sciences of the United States of America 104, 6602-6607
  30. Liu, H., Jiravanichpaisal, P., Cerenius, L., Lee, B. L., Soderhall, I. and Soderhall, K. (2007) Phenoloxidase is an important component of the defense against Aeromonas hydrophila Infection in a crustacean, Pacifastacus leniusculus. The Journal of Biological Chemistry 282, 33593-33598
  31. Tang, H., Kambris, Z., Lemaitre, B. and Hashimoto, C. (2006) Two proteases defining a melanization cascade in the immune system of Drosophila. The Journal of Biological Chemistry 281, 28097-28104
  32. Vass, E. and Nappi, A. J. (2000) Developmental and immunological aspects of Drosophila-parasitoid relationships. The Journal of Parasitology 86, 1259-1270
  33. Leclerc, V., Pelte, N., El Chamy, L., Martinelli, C., Ligoxygakis, P., Hoffmann, J. A. and Reichhart, J. M. (2006) Prophenoloxidase activation is not required for survival to microbial infections in Drosophila. EMBO Reports 7, 231-235
  34. Schnitger, A. K., Kafatos, F. C. and Osta, M. A. (2007) The melanization reaction is not required for survival of Anopheles gambiae mosquitoes after bacterial infections. The Journal of Biological Chemistry 282, 21884-21888
  35. De Gregorio, E., Han, S. J., Lee, W. J., Baek, M. J., Osaki, T., Kawabata, S., Lee, B. L., Iwanaga, S., Lemaitre, B. and Brey, P. T. (2002) An immune-responsive Serpin regulates the melanization cascade in Drosophila. Dev. Cell. 3, 581-592
  36. Ligoxygakis, P., Pelte, N., Ji, C., Leclerc, V., Duvic, B., Belvin, M., Jiang, H., Hoffmann, J. A. and Reichhart, J. M. (2002) A serpin mutant links Toll activation to melanization in the host defence of Drosophila. The EMBO Journal 21, 6330-6337
  37. Hashimoto, C., Kim, D. R., Weiss, L. A., Miller, J. W. and Morisato, D. (2003) Spatial regulation of developmental signaling by a serpin. Dev. Cell. 5, 945-950
  38. Ligoxygakis, P., Roth, S. and Reichhart, J. M. (2003) A serpin regulates dorsal-ventral axis formation in the Drosophila embryo. Curr. Biol. 13, 2097-2102
  39. Stein, D. S. and Stevens, L. M. (1991) Establishment of dorsal-ventral and terminal pattern in the Drosophila embryo. Curr. Opin. Genet. Dev. 1, 247-254

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