Progress 10/01/05 to 09/30/11
Outputs
OUTPUTS: We produced purified recombinant forms of multicopper oxidases from a mosquito, Anopheles gambiae, and determined enzymatic properties of these proteins, including substrate specificity. We used antibodies to examine the tissue location of multicopper oxidases in mosquitoes and in Drosophila. We continued to investigate the function of multicopper oxidases in iron metabolism of Drosophila and mosquitos. We produced a recombinant form of an insect protease inhibitor for stuctural studies using x-ray crystallography. We examined the structure and binding of microbial polysaccharides by recombinant carbohydrate-binding domains from insect hemolymph proteins that function as recognition factors to trigger immune responses. Dissemination: Lectures at conferences: Functional genomics of cuticle in Tribolium castaneum. The 3rd International Symposium on Insect Physiology, Biochemistry and Molecular Biology, Shanghai, China. Poster presentations at conferences: 1. Noh, M.Y., Kanost, M.R., Muthukrishnan, S., Beeman, R.W., Kramer, K.J., and Arakane, Y. (2011) RNAi-based functional study of yellow-e in Tribolium castaneum. Sixth International Symposium on Molecular Insect Science, Amsterdam. 2. Dai, H., Hiromasa, Y., Kanost, M.R. and Krishnamoorthi, R. (2011) Protein-carbohydrate interactions in insect innate immunity: structural and functional studies of an insect pathogen-recognition protein domain. 35th Steenbock Symposium, "Advances in Biomolecular NMR," Madison, WI. 3. Huber, P.A., Lomakin, J., Etsitty, J., Arakane, Y. Kramer, K.J., Beeman, R.W., Kanost, M.R. and Gehrke, S.H. (2011) Structure-function relationships in beetle elytral cuticle, a biological composite material. American Institute of Chemical Engineers, Annual meeting. 4. Sprouse, P., Lomakin, J., Arakane, Y., Kramer, K.J., Beeman, R.W., Dittmer, N.T., Kanost, M.R., and Gehrke, S.H. (2011) Structure-function relationships in beetle cuticle, a biological composite material. Polymers in Biology and Medicine 2011, American Chemical Society. PARTICIPANTS: Michael Kanost: PI; Ramaswamy Krishnamoorthi: coPI; Maureen Gorman: Research Assistant Professor; Neal Dittmer: Research Assistant Professor; Di Wu: Research Associate; Minglin Lang: Reasearch Associate; Daisuke Takahashi, Research Associate; Jayne Christen: GRA; Huaien Dai: GRA; Zeyu Peng: GRA; Zhen Li: GRA. Partner Organizations: NIH, NSF. Collaborators: Karl Kramer, Richard Beeman, Duy Hua, Kristin Michel. Training: training in biochemical research for three graduate students and three postdoctoral scientists. TARGET AUDIENCES: Life scientists and educators PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge: The functions of multicopper oxidases have been investigated in plants and fungi, but little is known about their roles in insects. We purified recombinant forms of multicopper oxidases from a mosquito, Anopheles gambiae and the fruit fly, Drosophila melanogaster and characterized their enzymatic activities and substrate specificity. We determinied the tissue localization of multicopper oxidase-1 in Drosophila and identified a potential role for this enzyme in metabolism of iron. We determined the tissue localization of multicopper oxidase 3 in Anopheles and determined that its enzymatic properties allow its classification as a laccase. Proteases function in immune responses in insect hemolymph. We used recombinant proteins and proteomics methods to investigate the interactions of proteases and protease inhibitors to regulate immune responses in Manduca sexta. We determined that serpin-3 regulates three different clip domain proteases, prophenoloxidase activating protease-1, prophenoloxidase activating protease-3, and hemolymph protease 8. The protease pathways are triggered by binding of glucan recognition proteins in hemolymph to microbial surfaces. We examined the structure and binding activities of carbohydrate recognition domains from a lepidopteran insect, Plodia interpunctella, using nuclear magnetic resonance experiments and began to define the binding surface for microbial polysaccharides and interactions between the protein domains. This research will lead to understanding of fundamental processes in insect biology, including the synthesis and structure of the exoskeleton and the molecular basis for insect immune responses. There is potential to identify new targets for use in biological or chemical control of insect populations. It will also develop fundamental understanding of molecules and biochemical processes common to humans and insects.
Publications
- An, C., Ragan, E.J. and Kanost, M.R. (2011) Serpin-1 splicing isoform J inhibits the proSpatzle-activating proteinase HP8 to regulate expression of antimicrobial hemolymph proteins in Manduca sexta Dev. Comp. Immunol. 35: 135-141.
- An, C., Budd, A., Kanost, M.R. and Michel, K. (2011) Characterization of a regulatory unit that controls melanization and affects longevity of mosquitoes. Cell. Mol. Life Sci. 68: 1929-1939.
