Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China.
Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China.
Fish Shellfish Immunol. 2014 Mar;37(1):115-21. doi: 10.1016/j.fsi.2013.12.011. Epub 2013 Dec 27.
Serine proteinase inhibitors represent an expanding superfamily of endogenous inhibitors that are regulate proteolytic events and involved in a variety of physiological and immunological processes. A five-domain Kazal-type serine proteinase inhibitor (poKSPI) was identified and characterized from pearl oyster Pinctada fucata based on expressed sequence tag (EST) analysis. The full-length cDNA was 737 bp with an open reading frame (ORF) 660 bp encoding a 219 amino acid protein a theoretical molecular weight (Mw) of 23.3 kDa and an isoelectric point (pI) of 8.40. A putative signal peptide of 19 amino acid residues and five tandem Kazal domains were identified. Four of the Kazal domains had the highly conserved motif sequences with six cysteine residues responsible for the formation of disulfide bridges. The deduced amino acid sequence of the poKSPI shared high homology with KSPIs from Hirudo medicinalis. The poKSPI mRNA could be detected in all examined tissues, the expression level of the poKSPI mRNA was the highest in mantle and gonad, while the lowest in haemocyte and intestine. After LPS challenge, the expression level of the poKSPI mRNA in digestive gland was significantly up-regulated at 4 h post-challenge and reached the peak at 12 h post-challenge, which was 4.23-fold higher than control group; the expression level of the poKSPI mRNA in gill was also significantly up-regulated at 8 and 12 h post-challenge, which were 4.48 and 2.26-fold higher than control group. After Vibrio alginolyticus challenge, the expression levels of the poKSPI mRNA in digestive gland were significantly up-regulated at 8, 12, 48 and 72 h post-challenge, which were 1.70, 1.79, 3.89 and 5.69-fold higher than control group, respectively; the expression level of the poKSPI mRNA in gill was significantly up-regulated at 24 h post-challenge, which was 5.30-fold higher than control group. The recombinant poKSPI protein could inhibit chymotrypsin and trypsin activities in dose-dependent manner, when the ratios of rpoKSPI to chymotrypsin and trypsin were 36:1 and 72:1, respectively, the proteinase activities of chymotrypsin and trypsin could be almost completely inhibited, but the rpoKSPI could not inhibit subtilisin.
丝氨酸蛋白酶抑制剂是一类内源性抑制剂超家族，可调节蛋白水解事件，并参与多种生理和免疫过程。本研究基于表达序列标签（EST）分析，从珍珠贝 Pinctada fucata 中鉴定并表征了一种具有五个结构域的 Kazal 型丝氨酸蛋白酶抑制剂（poKSPI）。全长 cDNA 为 737bp，开放阅读框（ORF）660bp，编码 219 个氨基酸的蛋白质，理论分子量（Mw）为 23.3kDa，等电点（pI）为 8.40。鉴定出一个推定的 19 个氨基酸残基的信号肽和五个串联的 Kazal 结构域。其中四个 Kazal 结构域具有高度保守的基序序列，6 个半胱氨酸残基负责形成二硫键。poKSPI 的推导氨基酸序列与来自医用水蛭的 KSPIs 具有高度同源性。在所有检测的组织中均能检测到 poKSPI mRNA，在套膜和性腺中 poKSPI mRNA 的表达水平最高，在血细胞和肠中最低。在 LPS 刺激后，poKSPI mRNA 在消化腺中的表达水平在攻毒后 4h 显著上调，并在攻毒后 12h 达到峰值，比对照组高 4.23 倍；poKSPI mRNA 在鳃中的表达水平也在攻毒后 8 和 12h 显著上调，分别比对照组高 4.48 和 2.26 倍。在哈维弧菌刺激后，poKSPI mRNA 在消化腺中的表达水平在攻毒后 8、12、48 和 72h 显著上调，分别比对照组高 1.70、1.79、3.89 和 5.69 倍；poKSPI mRNA 在鳃中的表达水平在攻毒后 24h 显著上调，比对照组高 5.30 倍。重组 poKSPI 蛋白能以剂量依赖的方式抑制胰凝乳蛋白酶和胰蛋白酶的活性，当 rpoKSPI 与胰凝乳蛋白酶和胰蛋白酶的比例分别为 36:1 和 72:1 时，胰凝乳蛋白酶和胰蛋白酶的酶活性几乎可以完全被抑制，但 rpoKSPI 不能抑制枯草杆菌蛋白酶。
