A zymogen form of masquerade-like serine proteinase homologue is cleaved during pro-phenoloxidase activation by Ca2+ in coleopteran and Tenebrio molitor larvae
A zymogen form of masquerade-like serine proteinase homologue is cleaved during pro-phenoloxidase activation by Ca2+ in coleopteran and Tenebrio molitor larvae
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DOI：
10.1046/j.1432-1033.2002.03155.x
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发表时间：
2002-09-01
期刊：
影响因子：
--
通讯作者：
Lee, BL
中科院分区：
文献类型：
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作者：
Lee, KY;Zhang, R;Lee, BL
为了阐明昆虫酚氧化酶原（pro-PO）系统的生化激活机制，我们从黄粉虫幼虫血淋巴中纯化了一个分子量为45 kDa的蛋白，并克隆了其cDNA。45-kDa蛋白的整体结构类似于果蝇伪装丝氨酸蛋白酶同源物，其是果蝇肌肉发育的重要组成部分。这种拟粉虫伪装样丝氨酸蛋白酶同源物（Tm-mas）在C-末端区域含有胰蛋白酶-埃克丝氨酸蛋白酶结构域，除了在活性位点三联体处Ser被Gly取代之外，在氨基末端区域含有二硫键结结构域。当纯化的45-kDa Tm-mas与含有pro-PO和其他pro-PO激活因子的CM-Toyopolysaccharide溶液孵育时，所得到的酚氧化酶（PO）活性显示不依赖于Ca ~（2+）。这表明纯化的45-kDa Tm-mas是pro-PO活化因子的活化形式。当PO活性不明显时，在血淋巴中检测到55-kDa酶原形式的Tm-mas。然而，当用Ca 2+孵育黄粉虫血淋巴时，79-kDa的黄粉虫pro-PO和55-kDa的酶原Tm-mas分别转化为76-kDa的PO和45-kDa的Tm-mas，具有可检测的PO活性。此外，当将粉虫血淋巴与Ca 2+和β-1，3-葡聚糖一起孵育时，PO前体转化为PO和55-kDa酶原Tm-mas转化为45-kDa蛋白质的速度比仅存在Ca 2+时更快。这些结果表明，通过有限的蛋白水解的55 kDa酶原的Tm-mas的切割是必要的PO活性，和Tm-mas是一个pro-PO激活辅因子。
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-Eke 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.