| dc.description.abstract | Melanization is insect acute-phase defense response to invading bacteria, fungi, protozoa, and endoparasitoids. Phenoloxidase (PO) participates in multiple steps of the melenization reaction. Produced as an inactive zymogen, prophenoloxidase (proPO), it is activated by a serine proteinase cascade upon recognition of the invaders. In Manduca sexta, the final step involves a proPO activating protease and a serine proteinase homolog (SPH) complex. After the proPO is activated, its activity is tightly regulated. I purified a ? -1,3-glucan Recognition Protein 2 (?GRP2) from Manduca cuticle extract. ?GRP2 specifically binds to laminarin, a soluble form of ? -1,3-glucan. This binding is linked with proPO activation. Based on this and other evidence, we conclude that ?GRP2 functions as a pattern recognition receptor for proPO activation in Manduca. Using recombinant proSPH1 and proSPH2 as substrates, I attempted to purify their activating enzymes. I observed cleavage of proSPH1 and proSPH2 by plasma fractions, and detected PO cofactor activity from the processed SPHs. More importantly, I found that the copresence of SPH1 and SPH2 is necessary for manifesting the cofactor activity. I expressed a Manduca PO inhibitor in E. coli and insect cells. The recombinant peptides moderately inhibit PO. My effort to isolate the natural inhibitor from the hemolymph was unsuccessful. Instead, I found a low molecular weight chemical with strong inhibitory activity to Manduca PO and mushroom tyrosinase. A clip-domain SPH, Vn50, from the endoparasitoid wasp Cotesia rubecula, venom, was found to inhibit PO from its host insect Pieris rapae. I used Manduca proPO system to study its inhibitory mechanism. I found that Vn50 down-regulated proPO activation by disrupting the protein-protein interactions among proPO, PAP, and SPH1/SPH2. | |
