A zymogen form of masquerade-like serine proteinase homologue is cleaved during pro- phenoloxidase activation by Ca2+ in coleopteran and Tenebrio molitor larvae.
To elucidate the biochemical activation mechanism of the insect pro- phenoloxidase (pro-PO) system, we purified a 45-kDa protein to homogeneity from the hemolymph of Tenebrio molitor (mealworm) larvae, and cloned its cDNA. The overall structure of the 45-kDa protein is similar to Drosophila masquerade serine proteinase homologue, which is an essential component in Drosophila muscle development. This Tenebrio masquerade-like serine proteinase homologue (Tm- mas) contains a trypsin-like serine proteinase domain in the C-terminal region, except for the substitution of Ser to Gly at the active site triad, and a disulfide-knotted domain at the amino-terminal region. When the purified 45-kDa Tm- mas was incubated with CM-Toyopearl eluate solution containing pro-PO and other pro-PO activating factors, the resulting phenoloxidase ( PO) activity was shown to be independent of Ca2+. This suggests that the purified 45-kDa Tm- mas is an activated form of pro-PO activating factor. The55-kDa zymogen form of Tm- mas was detected in the hemolymph when PO activity was not evident. However, when Tenebrio hemolymph was incubated with Ca2+, a 79-kDa Tenebrio pro-PO and the 55-kDa zymogen Tm- mas converted to 76-kDa PO and 45-kDa Tm- mas, respectively, with detectable PO activity. Furthermore, when Tenebrio hemolymph was incubated with Ca2+ and beta-1,3-glucan, the conversion of pro-PO to PO and the 55-kDa zymogen Tm- mas to the 45-kDa protein, was faster than in the presence of Ca2+ only. These results suggest that the cleavage of the 55-kDa zymogen of Tm- mas by a limited proteolysis is necessary for PO activity, and the Tm- mas is a pro-PO activating cofactor.[1]References
- A zymogen form of masquerade-like serine proteinase homologue is cleaved during pro-phenoloxidase activation by Ca2+ in coleopteran and Tenebrio molitor larvae. Lee, K.Y., Zhang, R., Kim, M.S., Park, J.W., Park, H.Y., Kawabata, S., Lee, B.L. Eur. J. Biochem. (2002) [Pubmed]
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