Role of O-glycosylation in Animal Development

O-糖基化在动物发育中的作用

• 批准号: 7733928
• 负责人: KELLY G TEN HAGEN
• 金额: $ 108.83万
• 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/7733928
• 关键词: Address; Adhesions; Adult; Affect; Affinity Chromatography; Animal Model; Animals; Apical; Biochemistry; Bioinformatics; Biological; Biological Models; Bulla; Carbohydrates; Cataloging; Catalogs; Cell Adhesion; Cell Communication; Cell Culture System; Cells; Characteristics; Cloning; Communication; Complex; Computer Systems Development; Cultured Cells; Data Set; Development; Disruption; Double-Stranded RNA; Drosophila genus; Drosophila melanogaster; Electron Microscopy; Embryo; Embryonic Development; Enzymes; Epithelial; Epithelial Cells; Epithelium; Eukaryota; Eukaryotic Cell; Event; Excision; Family; Family Sizes; Family member; Fishes; Future; Gene Expression; Generations; Genes; Genetic; Genetic Techniques; Gland; Glean; Goals; In Vitro; Insecta; Insertion Mutation; Integrins; Kidney; Knockout Mice; Laboratories; Link; Luminal region; Lung; Mammals; Mediating; Mesenchymal; Mesenchyme; Modeling; Modification; Morphogenesis; Mucins; Multigene Family; Mus; Mutation; Numbers; Organ; Organ Culture Techniques; Organism; Pattern; Phenotype; Play; Polypeptide N-acetylgalactosaminyltransferase; Polysaccharides; Post-Translational Protein Processing; Protein Glycosylation; Protein Isoforms; Proteins; RNA; RNA Interference; Role; Salivary Glands; Signal Transduction; Small Interfering RNA; Staging; Structure; Surface; System; Time; Tissues; Transcript; Transferase; Transferase Gene; Tube; Tubular formation; Wing; body system; fly; fungus; genome database; glycosylation; glycosyltransferase; imaginal disc; in vivo; interest; member; migration; mutant; neuronal cell body; polypeptide; preference; sugar