- Terenius, O., Papanicalou, A., Garbutt, J.S., Eleftherianos, I., Huvenne, H., Albrechtsen, M., An, C., Aymeric, J.-L., Barthel, A., Bebas P., Bitra, K., Bravo, A., Chevalier, F., Collinge, D.P., Crava, C.M., de Maagda, R.A., Duvic, B., Erlandson, M., Faye, I., Felfoldi, G., Fujiwara, H., Futahashi, R., Gandhe, A.S., Gatehouse, H.S., Gatehouse, L.N., Giebultowicz, J., Gomez, I., Grimmelikhuijzen, C.J., Groot, A.T., Hauser, F., Heckel, D.G., Hegedus, D.D., Hrycaj S.,Huang, Hull, J.J., Iatrou, K., Iga, M., Kanost, M.R., Kotwica, J., Li, C., Li, J., Lundmark, M., Matsumoto, S., Meyering-Vos, M., Millichap, P.J., Monteiro, A., Mrinal, N., Nagara, J., Niimi, T., Nowara, D., Ohnishi, A., Oostra, V., Ozaki, K., Papakonstandinou, M., Popadic, A., Rajam, M.V., Saenko, S., Simpson, R.M., Soberon, M., Strand, M.R., Tomita S., Toprak, U., Wang, P., We, C.W., Whyard, S., Zhang, W., ffrench-Constant, R.H., Herrero, S., Gordon, K., Swevers, L., and Smagghe, G. (2011) RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design. J. Insect Physiology. 57: 231-245.
- Lomakin, J., Huber, P.A., Eichler, C., Arakane, Y., Kramer, K.J., Beeman, R.W., Kanost, M.R., and Gehrke, S.H. (2011) Mechanical properties of the beetle elytron, a biological composite material. Biomacromolecules. 12: 321-335.
- An, C., Lovell, S., Kanost, M.R., Battaile, K.P., and Michel, K. (2011) Crystal structure of native Anopheles gambiae serpin-2, a negative regulator of melanization in mosquitoes. Proteins: Structure, Function and Bioinformatics. 79: 1999-2003.
- Kanost, M.R. and Clem, R.J. (2012) Insect Proteases. In Insect Molecular Biology and Biochemistry (L.I. Gilbert, ed) Elsevier, New York, pp. 346-364.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: We produced purified recombinant forms of multicopper oxidases from a beetle, Tribolium castaneum and a mosquito, Anopheles gambiae, and carried out experiments to investigate enzymatic properties of these proteins. We produced antibodies to several of these proteins, which we used to examine their tissue location in mosquitoes, beetles, and in Drosophila. We investigated the function of multicopper oxidases in iron metabolism of Drosophila and mosquiteos. We produced a recombinant form of an insect dopachrome conversion enzyme and produced crystals of this protein for ongoing structural studies. We produced recombinant forms of serine proteases and protease inhibitors from a moth, Manduca sexta, and carried out experiments to determine their functions in regulation of two immune responses, activation of phenoloxidase and activation of a signal transduction pathway that stimulates synthesis of antimicrobial proteins. Dissemination -Lectures at conferences: Protease pathways regulating insect immune responses. International Symposium on Silkworm Genome to Sustainable Agriculture, Tsukuba, Japan; Functional Genomics of insect cuticle tanning. Arthropod Genomics: New Approaches and Outcomes, Kansas City; Insect innate immune responses. Frontiers in Biomedical Research Symposium, Fargo, North Dakota. Poster presentations at conferences: Peng, Z., Gorman, M.J., Arakane, Y., and Kanost, M.R. (2010) Characterization of multicopper oxidase related protein (MCORP) in two insect species. American Society for Biochemistry and Molecular Biology, Annual Meeting. Anaheim, CA; Ragan, E. J. and Kanost, M.R. (2010) Proteomic identification of hemolymph proteins involved in early stages of immune response in the insect Manduca sexta. American Society for Biochemistry and Molecular Biology, Annual Meeting. Anaheim, CA; Lang, M., Gorman, M.J., Gardner, S.J., Yungeberg, S.L. and Kanost, M.R. (2010) Possible immune functions of two mosquito multicopper oxidases. American Society for Biochemistry and Molecular Biology, Annual Meeting. Anaheim, CA; Suwanchaichinda, C. and Kanost, M.R. (2010) Possible involvement of Manduca sexta hemolymph protease-1 in melanization. American Society for Biochemistry and Molecular Biology, Annual Meeting. Anaheim, CA; An, C., Budd, A., Kanost, M.R. and Michel, K. (2010) A regulatory unit of the melanization response affects the life span of mosquitoes. Arthropod Genomics: New Approaches and Outcomes, Kansas City; Peng, Z., Gorman, M.J., Arakane, Y., and Kanost, M.R. (2010) Characterization of multicopper oxidase related protein (MCORP) in two insect species. Arthropod Genomics: New Approaches and Outcomes, Kansas City; Lang, M., Gorman, M.J., Gardner, S.J., Yungeberg, S.L. and Kanost, M.R. (2010) Possible immune functions of two mosquito multicopper oxidases. Arthropod Genomics: New Approaches and