Calreticulin's functions in the adaptive immune response

钙网蛋白在适应性免疫反应中的功能

• 批准号: 7924278
• 负责人: MALINI RAGHAVAN
• 金额: $ 38.91万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7924278
• 关键词: Alanine; Antigen Presentation; Antigen Presentation Pathway; Antigen-Presenting Cells; Antigens; Apoptotic; Binding; Binding Sites; Biochemical; Calcium; Calnexin; Cell surface; Cells; Cellular Stress; Chickens; Complex; Cross Presentation; Cross-Priming; Defect; Dimerization; Disulfides; Endoplasmic Reticulum; Endosomes; Glycoproteins; Heating; Histocompatibility Antigens Class I; IgY; Immune response; Immunity; Immunofluorescence Immunologic; In Vitro; Lectin; Link; Location; Major Histocompatibility Complex; Mass Spectrum Analysis; Mediating; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Mutate; Oligosaccharides; Pathway interactions; Peptides; Phagocytes; Physiological; Polysaccharides; Property; Proteins; Proteolysis; Quality Control; Role; Route; Site; Structure; Substrate Interaction; Surface; T-Lymphocyte; Tunicamycin; Vaccine Design; Virus Diseases; Work; activated protein C receptor; base; calreticulin; conformational conversion; dimer; endoplasmic reticulum stress; in vitro activity; in vivo; insight; mutant; polypeptide; protein folding; receptor; research study; trafficking; trans-Golgi Network; uptake

DESCRIPTION (provided by applicant): Calreticulin is an endoplasmic reticulum (ER) chaperone that promotes folding and assembly of glycoproteins, including major histocompatibility complex (MHC) class I molecules. Calreticulin also has the capacity to direct exogenous antigens onto the MHC class I antigen presentation pathway, a phenomenon called cross-presentation. As a lectin, Calreticulin interacts with monoglucosylated core glycans on glycoproteins. Under certain conditions, Calreticulin is able to bind polypeptide components of substrates. Calcium depletion and heat-treatment expose calreticulin's polypeptide binding site and enhance Calreticulin binding to polypeptide substrates in vitro and in vivo cells. These treatments also induce Calreticulin dimerization and oligomerization. The formation of Calreticulin dimers is additionally induced by other types of ER stress, including virus infection and tunicamycin treatment. It is our hypothesis that these conformational transitions and polypeptide-binding properties are important for calreticulin's protein folding and cross-priming functions in cells. The first specific aim explores the role of polypeptide binding by Calreticulin during MHC class I folding and assembly in cells. We propose partial proteolysis and mass spectrometry-based approaches to identify Calreticulin sub-domains that are mobilized by calcium depletion. Conserved hydrophobic residues of Calreticulin, that are predicted to be surface-exposed, will be mutated to alanines. Mutants that display defects in interactions with polypeptide components of MHC class I heavy chains in vitro, as well as other mutants with defects in binding oligosaccharide substrates, will be expressed in calreticulin-deficient cells, and assessed for the ability to facilitate MHC class I folding and assembly. Together, these studies will allow us to refine our working model for the calreticulin-substrate interaction cycle, in which alternating interactions with oligosaccharide and polypeptide components of substrates are proposed. We will attempt to crystallize truncated versions of Calreticulin that have enhanced ability to bind polypeptide substrates, and also crystallize Calreticulin complexes with chicken IgY fragments. The second specific aim will explore mechanisms of calreticulin-mediated cross-presentation. Intracellular trafficking of Calreticulin and calreticulin-associated peptides during cross-presentation will be assessed, to investigate the hypothesis of an endosome-trans Golgi network-ER trafficking route. The requirement for Calreticulin for cross-presentation of antigens associated with apoptotic cells will also be assessed. Finally, the effects of ER stress on Calreticulin trafficking, cell surface expression, and interactions with receptors on antigen presenting cells will be assessed. Understanding the molecular mechanisms of calreticulin's functions, and elucidation of conditions that enhance calreticulin's T cell priming activities, will facilitate more effective design of vaccines.

描述（由申请人提供）：钙网蛋白是一种内质网（ER）伴侣蛋白，可促进糖蛋白（包括主要组织相容性复合体（MHC）I类分子）的折叠和组装。钙网蛋白还具有将外源性抗原引导到MHC I类抗原呈递途径上的能力，这种现象称为交叉呈递。作为凝集素，钙网蛋白与糖蛋白上的单葡萄糖基化核心聚糖相互作用。在一定条件下，钙网蛋白能够结合底物的多肽组分。钙耗竭和热处理暴露钙网蛋白的多肽结合位点，并增强钙网蛋白在体外和体内细胞中与多肽底物的结合。这些处理还诱导钙网蛋白二聚化和寡聚化。钙网蛋白二聚体的形成还由其他类型的ER应激诱导，包括病毒感染和衣霉素处理。这是我们的假设，这些构象转换和多肽结合特性是重要的钙网蛋白的蛋白质折叠和交叉启动功能的细胞。第一个具体的目标探讨了在细胞中MHC I类折叠和组装过程中钙网蛋白与多肽结合的作用。我们提出了部分蛋白水解和质谱为基础的方法来确定钙网蛋白的亚结构域，动员钙耗竭。钙网蛋白的保守疏水残基，预计是表面暴露的，将突变为丙氨酸。在体外与MHC I类重链的多肽组分相互作用中显示缺陷的突变体，以及在结合寡糖底物中具有缺陷的其他突变体，将在钙网蛋白缺陷细胞中表达，并评估促进MHC I类折叠和组装的能力。总之，这些研究将使我们能够完善我们的工作模型的钙网蛋白-底物相互作用周期，其中交替相互作用与寡糖和多肽成分的基板提出。我们将尝试结晶截短版本的钙网蛋白，具有增强的能力，结合多肽底物，也结晶钙网蛋白复合物与鸡IgY片段。第二个具体目标将探讨钙网蛋白介导的交叉呈递的机制。钙网蛋白和钙网蛋白相关肽在交叉呈递过程中的细胞内运输将被评估，以研究内体-高尔基体网络-ER运输途径的假设。还将评估与凋亡细胞相关的抗原交叉呈递对钙网蛋白的需求。最后，将评估ER应激对钙网蛋白运输、细胞表面表达以及与抗原呈递细胞上的受体的相互作用的影响。了解钙网蛋白功能的分子机制，并阐明增强钙网蛋白T细胞引发活性的条件，将有助于更有效的疫苗设计。