Kishore U, Leigh L E, Eggleton P, Strong P, Perdikoulis M V, Willis A C, Reid K B
Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
Biochem J. 1998 Jul 1;333 ( Pt 1)(Pt 1):27-32. doi: 10.1042/bj3330027.
The first step in the activation of the classical pathway of the complement system by immune complexes involves the binding of the six globular heads of C1q to the Fc regions of IgG or IgM. The globular heads of C1q are located C-terminal to the six triple-helical stalks present in the molecule; each head is considered to be composed of the C-terminal halves (3x136 residues) of one A-, one B- and one C-chain. It is not known if the C-terminal globular regions, present in each of the three types of chain, are independently folded modules (with each chain having distinct binding properties towards immunoglobulins) or whether the different binding functions of C1q are dependent upon a globular structure which relies on contributions from all three chains. As a first step towards addressing this question, we have expressed the globular head region (residues 87-226) of the C1q B-chain (ghB) as a soluble fusion protein with maltose-binding protein (MBP) in Escherichia coli. The affinity purified fusion protein, designated MBP-ghB, behaved as a dimer on gel filtration and bound preferentially to aggregated IgG rather than to IgM. It could also inhibit C1q-dependent haemolysis of both IgG- and IgM-sensitized erythrocytes. After its release from MBP, by use of Factor Xa, the free ghB exhibited a tendency to aggregate and come out of solution. Since MBP is known to be a monomeric molecule, the dimerization of the MBP-ghB fusion polypeptide is probably brought about by the ghB region, perhaps through hydrophobic interactions within the ghB region. The functional behaviour of MBP-ghB indicates that the globular regions of C1q may adopt a modular organization, i.e. each globular head of C1q may be composed of three structurally and functionally independent domains, thus retaining multivalency in the form of a heterotrimer.
免疫复合物激活补体系统经典途径的第一步涉及C1q的六个球状头部与IgG或IgM的Fc区域结合。C1q的球状头部位于分子中存在的六个三螺旋茎的C末端；每个头部被认为由一条A链、一条B链和一条C链的C末端半段（3×136个残基）组成。目前尚不清楚三种链中每一种链上存在的C末端球状区域是独立折叠的模块（每条链对免疫球蛋白具有不同的结合特性），还是C1q的不同结合功能依赖于一种球状结构，而这种结构依赖于所有三条链的贡献。作为解决这个问题的第一步，我们在大肠杆菌中表达了C1q B链（ghB）的球状头部区域（第87至226位残基），作为与麦芽糖结合蛋白（MBP）的可溶性融合蛋白。亲和纯化的融合蛋白，命名为MBP-ghB，在凝胶过滤中表现为二聚体，优先结合聚集的IgG而不是IgM。它还可以抑制IgG和IgM致敏红细胞的C1q依赖性溶血。通过使用因子Xa从MBP释放后，游离的ghB表现出聚集并从溶液中析出的趋势。由于已知MBP是单体分子，MBP-ghB融合多肽的二聚化可能是由ghB区域引起的，可能是通过ghB区域内的疏水相互作用。MBP-ghB的功能行为表明C1q的球状区域可能采用模块化组织，即C1q的每个球状头部可能由三个结构和功能独立的结构域组成，从而以异源三聚体的形式保留多价性。
