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

描述（由申请人提供）：记忆B细胞活化的分子研究由于其相对稀缺和缺乏有效的技术方法来揭示记忆细胞与幼稚B细胞所采用的独特活化途径而受到阻碍。大多数记忆B细胞表达含IgG的B细胞受体，而初始B细胞表达IgM/IgD。相对于IgM/IgD，IgG/IgE受体具有延长的胞质尾区，其含有保守的磷酸酪氨酸（pTyr）基序并赋予增强的存活和增殖。该提案利用尖端蛋白质组学方法、基因沉默方法和小鼠模型来阐明IgM/IgD与IgG基信号传导的差异。由于酪氨酸激酶/磷酸酶活性已知对BCR信号传导至关重要，我们的分析集中于确定IgG与IgM/IgD受体诱导的下游底物酪氨酸磷酸化的差异。根据我们的初步研究，我们预计会发现三类新型pTyr残基：（1）已知BCR信号传导效应物上的新型pTyr位点;（2）已知蛋白质上以前与BCR信号传导中间体无关的pTyr位点;和（3）新型蛋白质上的pTyr位点。将使用基因沉默方法在B细胞系和原代表达IgG的B细胞中对带有这些新pTyr位点的蛋白质进行功能表征。将应用进一步的质谱方法来表征与新型pTyr残基结合的蛋白质。确定这些效应物的功能重要性和信号传导特性将极大地帮助我们理解IgG表达如何指导记忆B细胞增殖和分化。 公共卫生相关性：许多疫苗的效力取决于长寿命记忆B细胞的抗原特异性激活，以在病原体再暴露时快速分化成高亲和力抗体产生细胞。在慢性自身免疫性疾病中，致病性抗体的产生也可能来源于记忆B细胞的增殖和随后的分化。尽管它们的重要性，记忆B细胞激活的分子研究已受到阻碍，他们的相对稀缺和缺乏有效的技术方法来揭示记忆与naove B细胞所采用的独特的激活途径。大多数记忆B细胞表达含IgG的B细胞受体，而幼稚B细胞表达IgM/IgD。该提案利用最先进的蛋白质组学方法，基因沉默方法和小鼠模型来鉴定和评估IgM/IgD与IgG信号传导的差异。由于酪氨酸激酶/磷酸酶活性已知对BCR信号传导至关重要，我们的分析集中于确定IgG与IgM/IgD受体诱导的下游底物酪氨酸磷酸化的差异。确定这些效应物的功能重要性和信号传导特性将极大地帮助我们理解IgG表达如何指导记忆B细胞增殖和分化。

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.