IN SITU TRANS LIGANDS OF CD22 IDENTIFIED BY GLYCAN-PROTEIN PHOTOCROSS-LINKING

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 CD22是一种B细胞信号调节因子，它识别糖蛋白多糖上的NeuAcalpha2-6Gal序列作为配体。CD22与同一细胞(顺式)和对向细胞(反式)上的糖蛋白相互作用，调节其在B细胞受体信号转导中的活性。虽然CD22主要识别邻近的CD22分子为B细胞上的顺式配体，但对相对细胞上的反式配体知之甚少。我们进行了一项蛋白质组学规模的研究，通过紫外光交联CD22-Fc嵌合体与B细胞糖蛋白结合，鉴定B细胞上CD22的候选反式配体。B细胞糖蛋白被设计为携带带有9-芳基叠氮的唾液酸。采用基于质谱学的定量蛋白质组学方法对交联产物进行分析，确定27种糖蛋白为候选反式配体。接下来，表达在一个细胞表面的CD22被光交联到相对的B细胞上的糖蛋白上，然后对带有候选配体抗体的产物进行免疫化学分析。在许多候选配体中，只有B细胞受体IgM被发现是CD22的主要原位反式配体，当与对面细胞上的CD22相互作用时，该配体选择性地重新分布到细胞接触部位。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. CD22, a regulator of B-cell signaling, is a siglec that recognizes the sequence NeuAcalpha2-6Gal on glycoprotein glycans as ligands. CD22 interactions with glycoproteins on the same cell (in cis) and apposing cells (in trans) modulate its activity in B-cell receptor signaling. Although CD22 predominantly recognizes neighboring CD22 molecules as cis ligands on B-cells, little is known about the trans ligands on apposing cells. We conducted a proteomics scale study to identify candidate trans ligands of CD22 on B-cells by UV photocross-linking CD22-Fc chimera bound to B-cell glycoproteins engineered to carry sialic acids with a 9-aryl azide moiety. Using mass spectrometry-based quantitative proteomics to analyze the cross-linked products, 27 glycoproteins were identified as candidate trans ligands. Next, CD22 expressed on the surface of one cell was photocross-linked to glycoproteins on apposing B-cells followed by immunochemical analysis of the products with antibodies to the candidate ligands. Of the many candidate ligands, only the B-cell receptor IgM was found to be a major in situ trans ligand of CD22 that is selectively redistributed to the site of cell contact upon interaction with CD22 on the apposing cell.