PNA RECEPTORS OF ACTIVATED LYMPHOCYTES

描述（由申请人提供）：本提案的目标是利用花生凝集素（PNA）识别在活化淋巴细胞上具有糖基化变化的糖蛋白，并了解CD45上相应碳水化合物变化的生物学功能。在初步实验中，申请人已经证明CD45是活化的B、CD4+和CD8+ T细胞上的主要PNA受体。研究表明，在活化的T细胞中，CD45二聚化抑制其磷酸酶活性，o -聚糖的唾液酰化可影响其二聚化。申请人希望了解唾液酸是否在活化的B细胞中CD45二聚化中起重要作用。第一个目标将是使用PNA沉淀来识别激活的B细胞和T细胞上的所有PNA受体。第二个目标是确定导致CD45变成PNA阳性的机制。代谢标记将用于确定PNA反应性CD45是现有CD45的重新合成还是脱氮化的结果。基因微阵列技术和实时PCR技术将用于研究候选基因的基因表达水平。第三个目标将集中于与Anne Dell博士的合作，以确定静止和激活B细胞上CD45的碳水化合物结构。在第四个目标中，CD45将被研究以确定它是否在B细胞系中二聚体化。实验还将使用ST3Gal 1敲除小鼠来确定去脱氮化是否是CD45形成二聚体的原因。与CD45相关的下游信号事件也将被研究。本研究将为理解CD45在B细胞中的功能调控提供依据。

DESCRIPTION (provided by applicant): The goal of this proposal is to identify glycoproteins that have glycosylation changes on activated lymphocytes using peanut agglutinin (PNA) and to understand the biological functions of the corresponding carbohydrate changes on CD45. In preliminary experiments the applicant has demonstrated that CD45 is the major PNA receptor on activated B, CD4+ and CD8+ T cells. It has been shown that in activated T cells, CD45 dimerizes to inhibit its phosphatase activity and sialylation of O-glycans can affect its dimerization. The applicant wishes to understand whether sialic acid plays an important role in CD45 dimerization in activated B cells. The first aim will be to use PNA precipitation to identify all PNA receptors on activated B and T cells. The second aim is to determine the mechanism that causes CD45 to become PNA positive. Metabolic labeling will be used to determine whether PNA reactive CD45 is the result of de novo synthesis or desialylation of existing CD45. Gene microarray technology and real-time PCR will be used to investigate the gene expression level of the candidate genes. Aim three will focus on a collaboration with Dr. Anne Dell to determine the carbohydrate structures of CD45 on resting and activated B cells. In the fourth aim, CD45 will be investigated to determine whether it dimerizes in a B cell line. Experiments will also be done to determine whether desialylation is the reason for CD45 to form dimers using ST3Gal 1 knock-out mice. Downstream signaling events that related with CD45 will also be investigated. This study will provide evidence for understanding the regulation of CD45 function in B cells.