Role of O-glycosylation in Animal Development

人体内的细胞被各种碳水化合物(糖)装饰，这些碳水化合物具有多种不同的功能。这些糖不仅作为细胞外部的保护性屏障，而且被发现参与了许多生物体中的细胞黏附、迁移、通讯和信号事件。事实上，最近在人类中描述的许多出生缺陷和综合征是负责调节、合成或合并细胞内碳水化合物的酶的缺陷(糖基化常见病或CDG)的结果。虽然糖被认为对胚胎发育和成人器官功能很重要，但我们仍然不知道它们是如何在分子水平上调节这些过程的。我们的团队研究了一种被称为粘蛋白O型连接的糖基化的蛋白质，它是由被称为多肽GalNAc转移酶(ppGaNTase或pgants)的酶家族启动的。它被称为粘液型？糖基化是因为它大量存在于被称为粘蛋白的蛋白质上，粘蛋白主导着唾液分泌、呼吸道、消化和生殖道。这种糖的添加存在于大多数高等生物中，包括哺乳动物、鱼类、昆虫、蠕虫和某些类型的真菌。这种蛋白质修饰在物种间的保守性表明，它在许多发育方面发挥着至关重要的作用(S)。目前已知哺乳动物中编码功能性ppGaNTase的家族成员多达24个。考虑到该家族的规模及其产生的复杂性，我们寻找了一种替代的、更简单的模型系统来帮助研究糖基化的生物学作用。对其他生物基因组数据库的分析表明，果蝇只有14个潜在成员，因此可能是一个更容易处理的实验系统。此外，果蝇提供了更复杂的遗传技术，更短的世代时间，以及丰富的特征良好的种群，可以在此基础上进行未来的研究。此外，果蝇在过去曾被成功地用来破译生物学问题，并将所学到的东西转化回更复杂的哺乳动物系统。 我们首先研究了果蝇体内负责O-连接糖基化的基因。我们已经证明了果蝇中至少有9个功能成员(可能总共14个)，并且至少有一个基因(Pgant35A)是生存所必需的(Ten Hagen and Tran，2002；Ten Hagen et al.，2003)。这些研究提供了第一个例子，表明在真核生物中，多基因家族的成员是发育和生存所必需的。为了阐明O-糖基化的生物学作用，我们专注于确定每个家庭成员在发育过程中何时何地被需要，以及确定失去这个家庭成员的后果。为了开始解决这些问题，我们进行了RNA原位杂交实验，以确定在胚胎发育期间每个基因的表达时间。这些信息将帮助我们确定在哪里寻找缺陷和受粘蛋白O-糖基化影响的发育途径。到目前为止，我们看到了该家族每个成员独特的时间和空间表达模式，其中一些在整个胚胎发育过程中在许多区域表达，而另一些在特定时间在特定区域表达(Tian和Ten Hagen提交)。一些家族成员在发育中的唾液腺和消化道的特定部位高度表达，表明O-糖基化在这些发育中的组织中发挥作用。此外，我们正在通过使用被称为凝集素的荧光标记糖结合蛋白来定义果蝇发育过程中存在的糖的种类。这些凝集素研究将证实我们关于pgant基因表达的数据，并告诉我们在细胞发育的每个阶段，细胞表面还存在哪些其他糖。为了应对失去一名家庭成员的后果，我们研究了含有缺陷pgant35A基因的果蝇的发育。我们已经确定，在发育过程中的多个不同时期，这种基因是生存所必需的。此外，我们还证明了重新添加pgant35A基因的功能形式将挽救所观察到的致死性，从而最终证明所观察到的缺陷是由于该特定基因的丢失所致。我们正在继续对胚胎发生的各个阶段进行仔细的分析，以确定在这些突变体中影响的确切生物学过程。我们在这里发现的结果将被翻译回哺乳动物系统，因为存在与pgant35A基因相对应的独特的哺乳动物。除了pgant35A的研究外，我们还在检查该家族其他成员的突变，以确定它们是否也是正常发育所必需的。像pgant35A一样，这些基因中的许多都有不同的哺乳动物对应基因，并且在体外显示出相似的酶活性，这表明在苍蝇身上的研究结果将揭示这些基因在老鼠和人类中的功能作用。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。我们目前正在构建缺乏苍蝇pgant35A基因的哺乳动物对应基因的小鼠，以确定它在哺乳动物发育中的作用。我们希望，上述研究的累积结果将阐明为什么O-连接的糖基化是必要的，以及糖在真核发育过程中的细胞通讯和相互作用中扮演什么角色。

Cells within the body are decorated with a variety of carbohydrates (sugars) that serve many diverse functions. These sugars not only act as a protective barrier on the outside of the cell, but have been found to be involved in cell adhesion, migration, communication and signaling events in many organisms. Indeed, many recently described birth defects and syndromes in humans are the result of defects in enzymes responsible for the regulation, synthesis or incorporation of carbohydrates in cells (Congential Disorders of Glycosylation or CDG). While sugars are recognized as being important for embryonic development and adult organ function, we still do not understand how they mediate these processes at the molecular level. Our group studies one type of sugar addition to proteins known as mucin-type O-linked glycosylation, which is initiated by the enzyme family known as the polypeptide GalNAc transferases (ppGaNTases or pgants). It is termed ?mucin-type? glycosylation because it is present in great abundance on proteins known as mucins, which dominate the salivary secretions, respiratory, digestive and reproductive tracts. This sugar addition is seen in most higher organisms including mammals, fish, insects, worms and some types of fungi. The conservation of this protein modification across species suggests that it plays a crucial role(s) during many aspects of development. It is known that there are as many as 24 family members encoding functional ppGaNTases in mammals. Given the size of the family and the complexity it generates, we sought an alternative, simpler model system to aid in investigating the biological role of glycosylation. Analysis of the genome databases from other organisms indicated that the fruit fly (Drosophila melanogaster) had only 14 potential members and may therefore be a more tractable experimental system. Additionally, the fruit fly offers more sophisticated genetic techniques, shorter