Profiling the enzymatic properties and inhibition of human complement factor B
Profiling the enzymatic properties and inhibition of human complement factor B
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DOI：
10.1074/jbc.m705646200
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发表时间：
2007-11-30
影响因子：
4.8
通讯作者：
Fairlie, David P.
中科院分区：
文献类型：
--
作者：
Le, Giang Thanh;Abbenante, Giovanni;Fairlie, David P.
人补体因子B是激活补体介导免疫的替代途径的C3转化酶的关键催化成分。虽然因子B本身是一种丝氨酸蛋白酶，但它作为一种无活性的酶原在人血清中循环，并且仅存在于因子B的无活性状态和各种片段的晶体结构。为了更深入地了解因子B的催化功能和性质，我们使用了4- 15残基肽的短对硝基苯胺衍生物作为底物来分析因子B的催化性质。影响因子B催化活性的因素是对ph的异常依赖。非生理碱性条件强烈促进了因子B对底物的裂解。与酶的ph可达构象一致，这可能对催化功能至关重要。传统六肽对硝基苯胺底物的小n端延伸显著提高了因子B的催化活性，其裂解位点比胰蛋白酶更具选择性。新的显色试验优化了催化条件，分析了不同底物序列，并开发了第一个可逆的竞争性底物B因子抑制剂。该抑制剂还被证明可以阻止C3由B因子、人工合成和天然补体活化途径的C3转化酶在体外形成C3a，并阻断膜攻击复合物的形成。一种稳定因子B活性构象的可逆底物抑制剂的可用性，结合ph促进的更高加工活性，可能为获得活性构象的因子B和C3转化酶的晶体结构提供新的途径。
Human complement factor B is the crucial catalytic component of the C3 convertase enzyme that activates the alternative pathway of complement-mediated immunity. Although a serine protease in its own right, factor B circulates in human serum as an inactive zymogen and there is a crystal structure only for the inactive state of factor B and various fragments. To provide greater insight to the catalytic function and properties of factor B, we have used short para-nitroanilide derivatives of 4-to 15-residue peptides as substrates to profile the catalytic properties of factor B. Among factors found to influence catalytic activity of factor B was an unusual dependence on pH. Non-physiological alkaline conditions strongly promoted substrate cleavage by factor B, consistent with a pH-accessible conformation of the enzyme that may be critical for catalytic function. Small N-terminal extensions to conventional hexapeptide para-nitroanilide substrates significantly increased catalytic activity of factor B, which was more selective for its cleavage site than trypsin. The new chromogenic assay enabled optimization of catalysis conditions, the profiling of different substrate sequences, and the development of the first reversible and competitive substrate-based inhibitor of factor B. The inhibitor was also shown to prevent in vitro formation of C3a from C3 by factor B, by synthetic and by natural C3 convertase of the alternative complement activation pathway, and to block formation of membrane attack complex. The availability of a reversible substrate-based inhibitor that could stabilize the active conformation of factor B, in conjunction with a pH-promoted higher processing activity, may offer a new avenue to obtain crystal structures of factor B and C3 convertase in an active conformation.