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 are also involved in cell adhesion, migration, communication and signaling events in many organisms. Our group studies one type of sugar addition to proteins, known as mucin-type O-linked glycosylation, which is initiated by the polypeptide GalNAc transferase (ppGalNAcT or PGANT) enzyme family. This sugar addition is seen in most eukaryotic organisms including mammals, fish, insects, worms and some types of fungi. The conservation of this protein modification across species suggests that it plays crucial roles during many aspects of development. It is known that there are as many as 20 family members encoding functional ppGalNAcTs in mammals. Given the size of the family and the complexity it generates, we sought an alternative, simpler model system to investigate 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. 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. These studies provided the first evidence that a member of this multigene family is required for development and viability in any eukaryote. Additionally, we defined the spatial and temporal patterns of expression of all the pgant family members throughout Drosophila development and elucidated the developmental profile of specific O-glycans. Of particular interest is the abundance of O-glycans along the presumptive apical and luminal regions of developing tubular structures in the fly. Examination of mutations in members of this glycosyltransferase family demonstrated that one gene is required at multiple distinct times during development. 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. 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. We are now defining the subcellular changes that are taking place in pgant35A mutants. Electron microscopy has revealed significant changes in the apical and luminal surfaces of the tracheal system, with loss of certain portions of the secreted cuticle and disruption of the taenidial folds that line the inside of the tracheal tube. We are currently performing these studies in a cell culture system to visualize subcellular changes more readily. In addition to pgant35A, we are also examining the developmental role of other members of this family. We have found that mutations in pgant3 alter epithelial cell adhesion in the Drosophila wing blade. A transposon insertion mutation in pgant3 or RNAi to pgant3 resulted in blistered wings, a phenotype characteristic of genes involved in integrin-mediated cell interactions. Precise excision of the tranposon restored pgant3 expression and wing integrity. Expression of wild type pgant3 in the mutant background also rescued the wing blistering phenotype, whereas expression of another family member did not, revealing a unique requirement for pgant3. pgant3 mutants displayed reduced O-glycosylation along the basal surface of wing imaginal discs, suggesting that reduced glycosylation of basal proteins in pgant3 mutants is responsible for disruption of adhesion in the adult wing blade. We have now identified one of the main O-glycosylated proteins in the wing disc using a combination of affinity purification, biochemistry and bioinformatics. This protein is specifically O-glycosylated in wild type wing discs but not in pgant3 mutants. Interestingly, this protein is known to mediate cell adhesion events during other stages of fly development as well. We are also investigating the role of each transferase using RNA interference (RNAi) in vivo to knockdown the transcript levels of each isoform. Expression of this dsRNA has recapitulated the phenotypes discussed above for pgant35A and pgant3 lending further support for the role of these genes in various aspects of epithelial tube formation and cell adhesion, respectively, as well as verifying the use of RNAi to specifically knockdown transferase gene expression in vivo. Interrogation of other isoforms by this approach indicates that additional pgant genes are required for viability. We are continuing to systematically analyze the consequences of loss of each pgant family member both in vivo as well as in cell culture. Given that this family is evolutionarily conserved and that mammalian and fly orthologues display similar enzymatic activities and substrates preferences in vitro, we are also investigating the role of O-glycans in mammalian organ system development. We are currently interrogating ppGaNAcT expression patterns during embryonic salivary gland development using microarray data sets generated by Dr. Matthew Hoffman as part of the NIDCR Salivary Gland Initiative. We are assessing the expression levels of each of the 18 mouse ppGalNAcTs during various stages of gland development to catalogue when each gene is expressed. We are also examining whether each gene is expressed in epithelial and/or mesenchymal tissue, to assess what unique roles each enzyme may be playing in the development of this organ. Preliminary analyses indicate expression of certain isoforms at key stages of glandular development. Additionally, there are a number of isoforms expressed specifically in either the mesenchyme or epithelium. From here, the functional role of isoforms will be interrogated using siRNA in submandibular organ culture. Additionally, mice deficient in a number of ppGalNAcT family members already exist. Because these lines are homozygous viable, we are using the organ culture system to examine the development of the salivary glands from these knockout mice. 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）。这些糖不仅充当细胞外部的保护屏障，而且还参与了许多生物体中细胞粘附，迁移，通信和信号事件。 我们的小组研究一种添加蛋白质的糖，称为粘蛋白型O-连接的糖基化，该糖基化是由多肽GalNAC转移酶（PPGALNACT或PGANT）酶家族引发的。在大多数真核生物中都可以看到这种加糖，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，它在发育的许多方面起着至关重要的作用。众所周知，在哺乳动物中编码功能性ppgalnacts的家庭成员多达20个家庭成员。鉴于家族的大小及其产生的复杂性，我们寻求一种替代，更简单的模型系统来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。 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（可能是12个成员），并且至少需要1个（PGANT35A）才能生存能力。这些研究提供了第一个证据，表明该多基因家族的成员在任何真核生物中都需要开发和生存能力。此外，我们定义了果蝇发育中所有pgant家族成员表达的空间和时间模式，并阐明了特定的O-聚糖的发育概况。特别令人感兴趣的是沿着蝇类型的肾小管结构的推定顶端和腔内区域的O-glycans。 检查该糖基转移酶家族成员的突变表明，在发育过程中需要多个不同的时间。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。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。现在，我们正在定义PGANT35A突变体中发生的亚细胞变化。 电子显微镜揭示了气管系统的顶端和腔表面​​的显着变化，并损失了分泌性角质层的某些部分，并破坏了taenidial褶皱，这些褶皱将气管内部的内部覆盖。 我们目前正在细胞培养系统中进行这些研究，以更容易地可视化细胞的变化。 除了PGANT35A，我们还正在研究该家族其他成员的发展作用。我们发现PGANT3中的突变改变了果蝇翼刀片中的上皮细胞粘附。 PGANT3或RNAI中的转座子插入突变导致翅膀起泡，这是涉及整联蛋白介导的细胞相互作用的基因的表型特征。 tranposon的精确切除恢复了PGANT3表达和机翼完整性。 野生型PGANT3在突变体背景中的表达也挽救了机翼起泡表型，而另一家族成员的表达则揭示了对PGANT3的独特要求。 PGANT3突变体显示沿翼想像盘的基底表面降低的O-糖基化，这表明PGANT3突变体中基底蛋白的糖基化降低是负责成年机翼刀片粘附的破坏。 现在，我们使用亲和力纯化，生物化学和生物信息学结合了机翼圆盘中的主要O-糖基化蛋白之一。该蛋白在野生型机翼盘中特异性O-糖基化，而不是PGANT3突变体中的O-糖基。 有趣的是，已知该蛋白在苍蝇发育的其他阶段也介导细胞粘附事件。 我们还使用RNA干扰（RNAi）在体内研究了每个转移酶的作用，以敲低每个同工型的转录水平。 该dsRNA的表达已概括了上述PGANT35A和PGANT3的表型，分别对这些基因在上皮管形成和细胞粘附的各个方面的作用进行进一步支持，并验证RNAi在VIVO中使用RNAi的使用特异性敲低转移酶基因表达。通过这种方法对其他同工型的询问表明可行性需要其他PGANT基因。 我们将继续系统地分析体内和细胞培养中每个pgant家族成员丧失的后果。 鉴于该家族在进化上是保守的，并且哺乳动物和苍蝇直系同源物在体外表现出相似的酶促活性和底物的偏好，因此我们还研究了O-聚糖在哺乳动物器官系统开发中的作用。 目前，我们正在使用Matthew Hoffman博士作为NIDCR唾液腺倡议的一部分生成的微阵列数据集在胚胎唾液腺发育过程中询问PPGANACT表达模式。 当表达每个基因时，我们正在评估在腺体发育到编目的各个阶段，在18个小鼠ppgalnacts中的每一种的表达水平。 我们还正在研究每个基因在上皮和/或间质组织中是否表达，以评估每个酶在该器官的发育中可能扮演的独特作用。 初步分析表明在腺发育的关键阶段表达了某些同工型。 此外，在间质或上皮中有许多同工型。 从这里开始，使用siRNA在下颌下器器官培养物中将询问同工型的功能作用。 此外，已经存在许多ppgalnact家族成员缺乏的小鼠。 由于这些线是纯合可行的，因此我们正在使用器官培养系统检查这些敲除小鼠的唾液腺的发展。 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。