SBIR TOPIC 294 PHASE I - DEVELOPMENT OF GLYCOSYLATION-SPECIFIC RESEARCH REAGENTS

蛋白质O-GlcNAc化（N-乙酰葡糖胺修饰），减少磷酸化，在细胞周期调节，凋亡和信号转导中起着关键作用。迄今为止，已发现约120种O-GlcNAc化的蛋白质，为了阐明O-GlcNAc化的功能，有必要研制针对O-GlcNAc化蛋白质的位点特异性抗体。许多用于O-GlcNAc化的泛特异性抗体是可用的。然而，很少有针对O-GlcNAc酰化蛋白质的位点特异性抗体已成功制备。 在I期研究期间，我们将合成具有针对c-myc和p53的O-GlcNAc化位点的各种特征的多种抗原肽，以测试特定肽设计对抗体的灵敏度和特异性的影响。将确定抗体的灵敏度和特异性以找出最佳肽设计。我们将使用人乳腺细胞系、正常细胞（MCF 10A）和肿瘤细胞（MCF-7和N-ras和H-ras MCF 10A）在用和不用各种O-GlcNAc酶抑制剂处理的情况下探索O-GlcNAc化位点特异性抗体的效用。

Protein O-GlcNAcylation (N-acetylglucosamine modification), which decreases phosphorylation, plays a critical role in cell-cycle regulation, apoptosis and signal transduction. So far, ~120 O-GlcNAcylated human proteins have been found. To elucidate functions of O-GlcNAcylation, it is necessary to develop site-specific antibodies for O-GlcNAcylated proteins. Many pan-specific antibodies for O-GlcNAcylation are available. However, few site-specific antibodies for O-GlcNAcylated proteins have been successfully produced. During the Phase I study, we will synthesize multiple antigen peptides with various features against O-GlcNAcylation sites of c-myc and p53 to test effects of a specific peptide design on the sensitivity and specificity of the antibodies. Sensitivity and specificity of the antibodies will be determined to find out the best peptide design. We will explore utility of the O-GlcNAcylation site-specific antibodies using human breast cell lines, normal (MCF10A) and tumor cells (MCF-7 and N-ras and H-ras MCF10A) with and without treatment with various O-GlcNAcase inhibitors.