FcαR的糖基化及其异形体的功能效应

发布时间：2018-03-07 21:18

本文选题：糖基　切入点：及其　出处：《北京协和医学院》2011年博士论文　论文类型：学位论文

【摘要】：本论文分为两部分,第一部分研究了FcαR的糖基化对其与IgA结合的影响；第二部分研究了FcαR异形体的表达及定位。第一部分 FcαR (CD89)是IgA的Fc受体,在IgA介导的免疫应答中发挥重要作用。FcαR是一个糖基化程度较高的蛋白质,它拥有六个潜在的N-糖基化位点。据文献报道,FcαR的异常糖基化与HIV感染、酒精性肝硬化和IgA肾病等疾病相关。本实验检测了N-糖基化对FcαR与IgA结合所发挥的作用。我们发现将稳定表达FcαR的CHO细胞用衣霉素进行去N-糖基化处理后,其与IgA的结合明显增强。为了明确FcαR引起与IgA结合增强的N-糖基化位点,我们利用点突变的方法分别把六个N-糖基化位点的天门冬酰胺(N)替换成谷氨酰胺(Q)。流式细胞分析显示N44、N120、N156、N165、N177位点去除糖基化不影响FcαR与IgA的结合,但是N58位点去除糖基化导致与IgA的结合显著增加。相似的结果也表现在稳定表达N58Q-FcαR的RBL2H3细胞(大鼠嗜碱性白血病细胞系)。除此以外,我们还发现FcαR经神经氨酸酶处理去除唾液酸后,其与IgA的结合也明显增强,而N58正是这一现象中起关键作用的位点。综上所述,我们的结果证明了N58位点的糖基化状态影响了FcαR与IgA的结合。第二部分 FcαR(本文代号为A1)是IgA的Fc受体,它在IgA介导的免疫应答中发挥着重要作用。以前的研究证实,在人的髓系细胞可以检测到△66EC2 fcαr(代号为A2)和△EC2 fcαr(代号为A3)的转录物,即EC2结构域近膜端缺失66个核苷酸的转录物和缺失完整的免疫球蛋白样EC2结构域的转录物。我们的实验主要研究了FcαR异形体(A2和A3)在CHO细胞、COS7细胞和RBL2H3细胞的表达和定位。与A1不同的是,A2和A3都不能在细胞表面表达。为了进一步探讨FcαR的胞外区影响其在细胞表面表达的氨基酸性质,我们构建了多种FcαR的缺失突变体和置换突变体。结果显示除第216位的苏氨酸以外,其余的FcαR缺失突变体均不能表达在细胞表面或者受体表达量明显降低。然而,将FcαR胞外区的一些氨基酸突变成丙氨酸,置换后的FcαR都能表达在细胞膜上另外,我们用Western Blot也没有检测到转染至CHO细胞的A2、A3蛋白条带。这一现象很有可能是由于A2和A3胞外区存在部分氨基酸的缺失,致使其三维结构发生了改变。因此,FcαR的EC2胞外区对维持蛋白质结构的稳定性和功能性发挥重要的作用。
[Abstract]:This thesis is divided into two parts: the first part studies the effect of FC 伪 R glycosylation on the binding of FC 伪 R to IgA, and the second part studies the expression and localization of FC 伪 R heteromorphism. Part one. FC 伪 R (CD89) is a FC receptor of IgA, which plays an important role in IgA mediated immune response. FC 伪 R is a highly glycosylated protein with six potential N-glycosylation sites. Abnormal glycosylation of FC 伪 R and HIV infection have been reported. The effects of N-glycosylation on the binding of FC 伪 R to IgA were studied. We found that CHO cells stably expressing FC 伪 R were treated with methionine to remove N-glycosylation. In order to clarify the N-glycosylation sites induced by FC 伪 R binding to IgA, By using point mutation method, the asparagine N of six N-glycosylation sites was replaced with glutamine QN. Flow cytometry analysis showed that the removal of glycosylation at the N156N165N177 site did not affect the binding of FC 伪 R to IgA, and flow cytometry (FCM) analysis showed that the glycosylation of six N-glycosylated sites at N156N165N177 site did not affect the binding of FC 伪 R to IgA. However, the removal of glycosylation at N58 site resulted in a significant increase in binding to IgA. Similar results were found in RBL2H3 cells that stably expressed N58Q-FC 伪 R (rat basophilic leukemia cell line). We also found that when FC 伪 R was treated with neuraminidase to remove sialic acid, the binding of FC 伪 R to IgA was significantly enhanced, and N58 was the key site in this phenomenon. Our results show that the glycosylation state of N58 affects the binding of FC 伪 R to IgA. Part two. FC 伪 R (codenamed A1) is the FC receptor of IgA, which plays an important role in the immune response mediated by IgA. Previous studies have confirmed that 66EC2 FC 伪 rand EC2 FC 伪 r (codenamed A2) and EC2 FC 伪 r (codenamed A3) can be detected in human myeloid cells. That is, the transcripts of 66 nucleotides and complete immunoglobulin-like EC2 domains are missing from the proximal end of the EC2 domain. Our experiment mainly studied the expression of FC 伪 R heteromorphism A _ 2 and A _ 3) in the CHO cells of COS7 cells and RBL2H3 cells. Different from A1, neither A _ 2 nor A _ 3 could be expressed on the cell surface. In order to further study the effect of extracellular domain of FC 伪 R on the amino acid properties of FC 伪 R, We constructed a variety of FC 伪 R deletion mutants and replacement mutants. The results showed that none of the FC 伪 R deletion mutants could express on the cell surface or the receptor expression level was significantly decreased except for the 216th position threonine. Some amino acids in the extracellular domain of FC 伪 R were mutated into alanine. We also did not detect the A2A3 protein band transfected to CHO cells by Western Blot. This phenomenon is probably due to the deletion of some amino acids in the extracellular domain of A2 and A3. Therefore, the extracellular domain of FC 伪 R plays an important role in maintaining the stability and functionality of protein structure.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392.1

【共引文献】