Role of O-glycosylation in Animal Development

发育糖生物学单元的首要目标是确定蛋白质O-糖基化调节基本生物学过程的机制，以更好地了解这种修饰在发育和疾病中的作用。粘蛋白型0-连接的糖基化是一种广泛的和进化上保守的蛋白质修饰，其由将糖N-乙酰半乳糖胺（GalNAc）转移到蛋白质中的丝氨酸和苏氨酸的羟基的酶家族（PGANTs或GalNAcTs）催化，所述丝氨酸和苏氨酸注定是膜结合的或分泌的。这种类型的糖基化缺陷是人类疾病家族性肿瘤性钙质沉着症和Tn综合征的原因。 此外，O-糖基化的变化与肿瘤进展和转移相关。 最近，全基因组关联研究已经确定了编码酶的基因，这些酶负责启动与HDL-胆固醇水平，甘油三酯水平，先天性心脏病，结肠癌和骨矿物质密度相关的O-糖基化。 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-聚糖在发育过程中的机制作用，以了解它们如何促进疾病易感性和进展。 我们小组以前的工作表明，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，证明这个多基因家族的成员是任何真核生物发育和生存所必需的。最近，我们进行了体内RNA干扰（RNAi），以确定其余的家庭成员，也是至关重要的生存能力。 我们已经发现，4个额外的家庭成员是必需的生存能力。 具体地，针对pgant 4、pgant 5、pgant 7或推定的糖基转移酶CG 30463的RNAi导致致死性。 此外，这些必需基因在特定组织（中胚层，消化系统和气管系统）中是必需的，这表明每个组织都具有独特的组织特异性功能。 最后，我们证明了这些新定义的必需基因之一负责适当的肠道功能。这种糖基转移酶的丧失导致O-糖基化蛋白分泌到肠腔中的减少和负责肠酸化的细胞中的形态学改变。这个家族成员的突变导致了不适当的肠道酸化。这些研究对这种蛋白质修饰在高等真核生物中的适当肠道功能中的作用具有影响，因为这些基因在小鼠和人类的胃、小肠和结肠中大量表达。 后续我们的果蝇研究证明了O-糖基转移酶在细胞外基质（ECM）形成中的作用，我们研究了哺乳动物O-糖基转移酶（Galnt 1）的丧失是否对基底膜形成和器官发生有影响，使用小鼠下颌下腺（SMG）作为模型系统。发育中SMG的基底膜是一系列复杂的成分，影响细胞信号传导、增殖和分化;此外，它富含O-糖基化蛋白。在这些研究中，我们证明了Galnt 1的缺失通过影响基底膜蛋白的分泌来影响哺乳动物SMG器官发生过程中FGF介导的细胞增殖。 缺乏酶Galnt 1（在SMG发育的早期阶段将糖添加到蛋白质中）的小鼠导致主要BM组分的细胞内蓄积沿着内质网（ER）应激增加。 沿着BM组成的变化，Galnt 1缺陷腺体显示FGF信号传导减少，AKT和MAPK磷酸化减少，上皮细胞增殖减少。 外源性添加的BM组件层粘连蛋白Galnt 1缺陷腺体救出FGF信号和生长缺陷的1-整合素依赖性的方式。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，从而影响细胞信号传导、增殖和器官生长。 这些结果突出了O-糖基化在建立细胞微环境中的保守作用，并对这种蛋白质修饰在发育和疾病中的作用产生了影响。 最后，我们研究了存在于同一蛋白质（α-肌营养不良蛋白聚糖）上的两种不同形式的糖基化之间的相互作用。 具体地，我们检查了α-肌营养不良聚糖上特定位点处的0-甘露糖基化的存在如何影响随后通过GalNAcT添加0-GalNAc。 通过使用酶和质谱方法的组合，我们发现，O-甘露糖的存在和特定的位置可以导致区域排斥或GalNAc添加的特定位置的变化。 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可能有助于疾病的严重程度，这在肌营养不良症中很常见。 总之，我们正在使用从果蝇中收集的信息，以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。我们的希望是，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起作用的机制。

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.