Outcomes, Kansas City; Huber, P.A., Lomakin, J., Thomas, R., Ettsity, J., Arakane, Y., Kramer, K.J., Beeman, R.W., Kanost, M.R., and Gehrke, S.H. (2010) Insights from biology for the development of tissue engineering scaffolds: the beetle exoskeleton. Biomedical Engineering Society Annual Meeting, Austin, TX. PARTICIPANTS: Michael Kanost: PI, Ramaswamy Krishnamoorthi: coPI, Maureen Gorman: Research Assistant Professor, Neal Dittmer: Research Associate, Chunju An: Research Associate, Minglin Lang: Reasearch Associate, Jayne Christen: GRA, Huaien Dai: GRA, Zeyu Peng: GRA. Partner Organizations: NIH, NSF. Collaborators: Karl Kramer, Richard Beeman, Duy Hua, Kristin Michel. Training: training in biochemical research for three graduate students and four postdoctoral scientists. TARGET AUDIENCES: Life scientists and educators PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge: Functions of multicopper oxidases have been investigated in plants and fungi, but little is known about their roles in insects. We purified recombinant forms of several multicopper oxidases from a mosquito, Anopheles gambiae, and a beetle, Tribolium castaneum and obtained new information about their enzymatic activities and substrate specificity. We also gained new knowledge about the regulation of expression of these enzymes in insects in different tissues and at different developmental stages. One multicopper oxidase gene (laccase-2) functions in oxidation of phenolic substrates, leading to tanning and hardening of the exoskeleton after each molt. Other multicopper oxidase genes in insect genomes have different functions, as they are expressed in tissues other than the epidermis, such as midgut, malpighian tubules, and ovaries. A conserved multicopper oxidase-like protein, which lacks key copper-binding amino acid residues needed for oxidation reactions, is expressed at highest levels at the pupal and adult stages, and may have a role in metamorphosis or reproduction. Research on protease functions in hemolymph of a caterpillar, Manduca sexta, has revealed two immune pathways involving series of proteases organized in a cascade. One pathway leads to activation of phenol oxidase, and the other pathway leads to activation of a cytokine, which stimulates synthesis of antimicrobial peptides. There is cross-talk between these two pathways, mediated by a protease, HP6, that participates in both innate immune responses. We identified serine protease inhibitors in hemolymph which specificially regulate the proteases, promoting a local response of limited duration. This research will lead to understanding of fundamental processes in insect biology, including the synthesis and structure of the exoskeleton and the molecular basis for insect immune responses. There is potential to identify new targets for use in biological or chemical control of insect populations. It will also develop fundamental understanding of molecules and biochemical processes common to humans and insects.
Publications
- An, C., Jiang, H. and Kanost, M.R. (2010). Proteolytic activation and function of the cytokine Spatzle in innate immune response of a lepidopteran insect, Manduca sexta. FEBS J. 277:148-162.
- Rayaprolu, S., Wang, Y., Kanost, M.R., Hartson, S., and Jiang, H. (2010). Functional analysis of four processing products from multiple precursors encoded by a lebocin-related gene from Manduca sexta. Dev. Comp. Immunol. 34:638-647.
- Suderman, R.J., Dittmer, N.D., Kramer, K.J., and Kanost, M.R. (2010). Model reactions for insect cuticle sclerotization: participation of amino groups in the cross-linking of Manduca sexta cuticle protein MsCP36. Insect Biochem. Mol. Biol. 40:252-258.
- Arakane, Y., Dittmer, N.T., Kramer, K.J., Muthukrishnan, S., Beeman, R.W. and Kanost, M.R. (2010). Identification, mRNA expression and partial functional analysis of the yellow family genes in Tribolium castaneum. Insect Biochem. Mol. Biol. 40:259-266.
- Ragan, E.J., An, C., Yang, C., and Kanost, M.R. (2010). Analysis of mutually-exclusive alternatively spliced serpin-1 isoforms and identification of serpin-1 proteinase complexes in Manduca sexta hemolymph. J. Biol. Chem. 285:29642-29650.
- Lomakin, J., Arakane, Y., Kramer, K.J., Beeman, R.W., Kanost, M.R., and Gehrke, S.H. (2010). Mechanical properties of elytra from Tribolium castaneum wild-type and body color mutant strains. J. Insect Physiol. 56:1901-1906.
- An, C. and Kanost, M.R. (2010). Manduca sexta serpin-5 regulates prophenoloxidase activation and the Toll signaling pathway by inhibiting hemolymph proteinase HP6. Insect Biochem. Mol. Biol. 40:683-689.
