Analysis of core 1-derived O-GalNAc modified glycoproteins in a conditional transgenic Cosmc knockout mouse in the pancreas.

伴随Tn抗原表达的O-GalNAc糖基化的改变是癌症和病理状态的标志。在该项目中，将检查Cosmc衍生的差异O-GalNAc糖基化（如在胰腺上皮内瘤形成（PanIN）、导管内乳头状粘液性肿瘤（IPMN）、胰腺腺癌（PDAC）和慢性胰腺炎（CP）中观察到的）对胰腺内分泌和外分泌功能的影响。产生条件性过表达多肽-GalNAc-转移酶2（GalNT 2）小鼠系，并与胰腺特异性Cre小鼠系Ptf 1a、Pdx 1和Sox 9交配。Cosmc基因敲除和GalNT 2过表达可能是导致胰腺癌和胰腺导管腺癌前体病变中Tn抗原表达的分子机制，本研究的基本目标是鉴定胰腺O-GalNAc聚糖，并研究不同O-糖基化对野生型和转基因小鼠胰腺外分泌功能的影响。将检查改变的O-GalNAc糖基化对蛋白质表达、定位和活性的影响，以及对腺泡细胞中的细胞生物学过程的影响，以及对炎症、致癌和代谢疾病、外分泌胰腺功能障碍和脂质代谢的发展的可能影响。将来自小鼠模型的结果与来自人PanIN、IPMN、PDAC和CP患者的样品进行比较。将通过Tn抗原特异性凝集素色谱法富集来自小鼠胰腺和患者样品的Tn抗原携带蛋白，随后通过质谱分析鉴定。通过检查Tn抗原小鼠模型中的胰腺依赖性临床相关参数，检测O-GalNAc糖基化改变的潜在影响。由于T-合酶活性的变化引起O-GalNAc糖基化在上位水平上的变化，因此将测定PDAC细胞系和PanIN、IPMN、PDAC和CP的人组织的T-合酶活性。因此，T-合成酶活性和Tn抗原表达之间的直接相关性可以执行。该项目将导致识别和改善的理解的生理和病理生理功能的O-GalNAc糖基化蛋白在胰腺。

Alterations in O-GalNAc glycosylation with expression of the Tn antigen is a hallmark of cancer and pathological states. In this project, the influence of Cosmc-derived differential O-GalNAc glycosylation, as observed in pancreatic intraepithelial neoplasias (PanIN), intraductal papillary mucinous neoplasms (IPMN), pancreatic adenocarcinomas (PDAC) and chronic pancreatitis (CP), on the function of the endo- and exocrine pancreas will be examined.To make such investigation possible a conditional Cosmc-KO, and a conditionally overexpressing polypeptide-GalNAc-transferase 2 (GalNT2) mouse line was generated and mated with the pancreas-specific Cre mouse lines Ptf1a, Pdx1 and Sox9. The molecular knockout of Cosmc, with overexpression of GalNT2 is probably the molecular mechanism resulting in expression of the Tn antigen in precursor lesions of pancreatic cancer and pancreatic ductal adenocarcinoma.A fundamental objective of this project should be the identification of pancreatic O-GalNAc glycans and the influence of differential O-glycosylation on the exocrine pancreatic function in wild type and transgenic mice. The influence of the altered O-GalNAc glycosylation on protein expression, localization and activity, as well as on cell biological processes in acinar cells, and a possible influence on inflammation, carcinogenesis and the development of metabolic diseases, exocrine pancreatic dysfunction and lipid metabolism will be examined. Results from the mouse models will be compared with samples from human PanIN, IPMN, PDAC and CP patients. Tn antigen-bearing proteins from the pancreas of mice and patient samples will be enriched via Tn antigen-specific lectin chromatography and subsequently identified by mass spectrometry analysis. The potential influence of altered O-GalNAc glycosylation is to be tested by examining pancreatic dependent clinically relevant parameters in the Tn antigen mouse model. The identified Tn antigen-bearing proteins will be characterized by biochemical and cell biological means to describe molecular effects of altered O-GalNAc glycosylation.Because changes in the T-synthase activity cause changes in the O-GalNAc glycosylation at a superordinated level, PDAC cell lines and human tissues of PanIN, IPMN and PDAC and CP will be assayed for T-sythase activity. Thus a direct correlation between T-synthase activity and Tn antigen expression can be performed.This project will lead to the identification and an improved understanding of the physiological and pathophysiological functions of O-GalNAc glycosylated proteins in the pancreas.