Role of Mucin-type O-glycans in Intestinal Inflammation

描述（由申请人提供）：粘蛋白型o -聚糖是结肠粘液凝胶层的主要成分。在溃疡性结肠炎（UC）患者中观察到肠道O-糖基化改变，但这种改变是否是病因尚不清楚。o -聚糖主要由核心1-和核心3-衍生的o -聚糖组成。这两种o -聚糖的生物合成分别由core11,3 -半乳糖基转移酶（T-synthase）和core31,3 -氨基葡萄糖基转移酶（C3GnT）控制。t合酶的功能需要一个特殊的伴侣，Cosmc。人类宇宙在x染色体上，增加了这个基因的体细胞突变的重要性。在初步研究中，我们从UC患者的结肠上皮细胞分离的DNA中检测到Cosmc基因的体细胞突变，表达异常的o -聚糖。我们假设改变的o -聚糖损害粘液屏障功能，这反过来使肠道菌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，我们开发了缺乏核心1-或核心1-和核心3-衍生的o -聚糖的小鼠（分别为IEC T-syn-/-和DKO小鼠）。此外，我们还培养了他莫昔芬（TM）诱导的肠上皮细胞T-syn缺失小鼠（TM- iec T-syn-/-）。这些小鼠发生自发性结肠炎，与大量粒细胞浸润和隐蔽性脓肿有关，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IEC T-syn-/-和Rag1-/-背景的DKO小鼠表现出类似的严重结肠炎，这表明先天性免疫细胞如粒细胞在结肠炎的发展中起重要作用。这支持了o -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改变的o -聚糖是否是人类UC发病的潜在分子机制。我们建议1)分析异常o -糖基化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 -聚糖异常表达的统计能力；2)确定O-聚糖在肠道屏障功能中的作用，通过系统发育分析研究O-聚糖缺陷小鼠发病前后细菌种类或密度的变化，并通过培养无菌O-聚糖缺陷小鼠，明确检测微生物群在结肠炎发生中的作用；3)明确粒细胞浸润的启动机制，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。

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.