兰州大学机构库 >生命科学学院
电镜单颗粒技术研究人源C反应蛋白与C1q的相互作用及其结构特征
Alternative TitleStudy on the Interaction and Structural Characteristics of Human C-reaction protein and C1q by Single Particle Electron Microscopy
李一方
Thesis Advisor吉尚戎
2017-05-01
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
KeywordCRP-C1q复合物 负染 电镜单颗粒技术
Abstract

    补体系统是先天免疫的重要组成部分,分为三条激活途径,其中C1q是经典补体途径中第一个蛋白成分C1的识别亚基,分子量为460kD,由A、B、C各6条共18条肽链组成,其C末端为6个异源三聚体A、B、C链形成的球状头部(gC1q),N端由6条ABC三螺旋形成胶原区。C1q可以识别多种自身或异源配体,其中包括急性期标识蛋白C反应蛋白(C-reactive protein, CRP)。CRP由5个相同亚基依靠氢键和疏水作用形成同源五聚体,分为A面(效应面)、B面(PC、Ca2+结合面)。当机体发生炎症反应时,CRP能够通过效应面与C1q结合激活经典补体途径,发挥抗炎、清除病原微生物等生物学功能。已有研究表明,gC1q的B链带有连续精氨酸(ArgB101, ArgB114和ArgB129),可与CRP位于五聚体中央孔附近的Asp112、Tyr175发生静电依赖性的互作,并且pH、盐浓度等因素对二者复合物的形成亦有影响,但仍未有对二者形成复合物的具体条件及其结构特征的报道。       本论文运用电镜负染技术探究不同pH值、盐浓度、摩尔比等因素对CRP-C1q复合物形成的影响,筛选出最有利于形成复合物的条件,且通过对不同孵育时间下复合物的电镜观察表明,复合物形成后具有一定的稳定性。通过应用电镜单颗粒技术对CRP-C1q复合物及相同制样条件下CRP、C1q的二维结构特征的分析比较,表明CRP-C1q复合物具有更为紧实的结构,二维类平均图显示其具有花边状轮廓,内部结构特征较为复杂,推断这与6个gC1q或胶原区与CRP效应面的随机性静电依赖性互作有关。综上,本论文对静息态CRP与C1q相互作用的条件进行优化,并首次运用电镜单颗粒技术观测到了CRP-C1q复合物的二维结构特征,为二者互作的可能形式提供了一定的实验依据。

Other Abstract

    Complement system, an important component of innate immune system, is composed of three activation pathways. C1q is the recognition subunit of the first protein component C1 in the classical complement pathway (CCP). It is made up of 18 polypeptide chains of three different types (6A, 6B, 6C), with a 460kD molecular weight comprising six ABC heterotrimeric C-terminal globular heads (gC1q), and an N-terminal heterotrimeric triple helices formed collagen region. C1q can recognize various self or non-self ligands, including acute phase protein like C-reactive protein (CRP). CRP, represented as a pentamer, is composed of five identical subunits related by hydrogen bonds and hydrophobic interaction. Its disc-like shape can be distinguished into A face (effector face) and B face (PC, Ca2+ binding face). Once the inflammatory reaction launched, the former can be recognized by C1q to further activate the classical complement pathway, which plays a role in anti-inflammation, pathogenic microorganisms clearance and other biological activities. The B chain of gC1q including continuous arginines (ArgB101, ArgB114和ArgB129) has been confirmed to interact with Asp112 and Tyr175 located close to the central pore of CRP and based on which an interaction model has been proposed. In addition, the pH value, salt concentration as well as other factors also have an impact on the formation of the complex as shown in the reported ELISA results. However, the direct structural information on the overall CRP-C1q complex has not been well characterized.     In this study, we investigated the binding property of CRP and C1q under different pH value, salt concentration and molar ratio by negative staining electron microscopy, and determined the condition that was most favorable for the complex formation. Based on the comparison and analysis of the 2D structural characteristics of individual CRP, C1q and CRP-C1q complex under the same sample preparation conditions, it showed that the CRP-C1q complex has a more compact structure, and the 2D class average appears a lace-like contour with a complicated internal density, which may derived from the randomly occurred electrostatic-dependent interaction between six globular heads or the collagen region of C1q and the CRP effector face. Above all, in this paper we optimized the CRP-C1q complex formation condition, and observed the 2D structural characteristic of CRP-C1q complex for the first time by using single particle electron microscopy, which provided some experimental basis for explaining the possible interaction forms between human CRP and C1q.

URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221537
Collection生命科学学院
Recommended Citation
GB/T 7714
李一方. 电镜单颗粒技术研究人源C反应蛋白与C1q的相互作用及其结构特征[D]. 兰州. 兰州大学,2017.
Files in This Item:
There are no files associated with this item.
Related Services
Recommend this item
Bookmark
Usage statistics
Export to Endnote
Altmetrics Score
Google Scholar
Similar articles in Google Scholar
[李一方]'s Articles
Baidu academic
Similar articles in Baidu academic
[李一方]'s Articles
Bing Scholar
Similar articles in Bing Scholar
[李一方]'s Articles
Terms of Use
No data!
Social Bookmark/Share
No comment.
Items in the repository are protected by copyright, with all rights reserved, unless otherwise indicated.