Defining the interaction of FCRLA with immunoglobulin

定义 FCRLA 与免疫球蛋白的相互作用

• 批准号: 8292374
• 负责人: PETER Daniel BURROWS
• 金额: $ 7.33万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-15 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8292374
• 关键词: Alleles; Antibodies; Antibody Formation; Antigens; Apoptosis; Autoimmunity; B Cell Proliferation; B cell differentiation; B-Cell Activation; B-Lymphocytes; Backcrossings; Binding; Biochemical; Biology; Cell Cycle; Cell Line; Cell Lineage; Cell surface; Cells; Complex; Confocal Microscopy; Defect; Disease; Endoplasmic Reticulum; Ensure; Evaluation; Fc Receptor; Fostering; Funding; Gene Expression Profile; Gene Family; Gene Targeting; Genetic Polymorphism; Glean; Glycoproteins; Goals; Golgi Apparatus; Guidelines; Human; Human Cell Line; Immune response; Immune system; Immunoglobulin A; Immunoglobulin Class Switching; Immunoglobulin Domain; Immunoglobulin G; Immunoglobulin Genes; Immunoglobulin Isotypes; Immunoglobulin M; Immunoglobulin Somatic Hypermutation; Immunoglobulin binding proteins; Immunoglobulins; Immunologic Deficiency Syndromes; In Vitro; J-Chain Immunoglobulins; Knockout Mice; Knowledge; Lead; Link; Membrane; Metabolism; Mission; Modeling; Molecular Chaperones; Mus; Mutagenesis; Nature; Negative Finding; Organelles; Paper; Pathway interactions; Peptide Signal Sequences; Phenotype; Play; Positioning Attribute; Process; Protein Isoforms; Protein Secretion; Proteins; Public Health; Publishing; Quality Control; Reporting; Research; Research Project Grants; Role; Site; Staging; Structure of germinal center of lymph node; System; Transmembrane Domain; United States National Institutes of Health; Up-Regulation; base; cell growth; embryonic stem cell; extracellular; glycosylation; innovation; insight; member; novel; overexpression; pathogen; promoter; protein distribution; receptor; small hairpin RNA; variable region gene

DESCRIPTION (provided by applicant): The intracellular assembly of immunoglobulins (Ig) in B lineage cells is essential for normal antibody responses and is stringently regulated at multiple levels by endoplasmic reticulum (ER) chaperones. FCRLA is an unusual member of the larger Fc receptor (FCR) and FCR-like (FCRL) gene family and its expression in hemopoietic cells is restricted to the B lineage. In marked contrast to FCRL1-6 and the "classical" Ig-binding FcR, which are transmembrane glycoproteins with extracellular immunoglobulin (Ig) domains, FCRLA is an intracellular protein. It lacks N-linked glycosylation sites and a transmembrane region, but contains a signal sequence to target it to the ER. FCRLA is a resident ER protein that associates with immunoglobulin in this organelle. Unique among Fc receptors, FCRLA can associate with multiple isotypes of Ig, IgM, IgG and IgA. The distinctive features of this protein lead to our central hypothesis: FCRLA is important in the initial metabolism of Ig in B cells and thus in normal immune system function. The significance of the proposal begins with the observation that FCRLA associates with Ig in the ER, which suggests a novel role for this protein in Ig assembly or degradation. Defects in human FCRLA expression may thus lead to autoimmunity or immunodeficiency diseases. The goal of our studies is to define the composition and functions of the Ig- FCRLA complexes. These functions could include aiding Ig folding, facilitating Ig assembly, regulating Ig transport, and/or contributing to the degradation f incompletely processed Ig molecules. FCRLA may thus play an essential role in the intracellular quality control system that ensures the correct production of antibodies. The biochemical and intracellular features of FCRL have so far only been defined in humans and the Fcrla knockout mouse has no perceptible phenotype, therefore the following studies will be done using human B cell lines: 1) Clarify the mechanism for the observed preferential association of FCRLA with the secretory isoform of IgM. 2) Determine the nature of the FCRLA-Ig complexes, including the degree of assembly of the associated Ig, identify any other Ig and/or FCRLA-associated proteins and define changes in the components of the complex that are dependent of the Ig isotype bound and/or the stage of B cell differentiation. 3) Define where along the secretory pathway FCRLA is released from Ig or if the FCRLA bound Ig is destined for destruction. 4) Pinpoint the regions of molecular interaction by mutagenesis of both FCRLA and Ig genes. 5) Ascertain FCRLA function by analysis of phenotypic changes induced by overexpression and shRNA depletion of FCRLA. The analysis here will include Ig assembly, pentamerization and J chain addition in the case of IgM, secretion, cell surface expression, and turnover. Changes in cellular growth rate, cell cycle distribution, and apoptosis will also be determined. These studies are innovative because of the novel features of FCRLA. No other FcR or Fc-like receptor has been shown to associate with IgM, IgG and IgA isotypes. Moreover, FCRLA is the only known Fc-like receptor that is an ER resident Ig-binding protein. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because the regulated assembly and transport of antibodies is essential for protective immune responses to most pathogens. The molecule we have identified and are studying, FCRLA, is positioned in a cellular compartment where it interacts with nascent antibodies and is expected to play an important role in antibody quality control. Therefore these studies are relevant to the NIH mission to foster pursuit of fundamental knowledge and reduce the burdens of human illness.

