The activation of pro-phenol oxidase is an important physiological process involved in defense mechanisms in insects. These include: cuticle sclerotization, wound healing, encapsulation and melanization of foreign materials, and generation of cytotoxic molecules. Knowledge of the activation process from model insects such as Manduca sexta will be useful for studying similar systems in blood-sucking insects that transmit human, diseases. Goals of this research are to characterize the pro-phenol oxidase activating proteinase and to study mechanisms that regulate the enzyme in M. sexta.
The specific aims of the project are: 1. Purification and characterization of the pro-phenol oxidase activating proteinase. The activating proteinase will be purified from both hemolymph and cuticle. Amino acid sequence and other biochemical characteristics of the enzyme will be studied. Enzymatic properties of the proteinase with and without a cofactor, named factor A, will be investigated to understand the activation mechanism of pro-phenol oxidase. 2. Molecular cloning and expression of the pro-phenol oxidase activating proteinase. With the amino acid sequence information and antiserum to the activating proteinase available, its cDNA will be cloned by screening M. sexta hemocyte, fat body or epidermis cDNA libraries. The proteinase will be expressed as a recombinant protein in its zymogen form for a study of upstream activators and eventual reconstitution of the pro-phenol oxidase activating system. Site, time, level, and inducibility of expression of the enzyme in M. sexta will be investigated. 3. Regulation of the pro-phenol oxidase activating proteinase by serine proteinase inhibitors. The inhibition reactions of the proteinase by M. sexta serpins and Kunitz-type trypsin inhibitors will be investigated to understand the physiological regulation of the enzyme. 4. Purification and characterization of factor A. Factor A which alters the activity and specificity of pro-phenol oxidase activating proteinase will be purified from cuticle and hemolymph. Its interactions with the activating proteinase and pro-phenol oxidase will be characterized. This research will contribute to our understanding of the biochemical process of pro-phenol oxidase activation, and will generate useful molecular probes for studying similar systems in insect vectors of human diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM058634-01
Application #
2730490
Study Section
Special Emphasis Panel (ZRG5-TMP (01))
Project Start
1999-03-01
Project End
2004-02-29
Budget Start
1999-03-01
Budget End
2000-02-29
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Kansas State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
City
Manhattan
State
KS
Country
United States
Zip Code
66506
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Cao, Xiaolong; Jiang, Haobo (2018) Building a platform for predicting functions of serine protease-related proteins in Drosophila melanogaster and other insects. Insect Biochem Mol Biol 103:53-69
Yang, Fan; Wang, Yang; Sumathipala, Niranji et al. (2018) Manduca sexta serpin-12 controls the prophenoloxidase activation system in larval hemolymph. Insect Biochem Mol Biol 99:27-36
Feng, Congjing; Zhao, Ya; Chen, Kangkang et al. (2018) Clip domain prophenoloxidase activating protease is required for Ostrinia furnacalis Guenée to defend against bacterial infection. Dev Comp Immunol 87:204-215
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Cao, Xiaolong; Gulati, Mansi; Jiang, Haobo (2017) Serine protease-related proteins in the malaria mosquito, Anopheles gambiae. Insect Biochem Mol Biol 88:48-62
He, Xuesong; Cao, Xiaolong; He, Yan et al. (2017) Hemolymph proteins of Anopheles gambiae larvae infected by Escherichia coli. Dev Comp Immunol 74:110-124
He, Yan; Wang, Yang; Zhao, Picheng et al. (2017) Serpin-9 and -13 regulate hemolymph proteases during immune responses of Manduca sexta. Insect Biochem Mol Biol 90:71-81
Cao, Xiaolong; Jiang, Haobo (2017) An analysis of 67 RNA-seq datasets from various tissues at different stages of a model insect, Manduca sexta. BMC Genomics 18:796
Wang, Yang; Jiang, Haobo (2017) Prophenoloxidase activation and antimicrobial peptide expression induced by the recombinant microbe binding protein of Manduca sexta. Insect Biochem Mol Biol 83:35-43

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