The insect metalloproteinase inhibitor gene of the lepidopteran Galleria mellonella encodes two distinct inhibitors
-
Marianne Wedde
, Christoph Weise , Rolf Nuck , Boran Altincicek and Andreas Vilcinskas
Abstract
The insect metalloproteinase inhibitor (IMPI) from the greater wax moth, Galleria mellonella, represents the first and to date only specific inhibitor of microbial metalloproteinases reported from animals. Here, we report on the characterization including carbohydrate analysis of two recombinant constructs encoded by impi cDNA either upstream or downstream of the furin cleavage site identified. rIMPI-1, corresponding to native IMPI purified from hemolymph, is encoded by the N-terminal part of the impi sequence, whereas rIMPI-2 is encoded by its C-terminal part. rIMPI-1 is glycosylated at N48 with GlcNAc2Man3, showing fucosylation to different extents. Similarly, rIMPI-2 is glycosylated at N149 with GlcNAc2Man3, but is fully fucosylated. rIMPI-1 represents a promising template for the design of second-generation antibiotics owing to its specific activity against thermolysin-like metalloproteinases produced by human pathogenic bacteria such as Vibrio vulnificus. In contrast, rIMPI-2 does not inhibit bacterial metalloproteinases, but is moderately active against recombinant human matrix metalloproteinases (MMPs). Both microbial metalloproteinases and MMPs induce expression of the impi gene when injected into G. mellonella larvae. These findings provide evidence that the impi gene encodes two distinct inhibitors, one inhibiting microbial metalloproteinases and contributing to innate immunity, the other putatively mediating regulation of endogenous MMPs during metamorphosis.
References
Ailor, E. and Betenbaugh, M.J. (1999). Modifying secretion and post-translational processing in insect cells. Curr. Opin. Biotechnol.10, 142–145.10.1016/S0958-1669(99)80024-XSearch in Google Scholar
Altincicek, B. and Vilcinskas, A. (2006). Metamorphosis and collagen-IV-fragments stimulate innate immune response in the greater wax moth, Galleria mellonella. Dev. Comp. Immunol.30, 1108–1118.10.1016/j.dci.2006.03.002Search in Google Scholar
Bode, W. and Maskos, K. (2003). Structural basis of the matrix metalloproteinases and their physiological inhibitors, the tissue inhibitors of metalloproteinases. Biol. Chem.384, 863–872.10.1515/BC.2003.097Search in Google Scholar
Brew, K., Dinakarpandian, D., and Nagase, H. (2000). Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim. Biophys. Acta1477, 267–283.10.1016/S0167-4838(99)00279-4Search in Google Scholar
Chaurand, P., Stoeckli, M., and Caprioli, R. (1999). Direct profiling of proteins in biological tissue sections by MALDI mass spectrometry. Anal. Chem.71, 5263–5270.10.1021/ac990781qSearch in Google Scholar
Clermont, A., Wedde, M., Seitz, V., Podsiadlowski, L., Lenze, D., Hummel, M., and Vilcinskas, A. (2004). Cloning and expression of an inhibitor of microbial metalloproteinases from insects contributing to innate immunity. Biochem. J.382, 315–322.10.1042/BJ20031923Search in Google Scholar
Culp, J., Johansen, H., Hellmig, B., Beck, J., Matthews, T., Delers, A., and Rosenberg, N. (1991). Regulated expression allows high level production and secretion of HIV-1 gp120 envelope glycoprotein in Drosophila Schneider cells. Biotechnology9, 173–177.10.1038/nbt0291-173Search in Google Scholar
Gohlke, M. (2002). Separation of N-glycans by HPLC. In: Posttranslational Modification of Proteins: Methods in Molecular Biology, Vol. 194, C. Kannicht, ed. (Totowa, NJ, USA: Humana Press), pp. 45–61.10.1385/1-59259-181-7:045Search in Google Scholar
