Modulation of MUC1 Mucin Trafficking

MUC1 粘蛋白运输的调节

• 批准号: 6517528
• 负责人: Rebecca P Hughey
• 金额: $ 26.63万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6517528
• 关键词: CHO cells; MDCK cell; biomarker; endocytosis; enzyme inhibitors; fatty acylation; gene expression; glycoprotein biosynthesis; glycoproteins; glycosylation; hexosyltransferase; immunoprecipitation; intracellular transport; isomerase; metastasis; molecular pathology; molecular site; mucins; palmitates; phosphorylation; protein localization; protein structure function; proteolysis; radiotracer; site directed mutagenesis; western blottings

DESCRIPTION (applicant's description): MUC1 is a well characterized marker for many cancers of epithelial origin that also directly contributes to the aggressive metastatic phenotype of tumors. Cumulative data from studies of these cancer patients indicates that high levels of particularly intracellularMUC1 in the tumor and in serum correlates with a strong potential for metastasis and a poor prognosis for the patient. The anti-adhesive property of this normally apical, transmembrane mucin (with 40-90 heavily 0-glycosylated tandem repeats of 20 aa) disrupts both cell-cell and cell-matrix interactions in non-polarized tumor cells, thus enhancing metastasis. The soluble MUC1 in patient serum and ascites also contributes to the aggressive phenotype of the cancer by acting as a tumor decoy by forming immunocomplexes with antibodies, by binding T-cells and blocking their proliferation, and by rebinding to the tumor and initiating signal transduction pathways. Since MUC1 has fewer and smaller 0-glycans in many tumors, preliminary studies were designed to understand how this contributes to the intracellular accumulation and shedding of MUC1 since the mechanisms for either event is unknown. Using normal and glycosylation-defective CHO cells, metabolic labeling and biotinylation, we find that cell surface expression of recombinant human MUC1 is dependent on 0-glycosylation. Surprisingly, we find that this enormous molecule is internalized by clathrin-mediated endocytosis indicating that the 40-90 tandem-repeat immunodominanat epitopes of MUCI are an ideal target for immunotoxin therapy. Thus, it is essential to understand what regulates MUC1 endocytosis and shedding to better target treatments to MUC1-positive tumors and avoid soluble MUCI in serum/ascites. We find that MUC1 endocytosis is blocked by mutation of a potential site for dual palmitoylation and enhanced by smaller 0-glycans, indicating that its trafficking may be modulated by homotypic clustering and association with lipid microdomains involved in signal transduction. Since we also find that MUCI shedding is endocytosis-dependent, our specific aims are designed to understand how MUCI trafficking and shedding is modulated by its 0-glycan structure, palmitoylation, and phosphorylation. Initial experiments will utilize non-polarized CHO cells while later experiments in new glycosylation-defective MDCK cells will define apical-specific modulation of MUCI trafficking.

描述（申请人的描述）：MUC 1是一种充分表征的标记物，用于 许多上皮来源的癌症也直接导致 肿瘤侵袭转移表型。研究的累积数据 这些癌症患者表明， 肿瘤和血清中的细胞内MUC 1与肿瘤细胞内MUC 1的表达密切相关。 转移的可能性和患者的不良预后。的 这种通常顶端的跨膜粘蛋白的抗粘附特性（具有40-90 20个氨基酸的高度0-糖基化串联重复序列）破坏细胞-细胞， 非极化肿瘤细胞中的细胞-基质相互作用，从而增强 转移患者血清和腹水中的可溶性MUC 1也有助于 癌症的侵袭性表型通过形成肿瘤诱饵而起作用， 与抗体的免疫复合物，通过结合T细胞并阻断其 增殖，并通过重新结合到肿瘤和启动信号转导 途径。由于MUC 1在许多肿瘤中具有更少和更小的0-聚糖， 初步研究旨在了解这如何有助于 MUC 1的细胞内积累和脱落，因为 事件未知。使用正常和糖基化缺陷的CHO细胞，代谢 标记和生物素化，我们发现重组体的细胞表面表达 人MUC 1依赖于O-糖基化。令人惊讶的是，我们发现， 巨大的分子通过网格蛋白介导的内吞作用内化， MUCI的40-90个串联重复的免疫优势表位是理想的 免疫毒素治疗靶点。因此，必须了解 调节MUC 1内吞和脱落，以更好地靶向治疗， MUC 1阳性肿瘤，避免血清/腹水中的可溶性MUCI。我们发现MUC 1 双重棕榈酰化潜在位点的突变阻止了内吞作用 并被较小的0-聚糖增强，表明其运输可能是 通过同型聚类和与脂质微区的关联来调节 参与信号传导。由于我们还发现MUCI脱落是 内吞依赖，我们的具体目标是了解MUCI如何 运输和脱落由其0-聚糖结构调节， 棕榈酰化和磷酸化。最初的实验将利用 非极化CHO细胞，而后来的实验中，新的糖基化缺陷 MDCK细胞将定义MUCI运输的顶端特异性调节。