Structure and function of recombinant cobra venom factor
Structure and function of recombinant cobra venom factor
复制标题
DOI：
10.1074/jbc.m403196200
复制
发表时间：
2004-07-16
影响因子：
4.8
通讯作者：
Vogel, CW
中科院分区：
文献类型：
--
作者：
Kock, MA;Hew, BE;Vogel, CW
眼镜蛇毒因子（CVF）是来自眼镜蛇毒的补体激活蛋白。它是补体成分C3的结构和功能类似物。CVF在功能上类似于C3的活化形式C3b。像C3 B一样，CVF结合因子B，其随后被因子D裂解以形成双分子复合物CVF，B B。CVF，Bb是C3/C5转化酶，其裂解补体组分C3和C5。CVF是一种三链蛋白，其结构类似于C3b降解产物C3c，其不能形成C3/C5转化酶。C3和CVF都是以单链前原蛋白的形式合成的。本研究报道了在两种昆虫细胞表达系统（杆状病毒感染的Sf9草地贪夜蛾细胞和稳定转染的S2果蝇细胞）中重组表达CVF前体。在这两种表达系统中，pro-CVF最初合成为单链pro-CVF分子，随后经蛋白水解加工成结构上类似于C3的双链形式的pro-CVF。C3样形式的pro-CVF可以进一步蛋白水解加工成另一种结构上类似于C3b的双链形式的pro-CVF。出乎意料的是，所有三种形式的前CVF都表现出成熟的天然CVF的功能活性。重组前CVF支持因子D和Mg 2+存在下因子B的活化，并像天然CVF一样消耗血清补体活性。双分子转化酶pro-CVF，Bb表现出C3裂解和C5裂解活性。pro-CVF和所得C3/C5转化酶的活性与CVF和CVF，Bb转化酶不可区分。重组产生活性形式的前CVF的能力确保了用于补体消耗的重要研究试剂的持续可用性，因为作为天然CVF来源的眼镜蛇毒液将越来越难以获得，因为印度眼镜蛇在濒危物种名单上。重组表达pro-CVF的实验系统对于描述CVF的结构和功能关系及其与C3的差异以及产生具有CVF样功能的人C3衍生物用于治疗性补体消耗（"人源化CVF"）的研究也是非常有价值的。
Cobra venom factor (CVF) is the complement-activating protein from cobra venom. It is a structural and functional analog of complement component C3. CVF functionally resembles C3b, the activated form of C3. Like C3b, CVF binds factor B, which is subsequently cleaved by factor D to form the bimolecular complex CVF, Bb. CVF, Bb is a C3/C5 convertase that cleaves both complement components C3 and C5. CVF is a three-chain protein that structurally resembles the C3b degradation product C3c, which is unable to form a C3/C5 convertase. Both C3 and CVF are synthesized as single-chain prepro-proteins. This study reports the recombinant expression of pro-CVF in two insect cell expression systems (baculovirus-infected Sf9 Spodoptera frugiperda cells and stably transfected S2 Drosophila melanogaster cells). In both expression systems pro-CVF is synthesized initially as a single-chain pro-CVF molecule that is subsequently proteolytically processed into a two-chain form of pro-CVF that structurally resembles C3. The C3-like form of pro-CVF can be further proteolytically processed into another two-chain form of pro-CVF that structurally resembles C3b. Unexpectedly, all three forms of pro-CVF exhibit functional activity of mature, natural CVF. Recombinant pro-CVF supports the activation of factor B in the presence of factor D and Mg2+ and depletes serum complement activity like natural CVF. The bimolecular convertase pro-CVF, Bb exhibits both C3 cleaving and C5 cleaving activity. The activity of pro-CVF and the resulting C3/C5 convertase is indistinguishable from CVF and the CVF, Bb convertase. The ability to produce active forms of pro-CVF recombinantly ensures the continued availability of an important research reagent for complement depletion because cobra venom as the source for natural CVF will be increasingly difficult to obtain as the Indian cobra is on the list of endangered species. Experimental systems to express pro-CVF recombinantly will also be invaluable for studies to delineate the structure and function relationship of CVF and its differences from C3 as well as to generate human C3 derivatives with CVF-like function for therapeutic complement depletion ("humanized CVF").