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

基于NMR数据阐明免疫球蛋白-Fc受体的分子识别和信号转导机制

• 批准号: 09672186
• 负责人: KATO Koichi
• 金额: $ 1.86万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09672186/
• 关键词: Immunoglobulin; Fc receptor; Molecular recognition; NMR; Protein Structure; Carbohydrafe chain; Hinge region; Stable isotope

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.

本项目中获得的关于小鼠可溶性Fc γ RII（sFc γ RII）与小鼠IgG 2b-Fc之间相互作用的结果总结如下：1.稳定同位素辅助的NMR分析显示sFc γ RII结合到Fc区的下铰链及其空间邻近处，其带负电荷并通过静电相互作用与sFc γ RII表面上的一些带正电荷的区域相互作用。一个无糖基化的Fc突变体，其中Asn 297被替换为Ala，已经完全失去了与sFc γ RII结合的能力。我们已经研究了通过用内切糖苷酶处理制备的各种Fc类似物与sFc γ RII的结合。已经显示，糖苷内切酶D处理的Fc类似物（其含有由一个GlcNAc和一个Fuc残基组成的碳水化合物链）对sFc γ RII具有显著降低的亲和力。NMR数据表明Fc区中的多肽-碳水化合物相互作用在糖苷内切酶D处理后变得不那么广泛，使碳水化合物链更加移动的，并且包含sFc γ RII结合位点的CH 2结构域的部分构象被扰乱。我们已经鉴定了sFc γ RII上的糖基化位点，确定了主要的糖型，并建立了用于修剪糖链的方案。通过凝胶过滤和超离心技术，我们已经表明，Fc-sFc γ RII相互作用的化学计量比为1：1。通过使用NMR光谱，还证明sFc γ RII与Fc上两个对称相关位点之一的结合诱导另一位点的构象变化，这排除了第二个sFc γ RII分子与Fc的结合。我们认为，通过这种构象调节，可以防止在不存在多价抗原的情况下，两个FcgammaRII分子与一个IgG分子自发交联，这可能会引发不期望的细胞反应。

项目成果

Y.Yamaguchi et al.: "Dynamics of the carbohydrate chains attached to the Fc portion of immunoglobulin G as studied by NMR spectroscopy assisted by selective ^<13>C labeling of the glycans" J.Biomol.NMR. 12. 385-39〓 (1998)

Y. Yamaguchi等人：“通过NMR光谱法通过聚糖的选择性13 C标记辅助研究连接至免疫球蛋白G的Fc部分的碳水化合物链的动力学”J.Biomol.NMR。 〓 (1998)

Y.Yamaguchi, K.kato, M.Shindo, S.Aoki, K.Furusho, K.Koga, N.Takahashi, Y.Arata, and I.Shimada: "Dynamics of the carbohydrate chains attached to the Fc portion of immunoglobulim G as Studied by NMR spectroscopy assisted by selective ^<13>C labeling of the

Y.Yamaguchi、K.kato、M.Shindo、S.Aoki、K.Furusho、K.Koga、N.Takahashi、Y.Arata 和 I.Shimada：“连接到免疫球蛋白 Fc 部分的碳水化合物链的动力学

T.Torizawa, K.Kato, Y.Kimura, T.Asada, H.Kobayashi, Y.Komatsu, H.Morioka, O.Nikaido, E.Ohtsuka, and I.Shimada: "^<31>PNMR study of the interactions between oligodeoxynucleotides containing (6-4) photoproduct and Fab fragments of monoclonal antibodies spec

T.Torizawa、K.Kato、Y.Kimura、T.Asada、H.Kobayashi、Y.Komatsu、H.Morioka、O.Nikaido、E.Ohtsuka 和 I.Shimada：“^<31>PNMR 研究

K.Hirayama et al.: "Complete and rapid peptide and glycopeptide mapping of mouse monoclonal antibody by LC/MS/MS using ion trap mass spectrometry" Anal.Chem.70. 2718-2725 (1998)

K.Hirayama 等人：“使用离子阱质谱法通过 LC/MS/MS 对小鼠单克隆抗体进行完整快速的肽和糖肽图谱分析”Anal.Chem.70。

K.Hirayama, R.Yuji, N.Yamada, K.Noguchi, Y.Yamaguchi, J.Enokizono, K.Kato, Y.Arata, and I.Shimada: "Convenient peptide mapping of immunoglobulin G2b and differentiation between leucine and isoleucine residues by mass spectrometry using ^2H-labeled leucine

K.Hirayama、R.Yuji、N.Yamada、K.Noguchi、Y.Yamaguchi、J.Enokizono、K.Kato、Y.Arata 和 I.Shimada：“免疫球蛋白 G2b 的便捷肽图分析以及亮氨酸和异亮氨酸的区分