Site-specific O-GlcNAc MAbs: New Tools for Glycoproteomics

描述（由申请人提供）：单个β - n -乙酰- d -氨基葡萄糖片段（O-GlcNAc）对细胞核和细胞质蛋白的丝氨酸和苏氨酸的o -糖基化是一种普遍存在的翻译后修饰，它是高度动态的，并且在细胞刺激下波动。O-GlcNAc经常与蛋白磷酸化发生竞争，这两种修饰在调控信号、转录以及癌基因和肿瘤抑制因子的功能方面存在广泛的串扰。糖尿病是这一应用的生物学驱动因素，是胰岛素抵抗水平升高、胰岛素抗凋亡作用抑制、循环O-GlcNAc改变已知会破坏正常信号的疾病的主要例子，并且与脂肪细胞因子水平的诱导、糖异生的解除和胰岛素基因转录的调节有关。我们将利用一种新的免疫原策略来开发位点特异性的O-GlcNac抗体，这些抗体针对三种在糖尿病信号抑制中起作用的O-GlcNac修饰蛋白的四个位点。因此，如果我们取得成功，除了为该策略提供测试系统外，在这项初步研究中产生的单克隆抗体将对糖尿病研究产生直接影响。我们预测，在第一阶段结束时，我们将展示一种策略，使糖科学公司能够以已知和可控的成本生产广泛的O-GlcNAc位点特异性单克隆抗体。我们认为这些将对疾病研究产生深远的影响。

DESCRIPTION (provided by applicant): O-glycosylation of serine and threonine of nuclear and cytoplasmic proteins by a single beta-N- acetyl-D-glucosamine moiety (O-GlcNAc) is a ubiquitous post-translational modification that is highly dynamic and fluctuates in response to cellular stimuli. O-GlcNAc often competes with protein phosphorylation, and these two modifications have extensive crosstalk in the regulation of signaling, transcription, and the functions of oncogenes and tumor suppressors. Diabetes, the biological driver for this application, is a prime example of a disease where increased levels of insulin resistance, inhibition of the anti-apoptotic action of insulin, alteration of circulating O-GlcNAc is known to disrupt normal signaling, and has been associated with the induction of adipocytokine levels, deregulation of gluconeogenesis, and modulation of insulin gene transcription. We will utilize a new immunogen strategy to develop site-specific O-GlcNac antibodies to four sites of O-GlcNAc modification on three proteins that play a role in signaling suppression in diabetes. Consequently, if we are successful, the mAbs generated in this initial study will have an immediate impact on diabetes research in addition to providing a test system for this strategy. We predict that at the end of phase 1, we will have demonstrated a strategy that will allow GlycoScientific to produce a wide range of O-GlcNAc site-specific MAbs, at a known and contained cost. We feel that these will have far reaching implications in disease research. PUBLIC HEALTH RELEVANCE: O-glycosylation of serine and threonine of nuclear and cytoplasmic proteins by a single beta-N- acetyl-D-glucosamine moiety (O-GlcNAc) is a ubiquitous post-translational modification that is highly dynamic and fluctuates in response to cellular stimuli. O-GlcNAc often competes with protein phosphorylation, and these two modifications have extensive crosstalk in the regulation of signaling, transcription, and the functions of oncogenes and tumor suppressors. Diabetes, the biological driver for this application, is a prime example of a disease where increased levels of insulin resistance, inhibition of the anti-apoptotic action of insulin, alteration of circulating O-GlcNAc is known to disrupt normal signaling, and has been associated with the induction of adipocytokine levels, deregulation of gluconeogenesis, and modulation of insulin gene transcription. We will utilize a new immunogen strategy to develop site-specific O-GlcNac antibodies to four sites of O-GlcNAc modification on three proteins that play a role in signaling suppression in diabetes. Consequently, if we are successful, the mAbs generated in this initial study will have an immediate impact on diabetes research in addition to providing a test system for this strategy.