In vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases from the Dengue vector mosquito Aedes aegypti.
In vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases from the Dengue vector mosquito Aedes aegypti.
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DOI：
10.1186/1471-2091-12-43
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发表时间：
2011-08-09
期刊：
影响因子：
--
通讯作者：
Miesfeld RL
中科院分区：
文献类型：
--
作者：
Rascón AA Jr;Gearin J;Isoe J;Miesfeld RL
主要的登革热病毒载体埃及伊蚊需要从血粉蛋白中获得的营养物质来完成生殖营养循环。虽然生物信息学分析的Ae。埃及中肠丝氨酸蛋白酶提供了进化的见解，但对这些消化酶的生物化学活性知之甚少。我们使用肽特异性抗体显示中肠丝氨酸蛋白酶表达为酶原前体，其在血液喂养后裂解为成熟形式。由于中肠蛋白质水平不足以直接从血液喂养的蚊子中纯化活性蛋白酶，我们工程化了编码异源肠激酶切割位点的重组蛋白，以允许产生四种中肠丝氨酸蛋白酶（AaET、AaLT、AaSPVI、AaSPVII）的真正成熟形式，用于酶动力学分析。显色胰蛋白酶底物BApNA的切割显示，AaET具有比牛胰蛋白酶高约30倍的催化效率（kcat/KM），并且比AaSPVI和AaSPVII高约2-3倍，然而，AaLT不切割BApNA。为了测量使用天然底物的蚊子中肠蛋白酶的酶活性，我们开发了基于白蛋白和血红蛋白蛋白的切割的定量切割测定。这些研究表明，重组AaLT酶确实具有催化活性，并以与AaET、AaSPVI和AaSPVII相当的效率切割白蛋白和血红蛋白。AaLT和AaSPVI成熟形式的结构建模表明AaLT与丝氨酸胶原酶最相似，而AaSPVI似乎是经典的胰蛋白酶。这些数据表明，含有异源肠激酶切割位点的重组丝氨酸蛋白酶的体外活化可用于研究生物学上重要的蚊子蛋白酶的酶动力学和底物切割特性。
The major Dengue virus vector Aedes aegypti requires nutrients obtained from blood meal proteins to complete the gonotrophic cycle. Although bioinformatic analyses of Ae. aegypti midgut serine proteases have provided evolutionary insights, very little is known about the biochemical activity of these digestive enzymes. We used peptide specific antibodies to show that midgut serine proteases are expressed as zymogen precursors, which are cleaved to the mature form after blood feeding. Since midgut protein levels are insufficient to purify active proteases directly from blood fed mosquitoes, we engineered recombinant proteins encoding a heterologous enterokinase cleavage site to permit generation of the bona fide mature form of four midgut serine proteases (AaET, AaLT, AaSPVI, AaSPVII) for enzyme kinetic analysis. Cleavage of the chromogenic trypsin substrate BApNA showed that AaET has a catalytic efficiency (kcat/KM) that is ~30 times higher than bovine trypsin, and ~2-3 times higher than AaSPVI and AaSPVII, however, AaLT does not cleave BApNA. To measure the enzyme activities of the mosquito midgut proteases using natural substrates, we developed a quantitative cleavage assay based on cleavage of albumin and hemoglobin proteins. These studies revealed that the recombinant AaLT enzyme was indeed catalytically active, and cleaved albumin and hemoglobin with equivalent efficiency to that of AaET, AaSPVI, and AaSPVII. Structural modeling of the AaLT and AaSPVI mature forms indicated that AaLT is most similar to serine collagenases, whereas AaSPVI appears to be a classic trypsin. These data show that in vitro activation of recombinant serine proteases containing a heterologous enterokinase cleavage site can be used to investigate enzyme kinetics and substrate cleavage properties of biologically important mosquito proteases.
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影响因子：
2.2
作者：
Brackney, Doug E.;Isoe, Jun;Black, W. C., IV;Zamora, Jorge;Foy, Brian D.;Miesfeld, Roger L.;Olson, Ken E.
通讯作者：
Olson, Ken E.
DOI：
10.1111/j.1432-1033.1985.tb09171.x
发表时间：
1985-01-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
LECROISEY, A;KEIL, B
通讯作者：
KEIL, B
影响因子：
3.9
作者：
ERLANGER, BF;COHEN, W;KOKOWSKY, N
通讯作者：
KOKOWSKY, N
DOI：
10.1016/s0097-8485(99)00044-3
发表时间：
2000-01-01
期刊：
COMPUTERS & CHEMISTRY
影响因子：
--
作者：
Cardozo, T;Batalov, S;Abagyan, R
通讯作者：
Abagyan, R
影响因子：
3.8
作者：
Bian, Guowu;Ralkhel, Alexander S.;Zhu, Jinsong
通讯作者：
Zhu, Jinsong