Role of Mucin-type O-glycans in Intestinal Inflammation

说明(申请人提供)：粘蛋白型O-葡聚糖是结肠粘液凝胶层的主要成分。溃疡性结肠炎(UC)患者已观察到肠道O-糖基化改变，但这种改变是否为病因尚不清楚。O-葡聚糖主要由核心1和核心3衍生的O-葡聚糖组成。这两类O-葡聚糖的生物合成分别受核心11，3-半乳糖基转移酶(T-合成酶)和核心31，3-氨基葡萄糖转移酶(C3GnT)控制。T-合成酶的功能需要一个特定的伴侣Cosmc。人类Cosmc位于X染色体上，增加了该基因体细胞突变的重要性。在初步研究中，我们从结肠上皮细胞分离的DNA中检测到Cosmc基因的体细胞突变，表达UC患者异常的O-糖链。我们推测，改变的O-葡聚糖损害粘液屏障功能，这反过来允许肠道微生物群与上皮和粘膜免疫细胞异常相互作用，从而导致结肠炎。为了验证这一假设，我们建立了缺乏核心1-或核心1-和核心3衍生O-多糖的组合的小鼠(分别为IEC T-syn-/-和DKO小鼠)。此外，我们还培育了他莫昔芬(TM)可诱导肠上皮细胞T-syn缺失的小鼠(TM-IEC T-syn-/-)。这些小鼠发展为自发性结肠炎，与大量粒细胞渗透和隐匿性脓肿有关，与活动性人类UC非常相似。值得注意的是，缺乏适应性免疫的Rag1-/-背景的IEC T-SYN-/-和DKO小鼠表现出类似的严重结肠炎，这表明粒细胞等先天免疫细胞在结肠炎的发展中起着至关重要的作用。这支持了O-葡聚糖在结肠炎中的病因学作用，并提供了一个独特的模型系统来测试O-葡聚糖的改变是否是人类UC发病机制中的一个潜在分子机制。我们建议1)分析O-糖基化异常如何损害肠粘蛋白的表达，并增加额外的患者样本，以提高我们初步观察到的COSMC突变导致UC患者结肠上皮O-糖链异常表达的统计能力；2)确定O-糖链在肠屏障功能中的作用，通过系统发育分析调查O-糖基化缺陷小鼠发病前后细菌种类或密度的变化，并通过建立无菌的O-糖链缺陷小鼠，明确微生物区系在结肠炎发生发展中的作用；3)确定启动粒细胞渗透的机制并确定粒细胞在结肠炎中的作用。我们提出的研究将揭示结肠炎发病机制的新见解，并可能导致新的治疗方法。 公共卫生相关性：溃疡性结肠炎(UC)是一种原因不明或治愈方法不明的慢性大肠炎症，可持续数年至数十年。UC通常会导致身体和心理上的不适，甚至残疾。UC患者大肠中被称为O-葡聚糖的大糖分子的表达发生了变化。然而，这种改变是否会导致这种疾病尚不清楚。这项拟议的项目有望为肠道粘蛋白O-葡聚糖在肠道粘液屏障功能和常见人类疾病发病机制中的作用提供新的见解。这项工作可能会为UC患者带来一种新的治疗方法。

DESCRIPTION (provided by applicant): Mucin-type O-glycans are primary components of the colonic mucus gel layer. Altered intestinal O- glycosylation has been observed in patients with ulcerative colitis (UC), but whether this alteration is an etiological factor is unknown. O-glycans consist mainly of core 1- and core 3-derived O-glycans. The biosynthesis of these two types of O-glycans is controlled by core 11,3-galactosyltransferase (T-synthase) and Core 31,3-glucosaminyltransferase (C3GnT), respectively. T-synthase function requires a specific chaperone, Cosmc. Human Cosmc is on the X-chromosome, increasing the significance of somatic mutations in this gene. In preliminary studies, we detected somatic mutations in the Cosmc gene in DNA, isolated from colonic epithelial cells, expressing abnormal O-glycans from UC patients. We hypothesize that altered O-glycans impair mucus barrier function, which in turn allows intestinal microflora to interact abnormally with epithelium and mucosal immune cells, thus causing colitis. To test this hypothesis, we developed mice lacking either core 1- or combined core 1- and core 3-derived O-glycans (IEC T-syn-/- and DKO mice, respectively). In addition, we have also developed mice with tamoxifen (TM) inducible deletion of T-syn in intestinal epithelial cells (TM-IEC T-syn-/-). These mice develop spontaneous colitis, which is associated with a massive granulocyte infiltration and cryptic abscesses, closely resembling active human UC. Significantly, IEC T-syn-/- and DKO mice in the Rag1-/- background, who lack adaptive immunity, manifested similarly severe colitis, suggesting an essential role for innate immune cells such as granulocytes in colitis development. This supports an etiological role for O-glycans in colitis and provides a unique model system to test whether altered O-glycans is a potential molecular mechanism in the pathogenesis of human UC. We propose to 1) analyze how abnormal O-glycosylation impairs the expression of intestinal mucins and add additional patient samples to improve the statistical power of our preliminary observations that Cosmc mutations cause abnormal expression of colon epithelial O-glycans in UC patients; 2) determine the role of O- glycans in intestinal barrier function, investigate changes in bacterial variety or density in O-glycan-deficient mice before and after disease onset by phylogenetic analysis, and test definitively the role of microbiota in colitis development by developing germ-free O-glycan-deficient mice; and 3) identify mechanisms initiating granulocyte infiltration and determine the role of granulocytes in colitis. Our proposed studies will reveal novel insights into the pathogenesis of colitis and may lead to new therapies. PUBLIC HEALTH RELEVANCE: Ulcerative colitis (UC) is a chronic inflammation of the large intestine with an unknown cause or cure that can last years to decades. UC often leads to physical as well as psychological discomfort and even disability. Altered expression of large sugar molecules called O-glycans in the large intestine is seen in UC patients. However, whether this alteration causes the disease is unknown. The proposed project is poised to provide novel insights into the role of intestinal mucin-type O-glycans in intestinal mucus barrier function and in pathogenesis of the common human disease. This work may lead to a novel therapy for patients with UC.