Functional distinctions of IgM vs. IgG-containing B cell receptors

描述(由申请人提供)：记忆B细胞激活的分子研究因其相对稀缺和缺乏有效的技术方法而受到阻碍，以揭示记忆B细胞与幼稚B细胞所使用的独特的激活途径。大多数记忆性B细胞表达含Ig G的B细胞受体，而幼稚B细胞表达Ig M/Ig D。相对于Ig M/Ig D，Ig G/Ig E受体有一个延伸的细胞质尾巴，含有保守的磷酸酪氨酸(PTyr)基序，促进了细胞的存活和增殖。这项建议利用先进的蛋白质组学方法、基因沉默方法和小鼠模型来阐明基于IgM/IGD和基于免疫球蛋白的信号传递的差异。由于酪氨酸激酶/磷酸酶活性对bcr信号转导至关重要，我们的分析集中在确定免疫球蛋白受体和免疫球蛋白M/免疫球蛋白D受体诱导下游底物酪氨酸磷酸化的差异。根据我们的初步研究，我们预计将发现三类新的pTyr残基：(1)已知BCR信号效应器上的新pTyr位点；(2)已知蛋白上以前未与BCR信号中间产物相关的pTyr位点；(3)新蛋白上的pTyr位点。含有这些新的pTyr位点的蛋白质将通过基因沉默方法在B细胞系和原代表达免疫球蛋白的B细胞中进行功能鉴定。进一步的质谱学方法将被应用于表征与新的pTyr残基结合的蛋白质。确定这些效应器的功能重要性和信号特性将极大地帮助我们理解免疫球蛋白表达如何指导记忆B细胞的增殖和分化。 公共卫生相关性：项目简介许多疫苗的效力依赖于长寿命记忆B细胞的抗原特异性激活，以便在病原体再次暴露时迅速分化为高亲和力抗体产生细胞。在慢性自身免疫性疾病中，致病抗体的产生也可能源于记忆B细胞的增殖和随后的分化。尽管它们很重要，但由于记忆B细胞的相对稀缺性以及缺乏有效的技术手段来揭示记忆B细胞与NAOVE B细胞所使用的独特激活途径，对记忆B细胞激活的分子研究一直受到阻碍。大多数记忆性B细胞表达含免疫球蛋白G的B细胞受体，而NAOVE B细胞表达IgM/IGD。这项建议利用先进的蛋白质组学方法、基因沉默方法和小鼠模型来识别和评估基于IgM/IGD和基于免疫球蛋白的信号的差异。由于酪氨酸激酶/磷酸酶活性对bcr信号转导至关重要，我们的分析集中在确定免疫球蛋白受体和免疫球蛋白M/免疫球蛋白D受体诱导下游底物酪氨酸磷酸化的差异。确定这些效应器的功能重要性和信号特性将极大地帮助我们理解免疫球蛋白表达如何指导记忆B细胞的增殖和分化。

DESCRIPTION (provided by applicant): Molecular studies of memory B cell activation have been hampered by their relative scarcity and lack of effective technical approaches to unveil the distinctive activation pathways employed by memory versus naive B cells. Most memory B cells express IgG-containing B cell receptors, while naive B cells express IgM/IgD. Relative to IgM/IgD, IgG/IgE receptors have an extended cytoplasmic tail that contains conserved phosphotyrosine (pTyr) motifs and confers enhanced survival and proliferation. This proposal utilizes cutting edge proteomic approaches, gene silencing methods and mouse models to elucidate differences in IgM/IgD- versus IgG-based signaling. As tyrosine kinase/phosphatase activity is known to be critical for BCR signaling, our analysis is focused on determining differences in induced tyrosine phosphorylation of downstream substrates by IgG versus IgM/IgD receptors. Based upon our pilot studies we expect to discover three classes of novel pTyr residues: (1) Novel pTyr sites on known BCR signaling effectors; (2) pTyr sites on known proteins not previously associated with BCR signaling intermediates; and (3) pTyr sites on novel proteins. Proteins bearing these novel pTyr sites will be functionally characterized using gene silencing methods in B cell lines and primary IgG-expressing B cells. Further mass spectrometry methods will be applied to characterize proteins binding to the novel pTyr residues. Determining the functional importance and signaling properties of these effectors should greatly aid our understanding of how IgG expression directs memory B cell propagation and differentiation. PUBLIC HEALTH RELEVANCE: Project Narrative The efficacy of many vaccines depends upon the antigen-specific activation of long-lived memory B cells to rapidly differentiate into high-affinity antibody producing cells upon pathogen re-exposure. In chronic autoimmune diseases, production of pathogenic antibody may also be derived from the propagation and subsequent differentiation of memory B cells. Despite their importance, molecular studies of memory B cell activation have been hampered by their relative scarcity and lack of effective technical approaches to unveil the distinctive activation pathways employed by memory versus naove B cells. Most memory B cells express IgG-containing B cell receptors, while naove B cells express IgM/IgD. This proposal utilizes cutting edge proteomic approaches, gene silencing methods and mouse models to identify and evaluate differences in IgM/IgD- versus IgG-based signaling. As tyrosine kinase/phosphatase activity is known to be critical for BCR signaling, our analysis is focused on determining differences in induced tyrosine phosphorylation of downstream substrates by IgG versus IgM/IgD receptors. Determining the functional importance and signaling properties of these effectors should greatly aid our understanding of how IgG expression directs memory B cell propagation and differentiation.