Serine protease P-IIc is responsible for the digestion of yolk proteins at the late stage of silkworm embryogenesis
Serine protease P-IIc is responsible for the digestion of yolk proteins at the late stage of silkworm embryogenesis
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丝氨酸蛋白酶 P-IIc 负责蚕胚胎发生后期卵黄蛋白的消化
DOI：
10.1016/j.ibmb.2016.03.003
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发表时间：
2016
影响因子：
3.8
通讯作者：
Zhao Ping
中科院分区：
文献类型：
--
作者：
Wang D;an;Zhang Yan;Dong Zhaoming;Guo Pengchao;Ma Sanyuan;Guo Kaiyu;Xia Qingyou;Zhao Ping
在家蚕中，卵黄蛋白包括卵黄蛋白、卵特异性蛋白和30 K蛋白，这些蛋白在胚胎发育过程中被严格调节的内源性蛋白酶依次降解。虽然这一过程已被广泛研究，但对家蚕胚胎发育最后两天卵黄蛋白降解的认识仍存在空白。在本研究中，我们分离并纯化了肠道丝氨酸蛋白酶P-IIc，其在25 °C和pH 11下表现出最佳活性。半定量RT-PCR结合Western blotting结果表明，P-IIc在胚胎发育的最后2天在肠道中表达活跃，并有显著的积累。天然卵黄蛋白与P-II体外孵育时，卵黄蛋白和ESP被选择性降解。P-IIc还表现出对30 K蛋白的活性，如通过BmLP 1的快速和完全消化以及BmLP 2和BmLP 3的部分消化所证明的。此外，RNAi敲低家蚕胚胎中的P-IIc显著降低胚胎10天残留卵黄蛋白的降解速率。综上所述，我们的研究结果表明，P-IIc代表了一种具有相对广泛的底物特异性的胚胎肠道蛋白酶，它在家蚕胚胎发育后期的卵黄蛋白降解中起着重要作用。
In silkworms, yolk proteins comprise vitellin, egg-specific protein and 30K proteins, which are sequentially degraded by endogenous proteases strictly regulated during embryogenesis. Although the process has been extensively investigated, there is still a gap in the knowledge about the degradation of silkworm yolk proteins on the last two days of embryonic development. In the present study, we isolated and purified a gut serine protease P-IIc, which demonstrated optimal activity at 25 °C and pH 11. Semi-quantitative RT-PCR combined with western blotting showed that P-IIc was actively expressed and significantly accumulated in the gut on the last two days of embryogenesis. When natural yolk proteins were incubated with P-IIcin vitro, vitellin and ESP were selectively degraded. P-IIc also demonstrated activity towards 30K proteins as evidenced by rapid and complete digestion of BmLP1 and partial digestion of BmLP2 and BmLP3. Furthermore, RNAi knockdown of P-IIc in silkworm embryos significantly reduced the degradation rate of residual yolk proteins on embryonic day 10. Taken together, our results indicate that P-IIc represents an embryonic gut protease with a relatively broad substrate specificity, which plays an important role in the degradation of yolk proteins at the late stage of silkworm embryogenesis.