Role of O-glycosylation in Animal Development

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，O-连接的糖基化对果蝇的生存能力是必不可少的。 此外，我们已经证明，负责启动O-糖基化的酶需要在特定的组织，这表明独特的组织特异性功能。例如，我们证明了一种必需的PGANT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。正在进行的工作集中在定义O-聚糖在消化系统内特定细胞中的机械作用。 我们有证据表明O-糖基化在消化道内不同细胞类型的正常功能中起着至关重要的作用。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。 在其他工作中，我们发现一个PGANT家族成员的丢失改变了ECM蛋白的分泌，从而影响BM组成并破坏果蝇翅膀发育过程中整合素介导的细胞粘附。同样，我们证明，O-糖基化也调节哺乳动物器官发育过程中的ECM的组成，影响整联蛋白和FGF信号传导，从而影响细胞增殖和发育中的唾液腺的生长。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。 我们目前正在与Tabak实验室合作，进一步研究O-糖基化丧失对哺乳动物发育其他方面的影响。具体来说，我们发现Galnt 1缺陷小鼠的心脏发育受到损害。我们目前正在定义Galnt 1缺陷心脏中发生的细胞和分子变化，并确定可能负责介导这些效应的Galnt的直接靶点。 最后，我们已经开发了一个系统，用于实时成像的O-糖基化和极化分泌在一个活的器官。 我们以前的研究表明，某些O-糖基转移酶的损失影响果蝇细胞的分泌和改变高尔基体的组织，这表明这些酶在维持适当的分泌器结构的作用。 由于O-糖基转移酶是分泌器的组成部分，并负责分泌和膜结合蛋白的修饰，我们假设它们在与蛋白质的分泌，运输，稳定性和/或功能相关的保守生物学功能中起着至关重要的作用。我们正在研究PGANTs如何通过在某些家族成员已发生突变的器官中真实的成像特定的分泌事件来介导对分泌和分泌器官结构的影响。 到目前为止，我们有证据表明，一些家庭成员在极化分泌的特定方面的作用。 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。我们还在活体器官内使用实时成像来确定O-糖基化影响分泌的具体过程。 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。

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 O-linked glycosylation is essential for viability in Drosophila. Moreover, we have demonstrated that the enzymes responsible for the initiation of O-glycosylation are required in specific tissues, suggesting unique tissue-specific functions. For example, we demonstrated that one essential PGANT 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. Ongoing work centers on defining the mechanistic roles for O-glycans in specific cells within the digestive system. We have evidence for crucial roles for O-glycosylation in the proper function of diverse cell types within the digestive tract. 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. In other work, we found that loss of one PGANT family member alters secretion of an ECM protein, thereby influencing BM composition and disrupting integrin-mediated cell adhesion during Drosophila wing development. Likewise, we demonstrated that O-glycosylation also modulates the composition of the ECM during mammalian organ development, influencing integrin and FGF signaling, thereby affecting cell proliferation and growth of the developing salivary glands. 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. We are currently collaborating with the Tabak laboratory to further investigate the effects of loss of O-glycosylation on other aspects of mammalian development. Specifically, we have found that heart development is compromised in Galnt1-deficient mice. We are currently defining the cellular and molecular changes occurring in the Galnt1-deficient hearts and identifying the direct targets of Galnt that may be responsible for mediating these effects. Finally, we have developed a system for real-time imaging of O-glycosylation and polarized secretion in a living organ. Our previous studies have demonstrated that the loss of certain O-glycosyltransferases affects secretion in Drosophila cells and alters Golgi organization, suggesting a role for these enzymes in maintaining proper secretory apparatus structure. As O-glycosyltransferases are components of the secretory apparatus and responsible for the modification of secreted and membrane bound proteins, we hypothesize that they play crucial roles in conserved biological functions related to secretion, transport, stability and/or function of proteins. We are investigating how the PGANTs are mediating effects on secretion and secretory apparatus structure by imaging specific secretory events in real time in organs where certain family members have been mutated. Thus far, we have evidence for the role of a number of family members in specific aspects of polarized secretion. 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. We are also using real-time imaging within living organs to define the specific processes by which O-glycosylation influences secretion. 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.