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由于其相对稀缺和缺乏有效的技术方法来揭示记忆与幼稚B细胞所采用的独特激活途径而受到阻碍。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受体有一个延长的细胞质尾部，包含保守的磷酸酪氨酸（pTyr）基序，并赋予增强的存活和增殖。本研究利用尖端的蛋白质组学方法、基因沉默方法和小鼠模型来阐明IgM/IgD与基于igg的信号传导的差异。由于已知酪氨酸激酶/磷酸酶活性对BCR信号传导至关重要，因此我们的分析重点是确定IgG与IgM/IgD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，我们希望发现三类新的pTyr残基：(1)已知BCR信号效应物上的新pTyr位点；(2)已知蛋白上的pTyr位点先前与BCR信号中间体无关；(3)新蛋白上的pTyr位点。携带这些新的pTyr位点的蛋白将在B细胞系和原代表达igg的B细胞中使用基因沉默方法进行功能表征。进一步的质谱分析方法将被用于表征结合新的pTyr残基的蛋白质。确定这些效应物的功能重要性和信号特性将极大地帮助我们理解IgG表达如何指导记忆B细胞的繁殖和分化。

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.