Role of O-glycosylation in Animal Development

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。我们小组以前的工作表明，O-连接的糖基化对于果蝇的生存是必不可少的。此外，我们已经证明，负责启动O-糖基化的酶在特定的组织中是必需的，这表明了独特的组织特异性功能。例如，我们证明了一种必要的PGANT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。正在进行的工作集中在确定O-葡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类型细胞的正常功能中起着至关重要的作用。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。 在另一项工作中，我们发现，在果蝇翅膀发育过程中，失去一个PGANT家族成员会改变ECM蛋白的分泌，从而影响BM组成并破坏整合素介导的细胞黏附。同样，我们也证明了O-糖基化还调节哺乳动物器官发育过程中细胞外基质的组成，影响整合素和成纤维细胞生长因子信号，从而影响细胞增殖和发育中的唾液腺的生长。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。我们目前正在与Tabak实验室合作，进一步研究O-糖基化缺失对哺乳动物发育的其他方面的影响。具体地说，我们发现Galnt1缺陷小鼠的心脏发育受到影响。我们目前正在定义Galnt1缺陷心脏中发生的细胞和分子变化，并确定可能负责调节这些影响的Galnt1的直接靶点。最后，我们开发了一个活体器官O-糖基化和极化分泌的实时成像系统。 我们以前的研究已经证明，某些O-糖基转移酶的缺失会影响果蝇细胞的分泌，并改变高尔基体组织，这表明这些酶在维持适当的分泌器官结构方面发挥了作用。由于O-糖基转移酶是分泌器官的组成部分，负责分泌和膜结合蛋白的修饰，我们推测它们在与蛋白质的分泌、运输、稳定性和/或功能有关的保守生物功能中发挥关键作用。我们正在通过实时成像某些家族成员突变的器官中的特定分泌事件，来研究这些植物是如何通过调节分泌和分泌器官结构的影响的。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发挥了作用。 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。我们还使用活体器官内的实时成像来确定O-糖基化影响分泌的具体过程。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。

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.