巨噬细胞表面特异糖链结构鉴定及功能研究

Glycans on the surface of immune cells are important components of the cell membrane. They are involved in multiple important biological processes of immune cells including cell proliferation, differentiation and immune response. Membrane glycans of immune cells are also reported to be closely related with the occurrence and development of many immune disorders. In our previous study, we found that the glycan recognition spectrum of CMIP was very special. We have verified that CMIP specifically bound to macrophages and induced them to differentiate into M1 phenotype by binding the glycans on membrane of macrophages. In this study, we intend to identify specific glycans on the surface of macrophages and study their function using CMIP. The research programme is as follows:1) Identify specific glycans on the surface of macrophages by developing a CMIP based binding assay, and combining with high-resolution mass spectrometry; 2) The cDNA microarray analyses are applied to select the key glycosyltransferase that specifically regulated the expressions of the identified glycans. Then the knock-out or knock-down of the selected genes will be performed in macrophages and changes of glycan on the surface of macrophages will be detected. After CMIP stimulating, the cell phenotype changes of the modified macrophages will be detected to study the function of membrane glycans; 3) LC-MS/MS are used to identify the possible glycoprotein that interact with CMIP on the membrane of immune cells. The related signal pathway will be detected to elucidate the important role of surface glycans involved in immune response. This study will provide new data to clarify the immune response mechanism of glycans on the cell surface of immune cells.

免疫细胞表面糖链是细胞膜的重要组成成分，参与细胞增殖、分化以及免疫应答等重要生命活动过程，与众多免疫类疾病的发生和发展也密切相关。我们前期研究发现，北虫草新凝集素CMIP糖识别谱较窄，且可选择性识别免疫细胞，通过结合细胞表面糖链诱导巨噬细胞M1型极化。本项目拟利用新凝集素CMIP研究巨噬细胞表面特异的糖链结构与功能，具体方案：1）利用CMIP富集并联合高分辨率质谱的方法，鉴定巨噬细胞表面特异糖链结构；2）通过表达谱基因芯片，筛选调控特异糖链合成的关键糖基转移酶，建立基因敲除/敲低巨噬细胞系，检测细胞膜糖链表达变化，并验证CMIP刺激后细胞表型变化；3）利用CMIP富集并结合质谱的方法，鉴定巨噬细胞表面CMIP的互作糖蛋白，对CMIP-糖蛋白相互作用后胞内信号通路进行验证，初步探究巨噬细胞表面糖链参与免疫应答的分子机制。本项目为阐明免疫细胞表面糖链在免疫应答中的功能机制提供新的数据。

糖基化修饰是生物体内广泛存在的蛋白质翻译后修饰。免疫细胞表面蛋白质的糖基化修饰被报道广泛参与细胞增殖、分化以及免疫应答等重要生命活动过程。本项目根据凝集素特异性识别与结合糖链结构的特性，结合质谱鉴定的方法，研究巨噬细胞表面的糖链结构与生物活性。主要研究内容分为以下4部分：(1)糖芯片技术解析北虫草新型凝集素的糖结合特异性；(2)北虫草凝集素与特异性糖配体亲和力参数测定；（3）凝集素共沉淀结合质谱鉴定解析巨噬细胞表面与北虫草凝集素特异性识别的糖蛋白；（4）北虫草凝集素诱导巨噬细胞M1极化分子机制验证。本项目的重要结果如下：（1）利用糖芯片技术获得北虫草凝集素的糖结合特异性。糖芯片结果解析表明CMIP特异性识别含 (Galβ1-4GlcNAcβ1-3)n的复杂N-糖链，CMRBL则对以Galβ1-4GlcNAc-结尾的糖链结构高度特异性地结合。（2）北虫草凝集素可特异性识别与结合巨噬细胞，且显著诱导巨噬细胞的M1型极化，而不影响或抑制M2型极化特征性分子的表达。（3）初步验证了CMIP 通过结合巨噬细胞表面Integrin β-1激活胞内MAPK，STAT1诱导巨噬细胞M1型极化的分子机制。本项目获得的关键数据如下：（1）凝集素CMIP与CMRBL的糖亲和谱。（2）CMIP与CMRBL诱导巨噬细胞M1型极化特征性分子的表达谱。（3）CMIP特异性结合巨噬细胞表面糖修饰蛋白。（4）CMIP特征性调控的巨噬细胞基因表达谱。本项目综合利用糖芯片、转录组与蛋白组学以及分子生物学技术，多层次地解析北虫草凝集素特异性识别与结合巨噬细胞表面糖蛋白，进而激活巨噬细胞M1型极化的分子机制。同时证明了细胞表面糖链结构在免疫细胞分化过程中的重要角色，为免疫细胞表面糖链结构的生物功能研究提供了坚实的数据基础。