Analysis of B cell memory and activation

我们先前发现激活诱导型胞苷脱氨酶(AID)是必需的，并控制两个基因改变系统，即免疫球蛋白类开关重组和体细胞超突变。然而，目前还不清楚艾滋病是如何发生的，并以不同的方式控制这两个事件。为了了解类开关重组和体细胞超突变是如何受AID调控的，我们试图确定AID的辅助因素。我们采取了两种方法来提高艾滋病病毒结合蛋白的候选水平。一种是以AID和AID突变体为诱饵的双杂交筛选。经过对文库的筛选，我们筛选出了30个在8个细胞中表达并在双杂交水平上与AID蛋白发生物理作用的候选基因。另一种方法是免疫共沉淀法，使用表达标志多肽标记的AID的哺乳动物细胞系。免疫沉淀法浓缩AID复合体，经SDS-PAGE电泳法分离，质谱法测定AID或AID突变体特异性沉淀物。以往的研究提供了两种类型的AID突变体(类开关缺陷突变体和体细胞高突变缺陷突变体)。类开关缺陷AID突变体在C末端没有16个氨基酸。另一方面，体细胞高突变缺陷AID突变体确实存在N末端的错义突变.将这两个突变体用于提取AID结合蛋白。根据其在B细胞中的表达、与AID/突变体的结合方式和基因信息，我们筛选出8个可能的AID辅助因子。现在，为了了解候选基因的功能意义，我们通过转导siRNA来限制候选基因的表达。当我们敲掉一个特定候选基因的表达时，我们观察到类转换诱导活性降低了90%。对这个候选基因的进一步研究应该知道为什么类转换抑制是通过该候选基因的还原酶表达水平来观察到的。

We previously revealed that activation induced cytidine deaminase (AID) is essential and control two genetic alteration systems namely immunoglobulin class switch recombination and somatic hypermutation. Still it is elusive how AID undergoes and differentially controls such two events. In order to understand how class switch recombination and somatic hypermutation are regulated by AID, we are trying to identify cofactors for AID. We took two approaches to raise possible candidates for AID binding protein. One is two hybrid screening using AID and AID mutants as baits. After cDNA screening of libraries, we isolated 30 candidates that are expressed in 8 cells and physically interact with AID protein at the level of two-hybrid assay. The other approach was co-immunoprecipitation using mammalian cell lines that express AID tagged with flag peptide. AID complex was enriched by the immunoprecipitation and resolved by SDS-PAGE, and then AID or AID mutant-specific precipitants were decided by mass analysis. Previous studies provided two kinds of AID mutants (class switch defective-and somatic hypermutation defective mutants). Class switch defective AID mutants do not have 16 amino acids at C-terminus. On the other hand, somatic hypermutation defective AID mutants do have missense mutation an N-terminus. The two mutants were applied to take AID binding protein. Based on expression in B cells, mode of binding with AID/mutants and cDNA information, we took eight candidates as reasonable and possible AID cofactors. Now to see functional significance for the candidates, we limit expression of candidates by transduction of siRNA. When we knock down expression of one particular candidate, we observed 90% reduction of class switch inducing activity. Further study of this candidate should be done to know why class switch inhibition is observed by reducina expression level of the candidate.