Role of O-glycosylation in Animal Development

发育糖生物学单位的首要目标是确定蛋白质O-糖基化调节基本生物过程的机制，以更好地了解这种修饰在发育和疾病中的作用。粘蛋白型O-连接糖基化是一种广泛存在的、在进化上保守的蛋白质修饰，由一系列酶(pgants或GalNActs)催化，将糖N-乙酰半乳糖胺(GalNAc)转移到蛋白质中注定要与膜结合或分泌的丝氨酸和苏氨酸的羟基。这种糖基化缺陷是导致人类疾病、家族性肿瘤、钙质沉着症和TN综合征的原因。此外，O-糖基化的改变与肿瘤的进展和转移有关。最近，全基因组相关研究已经确定了与高密度脂蛋白-胆固醇水平、甘油三酯水平、先天性心脏病、结肠癌和骨密度相关的基因中负责启动O-糖基化的酶的编码基因。从这些研究中，很明显，这种保守的蛋白质修饰具有多种生物学作用。我们研究小组的重点是阐明O-葡聚糖在发育过程中的机制作用，以了解它们如何促进疾病的易感性和进展。 我们课题组以前的工作表明，果蝇中至少有9个功能性转移酶基因，并且至少有一个是生存所必需的。这些研究提供了第一个证据，证明在任何真核生物中，这个多基因家族的成员都是发育和生存所必需的。最近，我们进行了体内RNA干扰(RNAi)，以确定剩余的家庭成员，这些成员也是生存所必需的。我们发现，要生存下去，需要额外的4名家庭成员。具体地说，pgant4、pgant5、pgant7或可能的糖基转移酶CG30463的RNAi导致了致死性。此外，这些必需基因是特定组织(中胚层、消化系统和气管系统)所必需的，这表明每个组织都具有独特的组织特定功能。最后，我们证明了这些新定义的必需基因之一负责正常的肠道功能。这种糖基转移酶的丢失导致O-糖基化蛋白分泌到肠腔的减少，以及负责肠道酸化的细胞的形态改变。这个家族成员的突变导致了不正确的肠道酸化。这些研究暗示了这种蛋白质修饰在高等真核生物肠道功能中的作用，因为这些基因在小鼠和人类的胃、小肠和结肠中大量表达。 继我们的果蝇研究表明O-糖基转移酶在细胞外基质(ECM)形成中的作用后，我们以小鼠下颌下腺(SMG)为模型系统，研究了哺乳动物O-糖基转移酶(Galnt1)的缺失是否对基底膜的形成和器官发生有影响。发育中的SMG的基底膜是一组复杂的成分，影响细胞信号传递、增殖和分化；此外，它还含有丰富的O-糖基化蛋白。在这些研究中，我们证明了在哺乳动物SMG器官发生过程中，Galnt1的缺失通过影响基底膜蛋白的分泌来影响成纤维细胞生长因子介导的细胞增殖。缺乏Galnt1酶(在SMG发育的早期向蛋白质添加糖)的小鼠，随着内质网(ER)压力的增加，导致主要BM成分在细胞内积累。随着BM成分的改变，Galnt1缺陷腺表现为成纤维细胞生长因子信号减弱，AKT和MAPK磷酸化减少，上皮细胞增殖减少。在Galnt1缺陷的腺体中外源性添加BM成分层粘连蛋白，以一种1-整合素依赖的方式拯救成纤维细胞生长因子信号和生长缺陷。我们的工作表明，O-糖基化影响哺乳动物器官发育过程中分泌的ECM的组成，从而对细胞信号、增殖和器官生长产生影响。这些结果强调了O-糖基化在建立细胞微环境中的保守作用，并暗示了这种蛋白质修饰在发育和疾病中的作用。 最后，我们研究了同一蛋白质(α-营养不良聚糖)上存在的两种不同糖基化形式之间的相互作用。具体地说，我们研究了α-肌营养不良聚糖上特定位点的O-甘露糖基化对GalNActs随后添加O-GalNAc的影响。通过结合酶和质谱学方法，我们发现O-甘露糖的存在和特定位置可以导致GalNAc加成的区域排斥或特定位置的改变。我们的研究表明，一种形式的糖基化可以影响另一种形式的糖基化的存在和/或位置，这表明两种类型的糖基化的变化都可能导致疾病的严重程度，就像在肌肉营养不良中常见的那样。 总而言之，我们正在利用从果蝇中收集的信息，更好地关注在更复杂的哺乳动物系统中受O-糖基化影响的发育的关键方面。我们希望上述研究的累积结果将阐明这种保守的蛋白质修饰在正常发育和疾病易感性中的作用机制。

The overarching goal of the Developmental Glycobiology Unit is to determine the mechanisms by which protein O-glycosylation regulates basic biological processes to better understand the role of this modification in development and disease. Mucin-type O-linked glycosylation is a widespread and evolutionarily conserved protein modification catalyzed by a family of enzymes (PGANTs or GalNAcTs) that transfer the sugar N-acetylgalactosamine (GalNAc) to the hydroxyl group of serines and threonines in proteins that are destined to be membrane-bound or secreted. Defects in this type of glycosylation are responsible for the human diseases familial tumoral calcinosis and Tn syndrome. Additionally, changes in O-glycosylation have been associated with tumor progression and metastasis. More recently, genome-wide association studies have identified the genes encoding the enzymes that are responsible for initiating O-glycosylation among those associated with HDL-cholesterol levels, triglyceride levels, congenital heart defects, colon cancer and bone mineral density. From these studies, it is apparent that this conserved protein modification has a multitude of biological roles. The focus of our research group is to elucidate the mechanistic role of O-glycans during development in order to understand how they contribute to disease susceptibility and progression. Previous work from our group demonstrated that there are at least 9 functional transferase genes in Drosophila and that at least one is required for viability. These studies provided the first evidence that a member of this multigene family is required for development and viability in any eukaryote. Most recently, we have performed in vivo RNA interference (RNAi) to identify the remaining family members that are also essential for viability. We have discovered that 4 additional family members are required for viability. Specifically, RNAi to pgant4, pgant5, pgant7 or the putative glycosyltransferase CG30463 