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: 7881344
• 负责人: MALINI RAGHAVAN
• 金额: $ 2.29万
• 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: 2009-07-14 至 2011-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7881344
• 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.

说明(申请人提供)：钙网蛋白是一种内质网(ER)伴侣蛋白，促进糖蛋白的折叠和组装，包括主要组织相容性复合体(MHC)I类分子。钙网蛋白还具有引导外源抗原进入MHC-I类抗原递呈途径的能力，这种现象称为交叉递呈。作为一种凝集素，钙网蛋白与糖蛋白上的单糖化核心糖链相互作用。在一定条件下，钙网蛋白能够结合底物的多肽成分。钙耗竭和热处理暴露了钙网织蛋白的多肽结合部位，并在体外和体内细胞中增强了钙网织蛋白与多肽底物的结合。这些处理还会诱导钙网蛋白二聚化和寡聚化。此外，其他类型的内质网应激，包括病毒感染和衣霉素处理，也可以诱导钙网织蛋白二聚体的形成。我们的假设是，这些构象转变和多肽结合特性对于钙网蛋白在细胞中的蛋白质折叠和交叉启动功能是重要的。第一个具体目的是探索多肽与钙网蛋白结合在细胞内MHC I类折叠和组装过程中的作用。我们提出了部分蛋白分解和基于质谱学的方法来识别钙缺乏所动员的钙网蛋白亚域。保守的钙网蛋白疏水残基，预计会暴露在表面，将突变为丙氨酸。在体外与MHC I类重链多肽成分相互作用出现缺陷的突变体，以及其他在结合寡糖底物方面存在缺陷的突变体，将在钙网蛋白缺陷的细胞中表达，并评估其促进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钙网蛋白-底物相互作用周期的工作模型，在该循环中，提出了与底物的寡糖和多肽成分的交替相互作用。我们将尝试结晶具有增强结合多肽底物能力的截短版本的钙网织蛋白，并结晶与鸡IgY片段的钙网织蛋白复合体。第二个具体目的是探索钙网蛋白介导的交叉呈现的机制。在交叉展示过程中，将评估钙网蛋白和钙网蛋白相关多肽在细胞内的运输，以调查内体-跨高尔基网络-内质网运输路线的假说。对钙网蛋白交叉呈递与凋亡细胞相关的抗原的需求也将进行评估。最后，将评估内质网应激对钙网蛋白运输、细胞表面表达以及与抗原提呈细胞上的受体相互作用的影响。了解钙网蛋白功能的分子机制，阐明增强钙网蛋白T细胞启动活性的条件，将有助于更有效地设计疫苗。