Elucidation of mechanisms of molecular recognition and signal transduction of immunoglobulin-Fc receptors on the basis of NMR data

本项目中关于小鼠可溶性FcGammaRII(SFcGammaRII)与小鼠IgG2b-Fc相互作用的研究结果总结如下：1.稳定同位素辅助的核磁共振分析表明，sFcGammaRII结合到Fc区域的下铰链及其空间邻近区域，这些区域带负电荷，并通过静电相互作用与sFcGammaRII表面的一些正电区域相互作用。一个糖基化的Fc突变体，其中Asn297被Ala取代，完全失去了与sFcGammaRII结合的能力。我们研究了通过内糖苷酶处理制备的各种Fc类似物与sFcGammaRII的结合。研究表明，内切糖苷酶D处理的Fc类似物含有一个GlcNAc和一个Fuc残基组成的碳水化合物链，显著降低了对sFcGammaRII的亲和力。核磁共振数据表明，经内切糖苷酶D处理后，Fc区的多肽-碳水化合物相互作用变得不那么广泛，使碳水化合物链更具流动性，CH2结构域的部分构象含有sFcGammaRII结合位点。我们已经确定了sFcGammaRII上的糖基化位点，确定了主要的糖形式，并建立了糖链的修剪方案。通过凝胶过滤和超速离心技术，我们证明了Fc-sFcGammaRII相互作用的化学计量比为1：1。利用核磁共振波谱还证明了sFcGammaRII与Fc上两个对称相关位点之一的结合引起了另一个位点的构象变化，从而排除了第二个sFcGammaRII分子与Fc的结合。我们认为，通过这种构象调节，可以防止在没有多价抗原的情况下，两个FcGammaRII分子由一个免疫球蛋白分子自发交联，这可能会引发不希望看到的细胞反应。

The results obtained in this projects concerning the interaction between mouse soluble FcgammaRII (sFcgammaRII) and mouse IgG2b-Fc are summarized as follows :1. The stable-isotope-assisted NMR analyses revealed that sFcgammaRII binds to the lower hinge and its spatial proximity of the Fc region, which are negatively charged and interact with some positively charged area on the sFcgammaRII surface via electrostatic interactions.2. An aglycosylated Fc mutant, in which Asn297 is substituted with Ala, has completely lost the ability to bind to sFcgammaRII.We have investigated binding to sFcgammaRII of a variety of Fc analogs prepared by the treatment with endoglycosidases. It has been shown that the endoglycosidase D-treated Fc analog, which contains the carbohydrate chains composed of one GlcNAc and one Fuc residues, has remarkably reduced affinity for sFcgammaRII.The NMR data indicate that the polypeptide-carbohydrate interactions in the Fc region become less extensive upon treatment by endoglycosidase D, rendering the carbohydrate chains more mobile and the conformation of parts of the CH2 domain containing the sFcgammaRII-binding site perturbed.3. We have identified the glycosylation sites on sFcgammaRII, determined the major glycoforms, and established the protocol for trimming of the carbohydrate chains.4. By gel-filtration and ultracentrifugation technique, we have shown that stoichiometry of the Fc-sFcgammaRII interaction is 1 : 1. By use of NMR spectroscopy, it has also been demonstrated that the binding of sFcgammaRII onto one of the two symmetrically related sites on Fc induces conformational change in the other site, which preclude the binding of second sFcgammaRII molecule to Fc. We suggest that, by this conformational regulation, spontaneous cross-linking of two FcgammaRII molecules by one IgG molecule in the absence of multivalent antigens, which might trigger undesirable cellular responses, is prevented.