- Zhu, Y., Ragan, E.J., and Kanost, M.R. (2010). Leureptin: a soluble, extracellular leucine-rich repeat protein from Manduca sexta that binds lipopolysaccharide. Insect Biochem. Mol. Biol. 40:713-722.
- Miyaji, T., Murayama, S., Kouzuma, Y., Kimura, N., Kanost, M.R., Kramer, K.J., and Yonekura, M. (2010). Molecular cloning of a multidomain cysteine protease and protease inhibitor precursor gene from the tobacco hornworm (Manduca sexta) and functional expression of the cathepsin-f like cysteine protease domain. Insect Biochem. Mol. Biol. 12:835-846.
- Dittmer, N.T. and Kanost, M.R. (2010). Insect multicopper oxidases: diversity, properties, and physiological roles. Insect Biochem. Mol. Biol. 40:179-188.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: We carried out experiments to determine the enzymatic properties of laccases from a moth, Manduca sexta, and a mosquito, Anopheles gambiae, and to investigate their function in the biology of these insects (Objectives 1 and 2). We carried out experiments to determine how carbohydrate-binding proteins in hemolymph of caterpillars (Plodia interpunctella and Manduca sexta) bind to the surface of microorganisms and trigger activation of protease cascades that activate immune responses (Objective 3). We initiated experiments to examine similar proteins in hemolymph of mosquitoes. We used purified recombinant proteins to investigate the regulation of serine proteases in insect hemolymph by serine protease inhibitors (serpins) (Objective 3). Dissemination: Lectures at conferences - Hemolymph protease cascades in Manduca sexta Symposium on Insect Infection and Immunity, Royal Entomological Society, Sheffield, UK; Hemolymph serine protease cascades in lepidopteran immune responses International Symposium on Bombyx mori functional genomics and modern silk road, Chongqing China; Functions and regulation of Manduca sexta hemolymph proteinases HP6 and HP8 in two innate immune pathways International Symposium on Bombyx mori functional genomics and modern silk road, Chongqing China; Evolution of insect immune systems Evolution Symposium 2009, University of Nebraska, Kearney. Poster presentations at conferences - Enzymatic pathways in the development of beetle elytral cuticle. American Chemical Society National Meeting, Salt Lake City; Mechanical properties of beetle elytral cuticle, a hierarchically ordered biomaterial. American Chemical Society National Meeting, Salt Lake City; Analysis of hemolymph proteinase 16 from an insect, Manduca sexta. American Society for Biochemistry and Molecular Biology, Annual Meeting; Regulation of Manduca sexta hemolymph proteinase 8 by serpin-1 isoforms. American Society for Biochemistry and Molecular Biology, Annual Meeting; Molecular cloning and biochemical characterization of Manduca sexta serpin-7, a regulator of prophenoloxidase activation. American Society for Biochemistry and Molecular Biology, Annual Meeting; Gene expression and functional characterization of two multicopper oxidase genes in the malaria mosquito, Anopheles gambiae. American Society for Biochemistry and Molecular Biology, Annual Meeting; Enzymatic Reactions and Mechanical Properties of Beetle Elytral Cuticle, a Multicomponent Biomaterial. American Institute of Chemical Engineers, Annual meeting; Proteomic identification of serpin-1 isoforms in the caterpillar Manduca sexta. Arthropod Genomics Symposium, Kansas City, MO; Roles of dopachrome-conversion enzyme (DCE) genes (yellow) in Tribolium castaneum. Arthropod Genomics Symposium, Kansas City, MO; Function of two abundant proteins in hard cuticle of Tribolium castaneum. Entomological Society of America, Annual Meeting, Indianapolis, IN; Recombinant expression and analysis of hemolymph proteinase 16 from an insect, Manduca sexta. Entomological Society of America, Annual Meeting, Indianapolis, IN PARTICIPANTS: Michael Kanost - PI, Ramaswamy Krishnamoorth - coPI, Jayne Christen - GRA, Huaien Dai - GRA; Partner Organizations: NIH, NSF; Collaborators: Karl Kramer, Richard Beeman, Duy Hua, Kristin Michel; Training: training in biochemical research for three graduate students and four postdoctoral scientists TARGET AUDIENCES: Life scientists and educators. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge: Laccases are multicopper oxidases, whose functions have been investigated in plants and fungi, but little is known about their roles in insects. We purified recombinant forms of several multicopper oxidases from a moth, Manduca sexta, and a mosquito, Anopheles gambiae, and obtained new information about their enzymatic activities and substrate specificity. We also gained new knowledge about the regulation of expression of these enzymes in insects in different tissues and at different developmental stages. These results indicate that one laccase gene has an important function in oxidation of phenolic substrates that leads to tanning and hardening of the exoskeleton after each molt and that additional multicopper oxidase genes in insect genomes have other functions still to be discovered. We obtained new understanding at a molecular level of how proteins in insect hemolymph (glucan-binding proteins) interact with polysaccharides on the surface of microorganisms. Such interactions trigger activation of proteases in hemolymph, which form signaling pathways that activate immune responses. We gained new knowledge of the molecular composition of these pathways in Manduca sexta and their regulation by protease inhibitors. This research will lead to understanding of fundamental processes in insect biology, including the synthesis and structure of the exoskeleton and the molecular basis for insect immune responses. There is potential to identify new targets for use in biological or chemical control of insect populations. It will also develop fundamental understanding of molecules and biochemical processes common to humans and insects.