resulted in lethality. Additionally, these essential genes were required in specific tissues (mesoderm, digestive system and tracheal system), suggesting unique tissue-specific functions for each. Finally, we demonstrated that one of these newly defined essential genes is responsible for proper gut function. Loss of this glycosyltransferase resulted in reduced secretion of O-glycosylated proteins into the lumen of the gut and morphological alterations in the cells responsible for gut acidification. Mutations in this family member resulted in improper gut acidification. These studies have implications for the role of this protein modification in the proper gut function in higher eukaryotes, as these genes are abundantly expressed in the stomach, small intestine and colon of mice and humans. Following up on our Drosophila studies demonstrating a role for an O-glycosyltransferase in extracellular matrix (ECM) formation, we investigated whether the loss of a mammalian O-glycosyltransferase (Galnt1) has an effect on basement membrane formation and organogenesis using the murine submandibular gland (SMG) as a model system. The basement membrane of the developing SMG is a complex array of components that influence cell signaling, proliferation and differentiation; additionally, it is rich in O-glycosylated proteins. In these studies, we demonstrate that the loss of Galnt1 affects FGF-mediated cell proliferation during mammalian SMG organogenesis by influencing the secretion of basement membrane proteins. Mice deficient for the enzyme Galnt1 (that adds sugars to proteins during early stages of SMG development) resulted in intracellular accumulation of major BM components along with increased endoplasmic reticulum (ER) stress. Along with changes in BM composition, Galnt1 deficient glands displayed decreased FGF signaling, reduced AKT and MAPK phosphorylation, and reduced epithelial cell proliferation. Exogenous addition of BM component laminin to Galnt1 deficient glands rescued FGF signaling and the growth defects in a β1-integrin-dependent manner. Our work demonstrated that O-glycosylation influences the composition of the secreted ECM during mammalian organ development, with resultant effects on cell signaling, proliferation and organ growth. These results highlight a conserved role for O-glycosylation in the establishment of cellular microenvironments and have implications for the role of this protein modification in both development and disease. Finally, we investigated the interplay between two different forms of glycosylation that are present on the same protein (alpha-dystroglycan). Specifically, we examined how the presence of O-mannosylation at specific sites on alpha- dystroglycan influences the subsequent addition of O-GalNAc by the GalNAcTs. By using a combination of enzymatic and mass spectroscopic methods, we found that the presence and specific location of O-mannose can result in either the regional exclusion or changes in the specific position of GalNAc addition. Our study demonstrates that one form of glycosylation can influence the presence and/or position of another form of glycosylation, suggesting that changes in both types of glycosylation may contribute to disease severity, as is commonly seen in muscular dystrophies. In summary, we are using information gleaned from Drosophila to better focus on crucial aspects of development affected by O-glycosylation in more complex mammalian systems. Our hope is that the cumulative results of the studies described above will elucidate the mechanisms by which this conserved protein modification operates in both normal development and in disease susceptibility.