Griesch, J., Wedde, M., and Vilcinskas, A. (2000). Recognition and regulation of metalloproteinase activity in the haemolymph of Galleria mellonella: a new pathway mediating induction of humoral immune responses. Insect Biochem. Mol. Biol.30, 461–472.10.1016/S0965-1748(00)00010-2Search in Google Scholar
Jarvis, D.L. and Finn, E.E. (1996). Modifying the insect cell N-glycosylation pathway with immediate early baculovirus expression vectors. Nat. Biotechnol.14, 1288–1292.10.1038/nbt1096-1288Search in Google Scholar PubMed
Johanson, K., Appelbaum, E., Doyle, M., Hensley, P., Zhao, B., Abdel-Meguid, S., Young, P., Cook, R., Carr, S., Matico, R., et al. (1995). Binding interactions of human interleukin 5 with its receptor α subunit-large scale production, structural, and functional studies of Drosophila-expressed recombinant proteins. J. Biol. Chem.270, 9459–9471.10.1074/jbc.270.16.9459Search in Google Scholar
Lauer-Fields, J. and Fields, G. (2002). Triple-helical peptide analysis of collagenolytic protease activity. Biol. Chem.383, 1095–1105.10.1515/BC.2002.118Search in Google Scholar
März, L., Altmann, F., Staudacher, E., and Kubelka, V. (1995). Protein glycosylation in insects. In: Glycoproteins, J. Montreuil, H. Schachter and J.F.G. Vliegenthart, eds. (New York, USA: Elsevier Science), pp. 543–563.Search in Google Scholar
Medzihradszky, K.F. (2002). Characterization of site-specific glycosylation. In: Posttranslational Modification of Proteins, Vol. 194, C. Kannnicht, ed. (Totowa, NJ, USA: Humana Press), pp. 191–225.10.1385/1-59259-181-7:101Search in Google Scholar
Miyoshi, S., Nakazawa, H., Kawata, K., Tomochika, K.-I., Tobe, K. and Shinoda, S. (1998). Characterization of the hemorrhagic reaction caused by Vibrio vulnificus metalloprotease, a member of the thermolysin family. Infect. Immun.66, 4851–4855.10.1128/IAI.66.10.4851-4855.1998Search in Google Scholar
Miyoshi, S., Sonoda, Y., Wakiyama, H., Rahman, M., Tomochika, K., Shinoda, S., Yamamoto, S., and Tobe, K. (2002). An exocellular thermolysin-like metalloprotease produced by Vibrio fluvialis: purification, characterization and gene cloning. Microb. Pathogen.33, 127–13410.1006/mpat.2002.0520Search in Google Scholar
Nagase, H. and Woessner, J.F. (1999). Matrix metalloproteinases. J. Biol. Chem.274, 2191–2194.10.1074/jbc.274.31.21491Search in Google Scholar
Nuck, R. (2002). Enzymatical hydrolysis of N-glycans from glycoproteins and fluorescent labeling by 2-aminobenzamide (2-AB). In: Posttranslational Modification of Proteins: Methods in Molecular Biology, Vol. 194, C. Kannicht, ed. (Totowa, NJ, USA: Humana Press), pp. 37–44.10.1385/1-59259-181-7:037Search in Google Scholar
Nuck, R. and Gohlke, M. (1997). Characterization of subnanomolar amounts of N-glycans by 2-aminobenzamide labelling, MALDI-TOF-MS and computer assisted sequence analysis. In: Methods in Glycobiology, R. Townsend and A. Hotchkiss, eds. (New York, USA: Marcel Dekker), pp. 491–508.Search in Google Scholar
Page-McCaw, A., Serano, J., Sante, J., and Rubin, GM. (2003). Drosophila matrix metalloproteinases are required for tissue remodelling, but not embryonic development. Dev. Cell4, 95–106.10.1016/S1534-5807(02)00400-8Search in Google Scholar
Rawlings, N., O'Brien, E., and Barrett, A. (2002). MEROPS: the protease database. Nucleic Acids Res.30, 343–346.10.1093/nar/30.1.343Search in Google Scholar PubMed PubMed Central
Rawlings, N., Tolle, D., and Barrett, A. (2004). Evolutionary families of peptidase inhibitors. Biochem. J.378, 705–716.10.1042/bj20031825Search in Google Scholar