描述（由申请人提供）：免疫球蛋白（IG）在B谱系细胞中的细胞内组装对于正常抗体应答是必需的，并且在多个细胞周期中受到严格调控。 内质网（ER）伴侣水平。FCRLA是较大的Fc受体（FCR）和FCR样（FCRL）基因家族的一个不寻常的成员，并且其在造血细胞中的表达限于B谱系。与FCRL 1 -6和“经典的”Ig结合FcR（它们是具有细胞外免疫球蛋白（IG）结构域的跨膜糖蛋白）形成鲜明对比，FCRLA是细胞内蛋白。它缺乏N-连接的糖基化位点和跨膜区，但含有将其靶向ER的信号序列。FCRLA是与该细胞器中的免疫球蛋白缔合的常驻ER蛋白。FCRLA在Fc受体中是独特的，它可以与多种同种型的IG、IgM、IgG和伊加结合。这种蛋白质的独特特征导致我们的中心假设：FCRLA在B细胞中IG的初始代谢中是重要的，因此在正常的免疫系统功能中是重要的。该建议的意义始于FCRLA与ER中的IG相关的观察，这表明该蛋白质在IG组装或降解中的新作用。因此，人FCRLA表达的缺陷可能导致自身免疫或免疫缺陷疾病。我们的研究目的是确定IG- FCRLA复合物的组成和功能。这些功能可以包括帮助IG折叠、促进IG组装、调节IG转运和/或促进不完全加工的IG分子的降解。因此，FCRLA可能在确保抗体正确产生的细胞内质量控制系统中发挥重要作用。FCRL的生物化学和细胞内特征迄今为止仅在人类中定义，并且Fcrla敲除小鼠没有可察觉的表型，因此将使用人类B细胞系进行以下研究：1）阐明所观察到的FCRLA与IgM的分泌亚型的优先结合的机制。2)确定FCRLA-IG复合物的性质，包括相关IG的组装程度，鉴定任何其他IG和/或FCRLA相关蛋白，并确定依赖于结合的IG同种型和/或B细胞分化阶段的复合物组分的变化。3)确定FCRLA在分泌途径的沿着从IG中释放的位置或FCRLA结合的IG是否注定要被破坏。4)通过对FCRLA和IG基因的突变来精确定位分子相互作用的区域。5)通过分析FCRLA过表达和shRNA缺失诱导的表型变化来确定FCRLA功能。这里的分析将包括IG装配、五聚化和在IgM的情况下的J链添加、分泌、细胞表面表达和周转。还将测定细胞生长速率、细胞周期分布和细胞凋亡的变化。这些研究 由于FCRLA的新颖特征，没有其他FcR或Fc样受体已显示与IgM、IgG和伊加同种型相关。此外，FCRLA是唯一已知的作为ER驻留Ig结合蛋白的Fc样受体。 公共卫生关系：拟议的研究与公共卫生有关，因为抗体的调节组装和运输对于大多数病原体的保护性免疫反应至关重要。我们已经鉴定并正在研究的分子FCRLA位于细胞区室中，在那里它与新生抗体相互作用，预计将在抗体质量控制中发挥重要作用。因此，这些研究与NIH的使命有关，即促进对基础知识的追求，减少人类疾病的负担。