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

O-GalNAc糖基化与Tn抗原表达的改变是癌症和病理状态的标志。在本项目中，将研究cosmc衍生的差异O-GalNAc糖基化对胰腺上皮内瘤变（PanIN）、导管内乳头状粘液瘤（IPMN）、胰腺腺癌（PDAC）和慢性胰腺炎（CP）对胰腺内分泌和外分泌功能的影响。为了使这项研究成为可能，我们产生了条件Cosmc-KO和条件过表达多肽- galnac -转移酶2 （GalNT2）小鼠系，并将其与胰腺特异性Cre小鼠系Ptf1a、Pdx1和Sox9配对。Cosmc基因敲除，GalNT2过表达可能是导致胰腺癌和胰管腺癌前体病变中Tn抗原表达的分子机制。本项目的一个基本目标应该是鉴定胰腺O-GalNAc聚糖，以及差异o -糖基化对野生型和转基因小鼠外分泌胰腺功能的影响。我们将研究O-GalNAc糖基化改变对腺泡细胞中蛋白质表达、定位和活性的影响，以及对细胞生物学过程的影响，以及对炎症、癌变、代谢疾病的发展、外分泌胰腺功能障碍和脂质代谢的可能影响。小鼠模型的结果将与人类PanIN、IPMN、PDAC和CP患者的样本进行比较。来自小鼠和患者胰腺样本的携带Tn抗原的蛋白将通过Tn抗原特异性凝集素层析富集，随后通过质谱分析鉴定。O-GalNAc糖基化改变的潜在影响将在Tn抗原小鼠模型中通过检查胰腺依赖的临床相关参数来检测。鉴定的携带Tn抗原的蛋白将通过生化和细胞生物学手段进行表征，以描述O-GalNAc糖基化改变的分子效应。由于t合酶活性的变化会引起O-GalNAc糖基化的超水平变化，因此将对PDAC细胞系和PanIN、IPMN、PDAC和CP的人体组织进行t合酶活性测定。因此，t合酶活性与Tn抗原表达之间可以直接相关。该项目将导致鉴定和提高对O-GalNAc糖基化蛋白在胰腺中的生理和病理生理功能的理解。

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.