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首页> 外文期刊>Biochemistry >Characterization and crystallization of soluble human Fc gamma receptor II (CD32) isoforms produced in insect cells.
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Characterization and crystallization of soluble human Fc gamma receptor II (CD32) isoforms produced in insect cells.

机译：昆虫细胞中产生的可溶性人Fcγ受体II（CD32）亚型的表征和结晶。

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摘要

Fc gamma RII (CD32), the receptor for the Fc part of IgG, is responsible for the clearance of immunocomplexes by macrophages and plays a role in the regulation of antibody production by B cells. To investigate the process of immunocomplex binding in terms of stoichiometry and stability of the Fc gamma RII:IgG complex, we produced both Fc gamma RII isoforms (Fc gamma RIIa and Fc gamma RIIb) as soluble proteins in insect cells. The expressed proteins could be purified in high yields and were biologically active as judged by their ability to bind IgG. Thus, the minor glycosylation performed by the insect cells is not crucial for the binding of the usually highly glycosylated Fc gamma RII to IgG. The dissociation constant of the sFc gamma RIIa:IgG-hFc complex was determined by fluorescence titration (KD = 2.5 x 10(-)7 M). Complementary sFc gamma RIIa antagonizes immunocomplex binding to B cells. Here sFc gamma RIIa showed a comparable dissociation constant (KD = 1.7 x 10(-)7 M) which was almost 10-fold lower than the constant for Fc gamma RIIb. The stoichiometry of the FcRIIa:IgG-hFc complex was determined by equilibrium gel filtration and shows that IgG is able to bind alternatively one or two Fc gamma RII molecules in a noncooperative manner. Furthermore, in an ELISA-based assay the isotype specificity of various anti-Fc gamma RII monoclonal antibodies was measured as well as their ability to interfere with the IgG recognition through its receptors. To further investigate the molecular basis of the Fc gamma RII-ligand interaction, we crystallized Fc gamma RIIb. Trigonal crystals diffracted to 3 A and the structure solution is in progress.
机译：FcγRII（CD32）是IgG Fc部分的受体，负责巨噬细胞清除免疫复合物，并在B细胞抗体产生的调节中发挥作用。为了研究化学复合物结合FcγRII：IgG复合物的化学计量和稳定性，我们在昆虫细胞中产生了两种FcγRII亚型（FcγRIIa和FcγRIIb）作为可溶性蛋白。所表达的蛋白可以高产率纯化，并且根据它们结合IgG的能力来判断其具有生物学活性。因此，昆虫细胞进行的少量糖基化对于通常高度糖基化的FcγRII与IgG的结合不是至关重要的。 sFcγRIIa：IgG-hFc复合物的解离常数通过荧光滴定法（KD = 2.5 x 10（-）7 M）确定。互补的sFcγRIIa拮抗免疫复合物与B细胞的结合。此处，sFcγRIIa显示出可比的解离常数（KD = 1.7 x 10（-）7 M），比FcγRIIb的常数低近十倍。 FcRIIa：IgG-hFc复合物的化学计量通过平衡凝胶过滤确定，显示IgG能够以非合作方式结合一个或两个FcγRII分子。此外，在基于ELISA的测定中，测量了各种抗FCγRII单克隆抗体的同种型特异性，以及它们通过其受体干扰IgG识别的能力。为了进一步研究FcγRII与配体相互作用的分子基础，我们结晶了FcγRIIb。三角晶体衍射至3 A，正在进行结构求解。

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