Rozen, S. and Skaletsky, H. (2000). Primer3 on the WWW for general users and for biologist programmers. In: Bioinformatics Methods and Protocols: Methods in Molecular Biology, Vol. 132, S. Krawetz and S. Misener, eds. (Totowa, NJ, USA: Humana Press), pp. 365–386.Search in Google Scholar
Schuhmann, B., Seitz, V., Vilcinskas, A., and Podsiadlowski, L. (2003) Cloning and expression of gallerimycin, an antifungal peptide expressed in immune response of greater wax moth larvae, Galleria mellonella. Arch. Insect Biochem. Physiol.53, 125–133.10.1002/arch.10091Search in Google Scholar
Seitz, V., Clermont, A., Wedde, M., Hummel, M., Vilcinskas, A., Schlatterer, K., and Podsiadlowski, L. (2003). Identification of immunorelevant genes from greater wax moth (Galleria mellonella) by a subtractive hybridization approach. Dev. Comp. Immunol.27, 207–215.10.1016/S0145-305X(02)00097-6Search in Google Scholar
Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996). Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem.68, 850–858.10.1021/ac950914hSearch in Google Scholar
Supuran, C., Scozzafava, A., and Mastrolorenzo, A. (2001). Bacterial proteases: current therapeutic use and future prospects for the development of new antibiotics. Expert Opin. Ther. Patents11, 221–259.10.1517/13543776.11.2.221Search in Google Scholar
Tomiya, N., Narang, S., Lee, Y.C., and Betenbaugh, M.J. (2004). Comparing N-glycan processing in mammalian cell lines to native and engineered lepidopteran insect cell lines. Glycoconj. J.21, 343–360.10.1023/B:GLYC.0000046275.28315.87Search in Google Scholar
Umezawa, H. (1976). Structures and activities of protease inhibitors of microbial origin. Methods Enzymol.45, 678–695.10.1016/S0076-6879(76)45058-9Search in Google Scholar
Vilcinskas, A. and Wedde, M. (2002). Insect inhibitors of metalloproteinases. IUBMB Life4, 339–343.10.1080/15216540216040Search in Google Scholar PubMed
Vorm, O., Roepstorff, P., and Mann, M. (1994). Matrix surfaces made by fast evaporation yield improved resolution and very high sensitivity in MALDI-TOF. Anal. Chem.66, 3281–3287.10.1021/ac00091a044Search in Google Scholar
Voss, T., Ergulen, E., Ahorn, H., Kubelka, V., Sugiyama, K., Maurer-Fogy, I., and Glossl, J. (1993). Expression of human interferon omega 1 in Sf9 cells. No evidence for complex-type N-linked glycosylation or sialylation. Eur. J. Biochem.217, 913–919.Search in Google Scholar
Vu, T. and Werb, Z. (2000). Matrix metalloproteinases: effectors of development and normal physiology. Genes Dev.14, 2123–2133.10.1101/gad.815400Search in Google Scholar PubMed
Wagner, R., Geyer, H., and Klenk, H.D. (1996). N-Acetyl-β-glucosaminidase accounts for differences in glycosylation of influenza virus hemagglutinin expressed in insect cells from a baculovirus vector. J. Virol.70, 4103–4109.10.1128/jvi.70.6.4103-4109.1996Search in Google Scholar
Watanabe, S., Kokuho, T., Takahashi, H., Takahashi, M., Kubota, T., and Inumaru, S. (2002). Sialylation of N-glycans on the recombinant proteins expressed by a baculovirus-insect cell system under β-N-acetylglucosaminidase inhibition. J. Biol. Chem.277, 5090–5093.10.1074/jbc.M110548200Search in Google Scholar PubMed
Wedde, M., Weise, C., Kopacek, P., Franke, P., and Vilcinskas, A. (1998). Purification and characterization of an inducible metalloprotease inhibitor from the hemolymph of greater wax moth larvae, Galleria mellonella. Eur. J. Biochem.255, 535–543.10.1046/j.1432-1327.1998.2550535.xSearch in Google Scholar PubMed
