Activity of fusion prophenoloxidase-GFP and its potential applications for innate immunity study.

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  • Author(s): Yang B;Yang B; Lu A; Peng Q; Ling QZ; Ling E
  • Source:
    PloS one [PLoS One] 2013 May 23; Vol. 8 (5), pp. e64106. Date of Electronic Publication: 2013 May 23 (Print Publication: 2013).
  • Publication Type:
    Journal Article; Research Support, Non-U.S. Gov't
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: Electronic-Print Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE
    • Publication Information:
      Original Publication: San Francisco, CA : Public Library of Science
    • Subject Terms:
      Immunity, Innate*; Catechol Oxidase/*biosynthesis ; Drosophila Proteins/*biosynthesis ; Drosophila melanogaster/*immunology ; Enzyme Precursors/*biosynthesis ; Recombinant Fusion Proteins/*biosynthesis; Animals ; Beauveria/immunology ; Bombyx/immunology ; Bombyx/microbiology ; Catechol Oxidase/genetics ; Cell Line ; Cloning, Molecular ; Copper/metabolism ; Drosophila Proteins/genetics ; Drosophila melanogaster/enzymology ; Drosophila melanogaster/genetics ; Enzyme Precursors/genetics ; Green Fluorescent Proteins/biosynthesis ; Green Fluorescent Proteins/genetics ; Larva/immunology ; Larva/microbiology ; Luminescent Proteins/biosynthesis ; Luminescent Proteins/genetics ; Micrococcus/immunology ; Molecular Sequence Data ; Protein Binding ; Proteolysis ; Recombinant Fusion Proteins/genetics ; Serine Proteases/metabolism ; Spores, Bacterial/immunology
    • 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.
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    • Molecular Sequence:
      GENBANK AAF57775; ADQ43426
    • Accession Number:
      0 (Drosophila Proteins)
      0 (Enzyme Precursors)
      0 (Luminescent Proteins)
      0 (Recombinant Fusion Proteins)
      0 (fluorescent protein 583)
      147336-22-9 (Green Fluorescent Proteins)
      789U1901C5 (Copper)
      EC 1.10.3.- (pro-phenoloxidase)
      EC 1.10.3.1 (Catechol Oxidase)
      EC 3.4.- (Serine Proteases)
    • Publication Date:
      Date Created: 20130530 Date Completed: 20140107 Latest Revision: 20240313
    • Publication Date:
      20240313
    • Accession Number:
      PMC3662757
    • Accession Number:
      10.1371/journal.pone.0064106
    • Accession Number:
      23717543