PNA RECEPTORS OF ACTIVATED LYMPHOCYTES

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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.