Publications
- Suwanchaichinda, C. and Kanost, M.R. (2009) The serpin gene family in Anopheles gambaie. Gene 442: 47-54.
- Dittmer, N.T., Gorman, M.J., and Kanost, M.R. (2009) Comparisons between an endogenous and recombinant laccase-like multicopper oxidase from the insect Manduca sexta. Insect Biochem. Mol. Biol. 39: 596-606.
- Arakane, Y., Lomakine, J., Beeman, R.W., Muthukrishnan, S., Gehrke, S.H., Kanost, M.R. and Kramer, K.J. (2009) Molecular and functional analyses of amino acid decarboxylases involved in cuticle tanning in Tribolium castaneum. J. Biol. Chem. 284: 16584-16594.
- An, C., Ishibashi, J., Ragan, E.J., Jiang, H., and Kanost, M.R. (2009) Functions of Manduca sexta hemolymph proteinases HP6 and HP8 in two innate immune pathways. J. Biol. Chem. 284: 19716-19726.
- Wang, X., Ribeiro. J.M.C., Broce, A.B., Wilkerson, M.J., and Kanost, M.R. (2009) An insight into the transcriptome and proteome of the salivary gland of the stable fly, Stomoxys calcitrans. Insect Biochem. Mol. Biol. 39: 607-614.
- Ragan, E.J., An, C., Jiang, H. And Kanost, M.R. (2009) Roles of hemolymph proteins in antimicrobial defences of Manduca sexta. In: Insect Infection and Immunity. (S. Reynolds and J. Rolff, eds.) Oxford University Press, pp. 34-48.
- Kanost, M.R. and Nardi, J.B (2009) Innate immune responses of Manduca sexta. In: Molecular Biology and Genetics of Lepidoptera. (M.R. Goldsmith and F. Marec, eds.) CRC Press, pp. 271-291.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The objectives of this project are: 1) Express and characterize recombinant laccases from a lepidopteran species, Manduca sexta; 2) Identify and characterize laccases in a mosquito, Anopheles gambiae; 3) Identify proteases in the pathway leading to activation of Manduca prophenoloxidase and study their regulation by serine protease inhibitors (serpins). Objective 2: We have focused on Manduca sexta hemolymph proteases HP6 and HP8, with goals of determining how these proteases are activated, identify their substrates in vivo, and which serpins regulate their activity. HP6 and HP8 are composed of an amino-terminal clip domain and a carboxyl-terminal proteinase domain. We expressed and purified recombinant proHP8 and a mutant of proHP8 with an activation site changed to facilitate zymogen activation by bovine factor Xa, using the Drosophila S2 cell expression system. We isolated a cDNA for a M. sexta homolog of Drosophila spatzle, and we found that M. sexta spatzle expression is upregulated after microbial challenge. We expressed and purfied recombinant pro-spatzle and demostrated that HP8 can cleave and activate recombinant pro-spatzle, producing a carboxyl-terminal fragment that is a disulfide-linked spatzle homodimer. Injection of spatzle into M. sexta larvae resulted in elevated mRNA levels for several antimicrobial peptide genes, including attacin, cecropin, lysozyme, gloverin, and moricin. Thus, we have identified a substrate for HP8 and found that proteolytically activated M. sexta spatzle can stimulate the synthesis of antimicrobial peptides. As our previous work demonstrated that HP6 activates proHP8, we are beginning to elucidate a cascade pathway for cytokine activation in this lepidopteran model. Objective 3: We expressed and purified recombinant Lac2A and 2B and determined the pH optima for oxidizing an artificial substrate (ABTS) and two physiologically relevant substrates (NBAD and NADA). We determined that Lac2A oxidizes four diphenols that we detected in larval and adult mosquitoes: dopa, dopamine, NBAD and NADA. We found that Lac2A also oxidizes a p-diphenol (hydroquinone), an activity that is characteristic of laccases but not another insect diphenoloxidase (tyrosinase). Preliminary data suggest that hydroquinone is a better substrate for Lac2A than the corresponding o-diphenol (catechol). We have generated homology models of Lac2A and 2B to help us to understand the differences in activity of these two enzymes. We have finished cloning full coding region cDNAs for all of the MCO genes, have cloned these cDNAs into the pFastBac vector and have begun making the recombinant viruses necessary for recombinant protein expression. We have generated antiserum specific to Lac3 and have started cloning cDNAs for expression of Lac4 and 5 antigens. We have been developing and optimizing methods for solubulizing laccases from insect tissues, immunohistochemistry, and recombinant enzyme purification. PARTICIPANTS: Michael Kanost: PI; Emily Ragan: GRA; Lucinda Sullivan: GRA; Jayne Christen: GRA Partner Organizations: NIH, NSF Collaborators: Karl Kramer, Richard Beeman, Duy Hua, Kristin Michel Training: training in biochemical research for three graduate students and four postdoctoral scientists TARGET AUDIENCES: Life scientists and educators PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This research will lead to understanding of fundamental processes in insect biology, including the synthesis and structure of the exoskeleton and the molecular basis for insect immune responses. There is potential to identify new targets for use in biological or chemical control of insect populations. It will also develop fundamental understanding of molecules and biochemical processes common to humans and insects
Publications
- Zhuang, S., Kelo. L., Nardi, J.B., and Kanost, M.R. (2008) Multiple alpha integrin subunits are involved in cell-mediated responses of the Manduca immune system. Dev. Comp. Immunol. 32: 365-379.