Wei, S., Xie, Z., Filenova, E., and Brew, K. (2003). Drosophila TIMP is a potent inhibitor of MMPs and TACE: similarities in structure and function to TIMP-3. Biochem.42, 12200–12207.10.1021/bi035358xSearch in Google Scholar PubMed
Yucel, T., Mutnal, A., Fay, K., Fligiel, S., Wang, T., Johnson, T., Baker, S., and Varani, J. (2005). Matrix metalloproteinase expression in basal cell carcinoma: relationship between enzyme profile and collagen fragmentation pattern. Exp. Mol. Pathol.79, 151–160.10.1016/j.yexmp.2005.05.003Search in Google Scholar PubMed
©2007 by Walter de Gruyter Berlin New York
Articles in the same Issue
- Accumulation of viroid-specific small RNAs and increase in nucleolytic activities linked to viroid-caused pathogenesis
- Characterisation of Plasmodium falciparum RESA-like protein peptides that bind specifically to erythrocytes and inhibit invasion
- Structural modifications to a high-activity binding peptide located within the PfEMP1 NTS domain induce protection against P. falciparum malaria in Aotus monkeys
- Characterisation of YtfM, a second member of the Omp85 family in Escherichia coli
- Presence of the propeptide on recombinant lysosomal dipeptidase controls both activation and dimerization
- Conformational studies on Arabidopsis sulfurtransferase AtStr1 with spectroscopic methods
- Glycine-assisted enhancement of 1,4-β-d-xylan xylanohydrolase activity at alkaline pH with a pH optimum shift
- Asef is a Cdc42-specific guanine nucleotide exchange factor
- Metal-binding sites at the active site of restriction endonuclease BamHI can conform to a one-ion mechanism
- Interaction of the cellular prion protein with raft-like lipid membranes
- Tissue-specific transcription factor HNF4α inhibits cell proliferation and induces apoptosis in the pancreatic INS-1 β-cell line
- Binding of aflatoxins to the 20S proteasome: effects on enzyme functionality and implications for oxidative stress and apoptosis
- The insect metalloproteinase inhibitor gene of the lepidopteran Galleria mellonella encodes two distinct inhibitors
- Activation profiles of the zymogen of aspergilloglutamic peptidase
Articles in the same Issue
- Accumulation of viroid-specific small RNAs and increase in nucleolytic activities linked to viroid-caused pathogenesis
- Characterisation of Plasmodium falciparum RESA-like protein peptides that bind specifically to erythrocytes and inhibit invasion
- Structural modifications to a high-activity binding peptide located within the PfEMP1 NTS domain induce protection against P. falciparum malaria in Aotus monkeys
- Characterisation of YtfM, a second member of the Omp85 family in Escherichia coli
- Presence of the propeptide on recombinant lysosomal dipeptidase controls both activation and dimerization
- Conformational studies on Arabidopsis sulfurtransferase AtStr1 with spectroscopic methods
- Glycine-assisted enhancement of 1,4-β-d-xylan xylanohydrolase activity at alkaline pH with a pH optimum shift
- Asef is a Cdc42-specific guanine nucleotide exchange factor
- Metal-binding sites at the active site of restriction endonuclease BamHI can conform to a one-ion mechanism
- Interaction of the cellular prion protein with raft-like lipid membranes
- Tissue-specific transcription factor HNF4α inhibits cell proliferation and induces apoptosis in the pancreatic INS-1 β-cell line
- Binding of aflatoxins to the 20S proteasome: effects on enzyme functionality and implications for oxidative stress and apoptosis
- The insect metalloproteinase inhibitor gene of the lepidopteran Galleria mellonella encodes two distinct inhibitors
- Activation profiles of the zymogen of aspergilloglutamic peptidase