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 β‐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.