Prophenoloxidase (proPO) activation in Manduca sexta: an analysis of molecular interactions among proPO, proPO-activating proteinase-3, and a cofactor.
Prophenoloxidase (proPO) activation in Manduca sexta: an analysis of molecular interactions among proPO, proPO-activating proteinase-3, and a cofactor.
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DOI：
10.1016/j.ibmb.2004.03.008
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发表时间：
2004-08
影响因子：
3.8
通讯作者：
Yang Wang;Haobo Jiang
中科院分区：
文献类型：
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作者：
Yang Wang;Haobo Jiang
酚氧化酶原（proPO）的蛋白水解活化是昆虫抵抗病原体和寄生虫感染的免疫系统的组成部分。该反应由烟草天蛾Manduca sexta中的proPO活化蛋白酶（PAP）及其辅因子介导（Proc. Natl. Acad. Sci. USA 95（1998）12220; J. Biol. Chem. 33（2003）1049）。辅因子由两种丝氨酸蛋白酶同系物（SPH）组成，其与免疫球蛋白-2（一种结合脂多糖的钙依赖性凝集素）缔合（Insect Biochem. Mol. 33（2003）197）。为了了解SPH-1和SPH-2在proPO活化中的辅助作用，我们开始研究proPO、PAP-3和蛋白酶样蛋白之间的分子相互作用。M.用羟基磷灰石、凝胶过滤、凝集素亲和层析和离子交换层析等方法，从包皮环切瘤血淋巴中分离纯化了sexta SPH-1和SPH-2。它们以平均分子量约为790 kDa的非共价低聚物形式存在。MALDI-TOF质谱指纹图谱分析显示SPH-1在Asp 85之前有一个新的切割位点。PAP辅因子并未显着改变PAP-3对合成底物乙酰基-Ile-Glu-Ala-Arg-p-硝基苯胺的米氏常数（KM）或kcatof，但极大地增强了PAP-3对proPO的激活。测定了proPO的表观Km值为9.4 μg/ml，接近其在幼虫血淋巴中的估计浓度。在存在过量proPO和设定量的PAP-3的情况下，随着添加更多SPH，检测到酚氧化酶（PO）活性水平增加。当PAP-3与SPH的摩尔比为1：1.4时，发生最大proPO活化的一半。凝胶过滤实验表明，proPO，PAP-3，和辅因子形成三元复合物。
Proteolytic activation of prophenoloxidase (proPO) is an integral part of the insect immune system against pathogen and parasite infection. This reaction is mediated by a proPO-activating proteinase (PAP) and its cofactor in the tobacco hornworm, Manduca sexta (Proc. Natl. Acad. Sci. USA 95 (1998) 12220; J. Biol. Chem. 278 (2003) 3552; Insect Biochem. Mol. Biol. 33 (2003) 1049). The cofactor consists of two serine proteinase homologs (SPHs), which associate with immulectin-2, a calcium-dependent lectin that binds to lipopolysaccharide (Insect Biochem. Mol. Biol. 33 (2003) 197). In order to understand the auxiliary effect of SPH-1 and SPH-2 in proPO activation, we started to investigate the molecular interactions among proPO, PAP-3, and the proteinase-like proteins. M. sexta SPH-1 and SPH-2 were purified from hemolymph of prepupae by hydroxylapatite, gel filtration, lectin-affinity, and ion exchange chromatography. They existed as non-covalent oligomers with an average molecular mass of about 790 kDa. MALDI-TOF mass fingerprint analysis revealed a new cleavage site in SPH-1 before Asp85. The PAP cofactor did not significantly alter Michaelis constant (KM) or kcatof PAP-3 towards a synthetic substrate, acetyl-Ile-Glu-Ala-Arg-p-nitroanilide, but greatly enhanced proPO activation by PAP-3. The apparent KMfor proPO was determined to be about 9.4 μg/ml, close to its estimated concentration in larval hemolymph. In the presence of excess proPO and a set amount of PAP-3, increasing levels of phenoloxidase (PO) activity were detected as more SPHs were added. Half of the maximum proPO activation occurred when the molar ratio of PAP-3 to SPH was 1:1.4. Gel filtration experiments suggested that proPO, PAP-3, and the cofactor formed a ternary complex.