generation times and a wealth of well-characterized stocks on which to build future studies. Moreover, the fruit fly has been used successfully in the past to decode biological problems and translate what has been learned back into the more complex mammalian systems. We began by characterizing the genes responsible for O-linked glycosylation in Drosophila melanogaster. We have demonstrated that there are at least 9 functional members in Drosophila (potentially 14 total) and that at least one gene (pgant35A) is required for viability (Ten Hagen and Tran, 2002; Ten Hagen et al., 2003). These studies provided the first example that a member of this multigene family is required for development and viability in a eukaryotic organism. In an effort to elucidate the biological role of O-glycosylation, we have focused on identifying when and where each family member is required during development as well as defining the consequences of the loss of a member of this family. To begin to address these issues, we performed RNA in situ hybridization experiments to define when during embryonic development each gene is expressed. This information will aid us in determining where to look for defects and developmental pathways influenced by mucin-type O-glycosylation. Thus far, we are seeing unique temporal and spatial expression patterns for each member of this family, with some being expressed in many regions throughout embryogenesis while others are expressed very specifically in certain regions at specific times (Tian and Ten Hagen, submitted). A number of family members are highly expressed in the developing salivary glands and specific portions of the digestive tract, suggesting a role for O-glycosylation in these developing tissues. Additionally, we are defining the variety of sugars present during fly development by employing fluorescent-labeled sugar binding proteins known as lectins. These lectin studies will corroborate our data on the expression of the pgant genes as well as tell us what other sugars are also present on the surface of cells at each stage of development. To address the consequences of the loss of a family member, we have examined the development of flies containing a defective pgant35A gene. We have established that this gene is required at multiple distinct times during development for viability. Additionally, we have shown that adding back the functional form of the pgant35A gene will rescue the lethality observed, conclusively demonstrating that the defects observed are due to the loss of this specific gene. We are continuing to do a careful analysis of the various stages of embryogenesis to identify the exact biological processes affected in these mutants. The results we find here will be translated back into the mammalian system, as a distinct mammalian counterpart to the pgant35A gene exists. In addition to the pgant35A studies, we are also examining mutations in other members of this family to determine whether or not they are required for proper development as well. Like pgant35A, many of these genes have distinct mammalian counterparts and display similar enzymatic activities in vitro, suggesting the results from studies in flies will shed light on the functional role of these genes in mice and humans. 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 currently constructing mice deficient in the mammalian counterpart of the fly pgant35A gene to determine its role in mammalian development. Our hope is that the cumulative results of the studies described above will elucidate why O-linked glycosylation is necessary and what role sugars play in cellular communication and interactions occurring during eukaryotic development.