- Ameri, M., Wang, X., Wilkerson, M.J., Kanost, M.R., and Broce, A.B. (2008) An immunoglobulin binding protein (Antigen 5) of the stable fly (Diptera: Muscidae) salivary gland stimulates bovine immune responses. J. Med. Entomol. 45: 94-101.
- Gorman, M.J., Dittmer, N.T., Marshall, J.L., and Kanost, M.R. (2008) Characterization of the multicopper oxidase gene family in Anopheles gambaie. Insect Biochem. Mol. Biol. 38: 817-824.
- Yu, X.-Q. and Kanost, M.R. (2008) Activation of Lepidopteran insect innate immune responses by C-type immulectins. In: Animal lectins: A Functional View (H.A. Ahmed and G.R. Vasta, eds.) CRC Press (Taylor and Francis), pp. 383- 395.
- Kanost, M.R. and Gorman, M.G. (2008) Phenoloxidases in insect immunity. In: Insect Immunology (N. Beckage, ed.) Academic Press/Elsevier, San Diego, pp. 69-96.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: This project involves the study of the functions of two types of oxidative enzymes, phenoloxidase (PO) and laccase, in insects. Both types of enzymes contain copper ions in their active site and use molecular oxygen to oxidize phenolic compounds, but they differ in catalytic mechanism, substrate specificity, and tissue localization. PO is present in hemolymph and functions in an innate immune response, in which infection stimulates a serine protease cascade that ultimately results in specific cleavage and activation of a prophenoloxidase zymogen. Laccase research in insects has been focused on cuticle sclerotization, although we believe that insect laccases may have additional functions. We completed kinetic analyses of purified recombinant forms of Manduca sexta laccase-2 and natural laccase-2 isolated from Manduca cuticle. The recombinant enzymes and the endogenous enzyme had similar kinetic properties (for example, pH optima of 5 and Km values of approximately 1 mM for
NADA and NBAD); however, the endogenous enzyme had two to four fold higher activity. Typical Michaelis-Menten kinetics were observed when NADA was the substrate, but substrate inhibition occurred with NBAD, suggesting an alternative site on the enzyme for interaction with NBAD. assays with NBAD suggested possible substrate inhibition. Similar experiments were carried out with recombinant proteins for two isoforms of Anopheles gambiae laccase-2, which result from alternative exon splicing. The two isoforms have similar optimum pH (~5) and activity toward NBAD, but the A isoform is approximately 4-fold more active than the B isoform toward NADA as a substrate. We have obtained complete cDNA clones for A.gambiae laccases 3-5 and will begin characterizing their expression patterns and enzymatic propoerties. We characterized the expression pattern and tyrosine hydroxylation activity of two enzymes from M. sexta, PO and tyrosine hydroxylase (TH). PO is constitutively expressed by hemocytes
and is present in plasma, TH is upregulated in hemocytes and the fat body in response to an immune challenge and remains intracellular. Our data suggest that TH is required for cuticle tanning and immunity in M. sexta. We that TH is a major producer of the DOPA required for both cuticle tanning and immune-associated melanization.
PARTICIPANTS: PI: Michael Kanost; GRAs: Emily Ragan, Lucinda Sullivan, Jayne Christen; Partner Organizations: NIH, NSF; Collaborators: Karl Kramer, Richard Beeman, Duy Hua, Kristin Michel; Training: training in biochemical research for three graduate students and four postdoctoral scientists
TARGET AUDIENCES: Life scientists and educators
Impacts
This research will lead to understanding of fundamental processes in insect biology, including the synthesis and structure of the exoskeleton and the molecular basis for insect immune responses. It will also develop fundamental understanding of molecules and biochemical processes common to humans and insects.
