Role of O-glycosylation in Animal Development

O-糖基化在动物发育中的作用

• 批准号: 7593386
• 负责人: KELLY G TEN HAGEN
• 金额: $ 91.84万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593386
• 关键词: Address; Adult; Affect; Animal Model; Animals; Antibodies; Apical; Back; Binding; Biological; Biological Models; Carbohydrates; Carrier Proteins; Cell Adhesion; Cell Shape; Cells; Cloning; Communication; Complex; Confocal Microscopy; Congenital Abnormality; Cytoplasmic Vesicles; Defect; Development; Diffusion; Disease; Disruption; Drosophila genus; Drosophila melanogaster; Embryo; Embryonic Development; Enzymes; Epithelial Cells; Eukaryota; Eukaryotic Cell; Event; Family; Family Sizes; Family member; Fishes; Future; Generations; Genes; Genetic; Genetic Techniques; Glean; Goals; Human; In Vitro; Insecta; Kidney; Label; Laboratories; Learning; Lectin; Light; Link; Luminal region; Lung; Mammals; Mediating; Modeling; Modification; Molecular; Morphogenesis; Mucins; Multigene Family; Mus; Organ; Organism; Pathway interactions; Pattern; Phenotype; Play; Polypeptide N-acetylgalactosaminyltransferase; Polysaccharides; Post-Translational Protein Processing; Process; Protein Glycosylation; Proteins; RNA; Regulation; Role; Salivary Glands; Signal Transduction; Staining method; Stains; Structure; Syndrome; System; Time; Tissues; Transferase Gene; Translating; Tube; body system; carbohydrate structure; fly; fungus; genome database; glycosylation; interest; member; migration; mutant; neuronal cell body; polypeptide; sugar; tool

Cells of 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 fully 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). 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 20 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 12 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 these studies by cloning and characterizing the genes responsible for O-linked glycosylation in Drosophila. We demonstrated that there are at least 9 functional transferase genes in Drosophila (potentially 12 members total) and that at least one (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 any eukaryote. Additionally, we have defined the spatial and temporal patterns of expression of all the pgant family members throughout Drosophila development (Tian and Ten Hagen, 2006). During this past year, we have also elucidated the developmental profile of specific O-glycans using a variety of fluorescent-labeled sugar binding lectins and antibodies that are specific for certain carbohydrate structures (Tian and Ten Hagen, 2007 in press). Using confocal microscopy, we were able to visualize the diverse array of O-glycans present on all developing structures and organs throughout embryogenesis. This information will aid us in determining what tissues and developmental pathways may require O-glycans as well as provide information as to what lectins would be most useful for identifying native proteins that contain O-glycans. This will further allow us to directly interrogate changes in carbohydrate composition in mutant strains during development. All of the studies mentioned above provide us with the background information and tools we will need to decipher the role these enzymes are playing during Drosophila development. This year, we have demonstrated that one gene is required at multiple distinct times during development for viability. Specifically, pgant35A is required during embryogenesis; homozygous mutants devoid of wild type maternal RNA show abnormal tracheal tube formation and migration of secondary branches in developing embryos (Tian and Ten Hagen, 2007). This is particularly interesting given that the Drosophila tracheal system serves as a model for branching morphogenesis in many mammalian organ systems, including the salivary gland, lung, kidney and vasculature. Specifically, we have found that pgant35A mutants have defects in epithelial cell shape, polarity and diffusion barrier formation within the tracheal system. We observed a decrease in apical staining of certain apical and luminal markers, concomitant with increased staining in cytoplasmic vesicles, suggesting that the phenotypes observed are the result of disruption of transport of proteins destined for the apical and luminal regions. We have also performed lectin staining of embryos and determined that the primary glycan in the tracheal system is GalNAc-Ser/Thr present in the apical and luminal regions; this glycan is severely reduced in pgant35A mutants. We have shown that adding back the functional form of the pgant35A gene will rescue the lethality, conclusively demonstrating that the defects observed are due to the loss of this specific gene. In addition to pgant35A, we are also examining whether other members of this family 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. To that end, we are also constructing mice deficient in the mammalian counterpart of the fly pgant35A gene to determine its role in mammalian development. 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 why O-linked glycosylation is necessary and what role sugars play in cellular communication and interactions occurring during eukaryotic development.

