A masquerade-like serine proteinase homologue is necessary for phenoloxidase activity in the coleopteran insect, Holotrichia diomphalia larvae
A masquerade-like serine proteinase homologue is necessary for phenoloxidase activity in the coleopteran insect, Holotrichia diomphalia larvae
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DOI：
10.1046/j.1432-1327.2000.01695.x
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发表时间：
2000-10-01
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：
Kwon, TH;Kim, MS;Lee, BL
在此之前，我们报道了在n端编码有二硫结基序和c端有胰蛋白酶样催化结构域的丝氨酸蛋白酶群成员的cDNA的分子克隆[Lee， S.Y, Cho， M.Y, Hyun, j.h., Lee， K.M., Homma， K.I, Natori， S., Kawabata， S.I, Iwanaga， S. & Lee， B.L.(1998)]。[j].中国生物医学工程学报，2016,33(2):444 - 444。ppaf - 1通过有限的蛋白水解直接参与前酚氧化酶（pro-PO）的激活，其整体结构与果蝇的东部丝氨酸蛋白酶高度相似，后者是正常胚胎发育过程中形成模式所必需的丝氨酸蛋白酶酶原。在这里，我们报道了另一种45 kda的新型PPAF的cDNA的纯化和分子克隆。该基因编码由415个氨基酸残基组成的丝氨酸蛋白酶同源物，分子量为45 256 Da。45 kda蛋白的整体结构类似于masquerade蛋白，masquerade是一种在果蝇胚胎发生、幼虫和蛹发育过程中表达的丝氨酸蛋白酶同源物。45kda蛋白在c端含有一个胰蛋白酶样丝氨酸蛋白酶结构域，除了活性位点的丝氨酸取代为甘氨酸外，在n端有一个二硫结结构域。从另一种鞘翅目黄斑蝶的幼虫cDNA文库中也克隆出了一个高度相似的45 kda丝氨酸蛋白酶同源物。通过体外重组实验，我们发现纯化的45 kda丝氨酸蛋白酶同源物、纯化的活性PPAF-I和纯化的pro-PO是在Holotrichia pro-PO体系中表达酚氧化酶活性所必需的。然而，与ppaf - 1或45-kDa蛋白孵育的亲po，没有观察到酚氧化酶活性。有趣的是，当45-kDa蛋白与ppaf - 1和pro-PO在Ca2+不存在的情况下孵育时，45-kDa蛋白被裂解为35-kDa蛋白。RNA印迹杂交发现，大肠杆菌攻毒后，Holotrichia血淋巴中45-kDa蛋白的表达增加。
Previously, we reported the molecular cloning of cDNA for the prophenoloxidase activating factor-I (PPAF-I) that encoded a member of the serine proteinase group with a disulfide-knotted motif at the N-terminus and a trypsin-like catalytic domain at the C-terminus [Lee, S.Y., Cho, M.Y., Hyun, J.H., Lee, K.M., Homma, K.I., Natori, S., Kawabata, S.I., Iwanaga, S. & Lee, B.L. (1998) Eur. J. Biochem. 257, 615-621]. PPAF-I is directly involved in the activation of pro-phenoloxidase (pro-PO) by limited proteolysis and the overall structure is highly similar to that of Drosophila easter serine protease, an essential serine protease zymogen for pattern formation in normal embryonic development. Here, we report purification and molecular cloning of cDNA for another 45-kDa novel PPAF from the hemocyte lysate of Holotrichia diomphalia larvae. The gene encodes a serine proteinase homologue consisting of 415 amino-acid residues with a molecular mass of 45 256 Da. The overall structure of the 45-kDa protein is similar to that of masquerade, a serine proteinase homologue expressed during embryogenesis, larval, and pupal development in Drosophila melanogaster. The 45-kDa protein contained a trypsin-like serine proteinase domain at the C-terminus, except for the substitution of Ser of the active site triad to Gly and had a disulfide-knotted domain at the N-terminus. A highly similar 45-kDa serine proteinase homologue was also cloned from the larval cDNA library of another coleopteran, Tenebrio molitor. By in vitro reconstitution experiments, we found that the purified 45-kDa serine proteinase homologue, the purified active PPAF-I and the purified pro-PO were necessary for expressing phenoloxidase activity in the Holotrichia pro-PO system. However, incubation of pro-PO with either PPAF-I or 45-kDa protein, no phenoloxidase activity was observed. Interestingly, when the 45-kDa protein was incubated with PPAF-I and pro-PO in the absence, but not in the presence of Ca2+, the 45-kDa protein was cleaved to a 35-kDa protein. RNA blot hybridization revealed that expression of the 45-kDa protein was increased in the Holotrichia hemolymph after Escherichia coli challenge.