Publications
- Waterhouse, R.M., Xi, Z.Y., Kriventseva, E., Meister, S., Alvarez, K.S., Bartholomay, L.C., Barillas-Mury, C., Bian, G., Blandin, S., Christensen, B.M., Dong, Y., Jiang, H., Kanost M.R., Koutsos, A.C., Levashina, E.A., Li, J., Ligoxygakis, P., MacCallum, R., Mayhew, G.F., Mendes, A., Michel, K., Osta, M., Paskewitz, S., Shin, S.W., Vlachou, D., Wang, L., Wei, W., Zheng, L., Zou, A., Severson, D.W., Raikhel, A.S., Kafatos, F.C., Dimopoulos, G., Zdobnov, E., and Christophides, G.K. (2007) Evolutionary dynamics of immune-related genes and pathways in disease vector mosquitoes. Science 316: 1738-1743.
- Zhuang, S., Kelo. L., Nardi, J.B., and Kanost, M.R. (2007) An integrin-tetraspanin interaction required for cellular innate immune responses of an insect, Manduca sexta. J. Biol. Chem. 282: 22563-22572.
- Zhuang, S., Kelo. L., Nardi, J.B., and Kanost, M.R. (2007) Neuroglian on hemocyte surfaces is involved in homophilic and heterophilic interactions of the innate immune system of Manduca sexta. Dev. Comp. Immunol. 31: 1159-1167.
- 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. J. Biol. Chem. 282: 11742-11749.
- Miyaji, T., Kouzuma, Y., Yaguchi, J., Matsumoto, R., Kanost, M.R., Kramer, K.J., Yonekura. M. (2007) Purification of a cysteine protease inhibitor from larval hemolymph of the tobacco hornworm (Manduca sexta) and functional expression of the recombinant protein. Insect Biochem. Molec. Biol. 37: 960-968.
- Zhuang, S., Kelo. L., Nardi, J.B., and Kanost, M.R. (2007) Multiple alpha integrin subunits are involved in cell-mediated responses of the Manduca immune system. Dev. Comp. Immunol. In press.
- Sotelo-Mundo, R.R., Lopez-Zavala, A.A., Garcia-Orozco, K.D., Arvizu-Flores, A.A., Velazquez-Contreras, E.F., Valenzuela-Soto, E.M., Rojo-Dominguez, A. And Kanost, M.R. (2007) The lysozyme from insect (Manduca sexta) is a cold-adapted enzyme. Protein and Peptide Letters 14:774-778.
- Gorman, M.J., An, C., and Kanost, M.R. (2007) Hydroxylation of tyrosine by phenoloxidase and tyrosine hydroxylase in Manduca sexta. Insect Biochem. Molec. Biol. 37: 1327-1337.
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Progress 01/01/06 to 12/31/06
Outputs
This project involves the study of the functions of two types of oxidative enzymes, phenoloxidase and laccase, in insects. Both types of enzymes contain copper ions in their active site and use molecular oxygen to oxidize phenolic compounds, but they differ in catalytic mechanism, substrate specificity, and tissue localization. PO is present in hemolymph and functions in an innate immune response, in which infection stimulates a serine protease cascade that ultimately results in specific cleavage and activation of a prophenoloxidase zymogen. Laccase research in insects has been focused on cuticle sclerotization, although we believe that insect laccases may have additional functions. The objectives of this project are: 1) Express and characterize recombinant laccases from a lepidopteran species, Manduca sexta; 2) Identify and characterize laccases in a mosquito, Anopheles gambiae; 3) Identify proteases in the pathway leading to activation of Manduca prophenoloxidase
and study their regulation by serine protease inhibitors (serpins). We purified recombinant forms of Manduca laccase-2 and natural laccase-2 isolated from Manduca cuticle. We demonstrated that laccase is not synthesized as a zymogen, and that all of the purified forms of laccase can hydrolyze the natural substrates N-acetyldopamine and beta-alanyldopamine. We purified recombinant proteins for two isoforms of Anopheles laccase-2, which result from alternative exon splicing and have begun to analyze their enzymatic properties and to understand the potential differences in their structure and activity through molecular modeling. We have defined pathways for activation of Manduca hemolymph proteases during immune responses. Hemolymph protease 14, which becomes active upon interaction with microbial surfaces and hemolymph pattern recognition proteins, activates hemolymph protease 21. Active hemolymph protease 21 then activates prophenoloxidase-activating protease-3, which in turn activates
prophenoloxidase. We have established a system for identifying the 12 splice variants of Manduca serpin-1 as a step toward determining which proteases each serpin isoform inhibits in vivo. We found that serpin-1K inhibits a chymotrypsin previously identified as a digestive enzyme in Manduca. We have shown that this same chymotrypsin is also expressed in hemocytes and is present in the hemolymph. We participated in analysis of the family of clip domain serine protease genes in two insect genomes, the honeybee Apis mellifera and the yellow fever mosquito Aedes aegypti and have found that A. mellifera has fewer such genes than the dipteran species that have been studied, whereas A. aegypti has a very large number of such genes, some with apparently complex domain structure. We analyzed the serpin gene family in the Anopheles gambiae genome and have experimentally determined sequences of most of the 18 identified A. gambiae serpins to aid in gene annotation and for producing recombinant
serpin proteins. Some of the serpin genes are expressed predominantly in larvae, whereas others are expressed predominantly in pupae and adults, suggesting stage-specific differences in the functions.