人体的细胞装饰有多种功能的碳水化合物（糖）。 这些糖不仅是细胞外部的保护性障碍，而且发现许多生物体中都参与细胞粘附，迁移，通信和信号事件。 实际上，许多最近描述的人类的先天缺陷和综合症是负责调节，合成或掺入碳水化合物（糖基化或CDG的疾病）的酶缺陷的结果。 尽管糖被认为对胚胎发育和成人器官功能很重要，但我们仍然不完全了解它们如何在分子水平上介导这些过程。我们的小组研究一种糖添加到称为粘蛋白型O-连接糖基化的蛋白质中，该糖基化是由称为多肽GalNAC转移酶（PPGANTase或PGANTS）的酶家族引发的。 在大多数较高的生物中都可以看到这种添加糖，包括哺乳动物，鱼类，昆虫，蠕虫和某些类型的真菌。 跨物种的这种蛋白质修饰的保护表明，在发育的许多方面，它在许多方面都起着至关重要的作用。 众所周知，有多达20个家庭成员编码哺乳动物的功能性ppgantases。鉴于家族的大小及其产生的复杂性，我们寻求一种替代，简单的模型系统，以帮助研究糖基化的生物学作用。对来自其他生物体的基因组数据库的分析表明，果蝇（果蝇Melanogaster）只有12个潜在成员，因此可能是一个更可触及的实验系统。 此外，果蝇还提供了更复杂的遗传技术，较短的生成时间以及大量良好的特征库存，可以在这些股票上建立未来的研究。此外，过去已经成功地使用了果蝇来解码生物学问题，并将所学的东西转化为更复杂的哺乳动物系统。 我们通过克隆和表征负责果蝇中O连接糖基化的基因开始了这些研究。 我们证明了果蝇中至少有9个功能转移酶基因（可能有12个成员），并且至少需要一个（Pgant35a）才能生存能力（Ten Hagen and Tran，2002; Ten Hagen等，2003）。这些研究提供了第一个例子，即任何真核生物的发展和生存能力都需要这个多基因家族的成员。 此外，我们定义了果蝇发展中所有PGANT家族成员表达的空间和时间模式（Tian and Ten Hagen，2006）。 在过去的一年中，我们还使用各种针对某些碳水化合物结构的特定的荧光糖结合凝集素和抗体阐明了特定的O-聚糖的发育曲线（Tian and Ten和Ten Hagen，2007年，印刷中）。 使用共聚焦显微镜，我们能够可视化在整个胚胎发生上所有发育中的结构和器官上存在的多种O-聚糖阵列。 这些信息将有助于我们确定哪些组织和发育途径可能需要O-聚糖，并提供有关凝集素最有用的信息，可用于鉴定含有O-聚糖的天然蛋白。这将进一步使我们能够直接询问发育过程中突变菌株中碳水化合物组成的变化。 上面提到的所有研究为我们提供了背景信息和工具，我们将需要这些酶在果蝇开发中扮演的作用。 今年，我们已经证明，在开发过程中，在多个不同的时间内需要一个基因才能生存能。 具体而言，胚胎发生过程中需要pgant35a。没有野生型母体RNA的纯合突变体显示出异常的气管管形成和次级分支的迁移（Tian and Ten Hagen，2007）。 鉴于果蝇气管系统是许多哺乳动物器官系统（包括唾液腺，肺，肾脏和脉管系统）分支形态发生的模型，这一点尤其有趣。 具体而言，我们发现PGANT35A突变体在气管系统内的上皮细胞形状，极性和扩散屏障形成中具有缺陷。 我们观察到某些顶端和腔内标记的顶端染色降低，这与细胞质囊泡中的染色增加同时，这表明观察到的表型是蛋白质破坏原来针对顶端和腔内区域的蛋白质转运的结果。 我们还对胚胎进行了凝集素染色，并确定气管系统中的主要聚糖是根尖和腔内区域中存在的galnac-ser/th。在PGANT35A突变体中，该聚糖大大降低。 我们已经表明，添加pgant35a基因的功能形式将挽救杀伤力，最终证明观察到的缺陷是由于该特定基因的丧失所致。 除了PGANT35A，我们还研究了该家族的其他成员是否也需要适当的发展。 像pgant35a一样，这些基因中的许多基因具有不同的哺乳动物对应物，并且在体外表现出相似的酶促活性，这表明苍蝇研究的结果将揭示这些基因在小鼠和人类中的功能作用。 为此，我们还在pgant35a基因的哺乳动物对应物中构建小鼠，以确定其在哺乳动物发育中的作用。 总而言之，我们正在使用从果蝇收集的信息，以更好地专注于更复杂的哺乳动物系统中O-糖基化影响的发展的关键方面。 我们的希望是，上述研究的累积结果将阐明为何需要O连接的糖基化以及糖在真核发育过程中发生的细胞交流和相互作用中扮演什么角色。