核心岩藻糖修饰对成熟B细胞抗原识别、活化及抗体产生的调节作用机理研究

In the germinal center microenvironment, B cell recognition of antigens leads to the formation of an immunological synapse (IS) which could induce the efficient B cell activation and antibody production. During the formation of IS, the B cell receptor (BCR) gathers antigen into a central cluster, surrounded by a peripheral ring of very late antigen-4 (VLA-4) and vascular adhesion molecule-1 (VCAM-1). The immune molecules, such as BCR, VLA-4 and VCAM-1, are heavily core fucosylated. Core fucosyltransferase (Fut8) catalyzes the transfer of a fucose residue from GDP-fucose to the innermost N-acetylglucosamine (GlcNAc) residue of hybrid and complex N-glycans via an? alpha 1,6-linkage (core fucosylation) in the Golgi apparatus in mammals. The core fucosylation on N-glycans of glycoprotein is only catalyzed by Fut8. Fut8 is able to modify the function of BCR, VLA-4 and VCAM-1 with their core fucosylation. Contrast to wild mice, mature B cell population in the spleen was impaired in Fut8 knockout (Fut8-/-) mice. In enzyme-linked immunospot (ELISPOT) assay, the number of IgG-secreting splenic lymphocytes after immunizations with lipopolysaccharide (LPS) was significantly lower than those of Fut8+/+ mice, suggesting that that core fucose plays a key role in the antigen recognition and activation of mature B cells. To determine the underlying mechanism of the impaired population of mature B cells seen in Fut8-/- mice, we established a Fut8 knockdown mature B cell line (3-83-KD) by a siRNA technique using matue B cell line (3-83), and a Fut8 restored mature B cell line (3-83-KD-Re) by re-introduction of the Fut8 gene to 3-83-KD cells. In this program, considerable attention is focused on the research process, such as antigen recognition, IS formation, B cell signal transduction, cytoskeleton reorganizations, antibody production and IgG2 N-glycan structure analysis, as well as on the mechanisms of core fucosylation in mature B cell recognition of antigens, activation and antibody production. These results will lay a foundation for significantly understanding the process of humoral immune response from the field of glycosylation.

在生发中心微环境中成熟B细胞识别抗原时B细胞与滤泡树突状细胞相互接触界面形成免疫突触(IS)，这是B细胞活化及抗体产生的关键步骤。参与IS形成的关键免疫分子，如B细胞抗原受体(BCR)、VLA-4以及VCAM-1均是核心岩藻糖基化的糖蛋白。核心岩藻糖基转移酶(Fut8)是催化核心岩藻糖合成的唯一的糖基转移酶，其对BCR、VLA-4及VCAM-1的功能具有重要调节作用。申请人前期工作发现：与野生型小鼠相比，Fut8基因敲除小鼠脾脏中成熟B细胞数量明显减少及其抗体产生能力明显减弱，说明核心岩藻糖修饰对B细胞活化及抗体产生至关重要。本课题拟建立Fut8基因沉默以及Fut8基因重新恢复B细胞模型，开展B细胞抗原识别、IS形成、细胞信号传导、抗体产生及IgG2 N-糖链结构分析等研究，探讨核心岩藻糖修饰对B细胞抗原识别、活化及抗体产生的调节作用机理，从糖基化角度为加深对体液免疫应答认识提出新的见解。

阐明B细胞抗原（Ag）识别，B细胞活化及抗体产生的调节机理是特异性体液免疫应答的关键科学问题。B细胞受体（BCR）与Ag结合后互相交联形成多聚体(polymerization)，并激活Fyn、Syk等Src家族的蛋白酪氨酸激酶，启动信号转导的级联反应使B细胞活化，并进一步分化成浆细胞产生抗体。.核心岩藻糖基转移酶 (Fut8)是催化核心岩藻糖合成的唯一的糖基转移酶，在高尔基体内以GDP-岩藻糖为供体，以1,6糖苷键的形式把岩藻糖基转移到与天冬酰胺相邻的N-糖链还原末端乙酰葡糖胺，形成核心岩藻糖基。核心岩藻糖基修饰对于维持蛋白质正常结构与功能发挥重要调节作用。BCR、VLA-4以及VCAM-1均是富含核心岩藻糖基的糖蛋白。本课题中我们利用逆转录病毒包装Fut8-siRNA技术及cDNA点突变技术，建立成熟B细胞株（3-83）、Fut8基因沉默成熟B细胞株（3-83-KD）、Fut8恢复成熟B细胞株（3-83-KD-re）。再利用CRISPR/Cas9 技术建立Fut8基因敲除成熟B细胞株（3-83-KD）及Fut8-/-小鼠模型，系统地阐明了Fut8调节BCR抗原识别，多聚体形成，BCR内在化，细胞信号传导，脂筏形成，免疫突触形成及抗体产生的作用机理。利用质谱分析技术确定了BCR的糖链结构及糖型（Journal of Immunology. 2015, 15;194:2596-606）。.本课题实现了糖生物学、分子免疫学及分子生物学等多学科交叉，不仅为揭示核心岩藻糖修饰对成熟B细胞Ag识别、活化及抗体产生的调节作用提供科学依据，而且从糖基化角度为加深对体液免疫应答的认识提出新的见解。我们在临床样品的分析中发现，自身免疫性疾病及感染患者血清中核心岩藻糖基化水平明显增高，提示Fut8是自身免疫性相关疾病治疗及诊断的潜在靶点。