Impacts
This research will lead to understanding of fundamental processes in insect biology, including the synthesis and structure of the exoskeleton and the molecular basis for insect immune responses. It will also develop fundamental understanding of molecules and biochemical processes common to humans and insects
Publications
- Evans, J.D., Aronstein. K., Chen, Y.P., Hetru, C., Imler, J-L., Jiang, H., Kanost, M.R., Thompson, G., Zhou, Z. Hultmark, D. 2006. Immune pathways and defense mechanisms in honey bees, Apis mellifera. Insect Molecular Biology 15:645-656.
- Zou, Z., Lopez, D. Kanost, M.R., Evans, J. and Jiang, H. 2006. Comparative analysis of serine protease-related genes in the honey bee genome: possible involvement in embryonic development and innate immunity. Insect Molecular Biology 15:603-614..
- Michel, K., Suwanchaichinda, C., Morlais, I., Lambrechts, L., Cohuet, A., Awono-Ambene, P.H., Simard, F., Fontenille, D., Kanost, M.R. and Kafatos, F.C. 2006. Increased melanizing activity in An. gambiae due to SRPN2 depletion does not affect development of Plasmodium falciparum. Proc. Natl Acad Sci. USA. 103:16858-16863
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Progress 01/01/05 to 12/31/05
Outputs
Two types of phenoloxidases are important in insect physiology: tyrosinase, simply known as phenoloxidase (PO), and laccase. Both PO and laccase rely on bound copper ions to act as intermediate electron acceptors as they oxidize a phenolic substrate and reduce molecular oxygen, although they differ in details of catalytic mechanism, substrate specificity, and tissue localization. PO is present in hemolymph and functions in an innate immune response, in which infection stimulates a serine protease cascade that ultimately results in specific cleavage and activation of a prophenoloxidase zymogen. Laccase research in insects has been focused on cuticle sclerotization, although we believe that insect laccases may have additional functions. The objectives of this project are: 1) Express and characterize recombinant laccases from a lepidopteran species, Manduca sexta; 2) Identify and characterize laccases in a mosquito, Anopheles gambiae; 3) Identify proteases in the pathway
leading to activation of Manduca prophenoloxidase and study their regulation by serine protease inhibitors (serpins). We have produced recombinant forms of Manduca laccase-2, using a baculovirus expression system and have purified the recombinant proteins as well as natural laccase-1 isolated from Manduca cuticle. Experiments have been conducted to determine kinetic constants for the recombinant and natural laccases with N-acetyldopamine and beta-alanyldopamine as substrates. We have identified five laccase genes in the Anopheles genome and have cloned full length cDNAs encoding these enzymes. We constructed baculoviruses for recombinant expression of two isoforms of Anopheles laccase 2, which result from alternative exon splicing. RT-PCR experiments have been carried out to analyze expression patterns for the five Anopheles laccase genes during development and after blood-feeding. We have produced several Manduca hemolymph proteases as recombinant proteins, using baculovirus or
Drosophila S2 cell expression systems and developed methods to purify the proteins. These include prophenoloxidase activating protease-3, hemolymph proteases 1, 2, 6, 8, and 21. We have purified hemolymph protease 14 from Manduca plasma. Experiments with recombinant protease 1 indicate that it can stimulate activation of prophenoloxidase when mixed with Manduca plasma, suggesting that it takes part in the activation pathway. We have begun to characterize by 2D gel electrophoresis and mass spectrometry the compenents of a high molecular weight protein complex, which forms in Manduca hemolymph concurrent with activation of the prophenoloxidase cascade. This complex includes active phenoloxidase, immulectins, glucan-binding proteins, and serine protease homologs. A new Manduca serpin gene, serpin-7 was identified and its cDNA was cloned. Recominant forms of the serpin-1 isoforms generated by alternative exon splicing have been produced for testing their function as regulators of
proteases in the phenoloxidase activation cascade. U.S. Provisional Patent Application: Inhibitors of insect laccase, filed 07/26/05 by Kansas State University Research Foundation, Neal Dittmer, Karl Kramer, and Michael R. Kanost.
Impacts
This research will lead to understanding of fundamental processes in insect biology, including the synthesis and structure of the exoskeleton and the molecular basis for insect immune responses.
Publications
- Arkane, Y., Muthukrishnan, S., Beeman, R.W., Kanost, M.R., and Kramer, K.J. 2005. Laccase 2 is the phenoloxidase gene required for beetle cuticle tanning. Proc. Natl. Acad. Sci. USA, 102: 11337-11342.
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