Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy
Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy
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DOI：
10.1016/j.bbagen.2005.10.002
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发表时间：
2006-04-01
影响因子：
3
通讯作者：
Kato, K
中科院分区：
文献类型：
--
作者：
Yamaguchi, Y;Nishimura, M;Kato, K
免疫球蛋白G（IgG）的Fe部分在每个重链的C（H）2结构域的Asn 297处表达双触角复合型寡糖，其微异质性取决于生理和病理状态。已知这些N-聚糖对于促进IgG的适当效应子功能（如补体激活和Fc γ受体（Fc γ R）介导的活性）至关重要。为了更好地理解Fc糖基化的作用，我们制备了一系列截短的人IgGl-Fe糖型，并通过表面等离子体共振和核磁共振（NMR）方法分析了它们与人可溶性Fc γ RIIIa（sFc γ RIIIa）和葡萄球菌蛋白A的相互作用。由于半乳糖苷酶和随后的N-乙酰氨基己糖苷酶处理IgGI-Fc，观察到对sFc γ RIIIa的亲和力逐渐但不太明显的降低。以下糖苷内切酶D处理产生由岩藻糖基化GlcNAc组成的二糖结构，消除了IgGI-Fc对sFc γ RIIIa的亲和力。另一方面，这些糖苷酶处理没有显著影响IgGI-Fc对蛋白A的亲和力。对一系列Fc糖型的稳定同位素辅助核磁共振数据的检查表明，逐步修剪碳水化合物残基会导致CH 2结构域中受干扰的氨基酸残基数量随之增加。此外，GlcNAc β 1-4GlcNAc糖苷键的裂解诱导了下铰链区部分的构象改变，其不与碳水化合物部分直接接触并形成主要的Fc γ R结合位点，而C（H）2/C（H）3界面的构象几乎没有受到干扰，即蛋白A结合位点。这些结果表明，碳水化合物部分是维持Fc γ R结合位点结构完整性所必需的。(c)2005 Elsevier B. V.保留所有权利。
The Fe portion of immunoglobulin G (IgG) expresses the biantennary complex type oligosaccharides at Asn297 of the C(H)2 domain of each heavy chain with microheterogeneities depending oil physiological and pathological states. These N-glycans are known to be essential for promotion of proper effector functions of IgG such as complement activation and Fc gamma receptor (Fc gamma R)-mediated activities. To gain a better understanding of the role of Fc glycosylation, we prepared a series of truncated glycoforms of human IgGl-Fe and analyzed their interactions with human soluble Fc gamma RIIIa (sFc gamma RIIIa) and with staphylococcal protein A by surface plasmon resonance and nuclear magnetic resonance (NMR) methods. Progressive but less pronounced reductions in the affinity for sFc gamma RIIIa were observed as a result of the galactosidase and subsequent N-acetylhexosaminidase treatments of IgGI-Fc. The following endoglycosidase D treatment, giving rise to a disaccharide structure composed of a fucosylated GlcNAc, abrogated the affinity of IgGI-Fc for sFc gamma RIIIa. Oil the other hand, those glycosidase treatments did not significantly affect the affinity of IgGI-Fc for protein A. Inspection of stable-isotope-assisted NMR data of a series of Fc glycoforms indicates that the stepwise trimming Out of the carbohydrate residues results in concomitant increase in the number of amino acid residues perturbed thereby in the CH2 domains. Furthermore, the cleavage at the GlcNAc beta 1-4GlcNAc glycosidic linkage induced the conformational alterations of part of the lower hinge region, which makes no direct contact with the carbohydrate moieties and forms the major Fc gamma R-binding site, while the conformation of the C(H)2/C(H)3 interface was barely perturbed that is the protein A-binding site. These results indicate that the carbohydrate moieties are required for maintaining the structural integrity of the Fc gamma R-binding site. (c) 2005 Elsevier B.V. All rights reserved.