title: Activity of fusion prophenoloxidase-GFP and its potential applications for innate immunity study.
abstract: Insect prophenoloxidase (PPO) is essential for physiological functions such as melanization of invading pathogens, wound healing and cuticle sclerotization. The insect PPO activation pathway is well understood. However, it is not very clear how PPO is released from hemocytes and how PPO takes part in cellular immunity. To begin to assess this, three Drosophila melanogaster PPO genes were separately fused with GFP at the C-terminus (rPPO-GFP) and were over-expressed in S2 cells. The results of staining and morphological observation show that rPPO-GFP expressed in S2 cells has green fluorescence and enzyme activity if Cu(2+) was added during transfection. Each rPPO-GFP has similar properties as the corresponding rPPO. However, cells with rPPO-GFP over-expressed are easier to trace without PO activation and staining. Further experiments show that rPPO1-GFP is cleaved and activated by Drosophila serine protease, and rPPO1-GFP binds to Micrococcus luteus and Beauveria bassiana spores as silkworm plasma PPO. The above research indicates that the GFP-tag has no influence on the fusion enzyme activation and PPO-involved innate immunity action in vitro. Thus, rPPO-GFP may be a convenient tool for innate immunity study in the future if it can be expressed in vivo.
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People's Republic of China.
authors.forename: Bing
authors.initials: B
authors.lastname: Yang
authors.forename: Anrui
authors.initials: A
authors.lastname: Lu
authors.forename: Qin
authors.initials: Q
authors.lastname: Peng
authors.forename: Qing-Zhi
authors.initials: QZ
authors.lastname: Ling
authors.forename: Erjun
authors.initials: E
authors.lastname: Ling
mesh_terms: D000818:Animals; D052982:Beauveria / Q000276:immunology; D012831:Bombyx / Q000276:immunology / Q000382:microbiology; D004156:Catechol Oxidase / Q000096:biosynthesis* / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism; D029721:Drosophila Proteins / Q000096:biosynthesis* / Q000235:genetics; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics / Q000276:immunology*; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D007113:Immunity, Innate; D007814:Larva / Q000276:immunology / Q000382:microbiology; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008837:Micrococcus / Q000276:immunology; D008969:Molecular Sequence Data; D011485:Protein Binding; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D057057:Serine Proteases / Q000378:metabolism; D013171:Spores, Bacterial / Q000276:immunology
chemical_list: D029721:Drosophila Proteins; D004792:Enzyme Precursors; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins; D003300:Copper; C038451:pro-phenoloxidase; D004156:Catechol Oxidase; D057057:Serine Proteases