Role of O-glycosylation in Animal Development

O-糖基化在动物发育中的作用

基本信息

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
项目类别：
财政年份：
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

项目摘要

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.

身体的细胞被各种碳水化合物（糖）装饰，具有多种不同的功能。这些糖不仅充当细胞外部的保护屏障，而且还参与许多生物体中的细胞粘附、迁移、通讯和信号传导事件。我们的小组研究了一种类型的蛋白质糖添加，称为粘蛋白型 O 连接糖基化，它是由多肽 GalNAc 转移酶（ppGalNAcT 或 PGANT）酶家族启动的。这种糖添加存在于大多数真核生物中，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发育的许多方面发挥着至关重要的作用。据了解，哺乳动物中有多达20个编码功能性ppGalNAcT的家族成员。考虑到该家族的规模及其产生的复杂性，我们寻求一种替代的、更简单的模型系统来研究糖基化的生物学作用。对其他生物体基因组数据库的分析表明，果蝇 (Drosophila melanogaster) 只有 12 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，可供未来研究使用。我们通过克隆和表征果蝇中负责 O-连接糖基化的基因来开始这些研究。我们证明果蝇中至少有 9 个功能性转移酶基因（总共可能有 12 个成员），并且至少有一个 (pgant35A) 是活力所必需的。这些研究提供了第一个证据，证明任何真核生物的发育和生存都需要这个多基因家族的成员。此外，我们定义了所有 pgant 家族成员在果蝇发育过程中的空间和时间表达模式，并阐明了特定 O-聚糖的发育概况。特别令人感兴趣的是，沿着果蝇中正在发育的管状结构的假定顶端和管腔区域存在丰富的O-聚糖。对这一糖基转移酶家族成员突变的检查表明，在发育过程中的多个不同时间需要一个基因。具体来说，pgant35A在胚胎发生过程中是必需的；缺乏野生型母体RNA的纯合突变体在发育中的胚胎中表现出异常的气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、肾和脉管系统）分支形态发生的模型，这一点特别有趣。我们现在正在定义 pgant35A 突变体中发生的亚细胞变化。电子显微镜显示气管系统的顶端和管腔表面发生了显着变化，分泌角质层的某些部分丢失，气管导管内部的带状褶皱被破坏。我们目前正在细胞培养系统中进行这些研究，以便更容易地观察亚细胞变化。除了pgant35A之外，我们还在研究该家族其他成员的发育作用。我们发现 pgant3 的突变改变了果蝇翼片的上皮细胞粘附。 pgant3 中的转座子插入突变或 pgant3 的 RNAi 导致翅膀起泡，这是参与整合素介导的细胞相互作用的基因的表型特征。转座子的精确切除恢复了 pgant3 的表达和翼的完整性。野生型 pgant3 在突变体背景中的表达也挽救了翅膀起泡表型，而另一个家族成员的表达却没有，这揭示了对 pgant3 的独特要求。 pgant3突变体沿着翅成虫盘的基底表面表现出O-糖基化减少，这表明pgant3突变体中基底蛋白糖基化的减少是导致成虫翅片粘附破坏的原因。现在，我们结合亲和纯化、生物化学和生物信息学，鉴定出了翼盘中主要的 O-糖基化蛋白之一。该蛋白在野生型翼盘中被特异性 O-糖基化，但在 pgant3 突变体中则不然。有趣的是，已知这种蛋白质也在果蝇发育的其他阶段介导细胞粘附事件。我们还在体内使用 RNA 干扰 (RNAi) 来研究每种转移酶的作用，以降低每种亚型的转录水平。该 dsRNA 的表达概括了上面讨论的 pgant35A 和 pgant3 的表型，进一步支持了这些基因分别在上皮管形成和细胞粘附的各个方面的作用，并验证了使用 RNAi 特异性敲低转移酶基因表达体内。通过这种方法对其他亚型的询问表明，生存能力需要额外的 pgant 基因。我们正在继续系统地分析每个 pgant 家族成员在体内和细胞培养中损失的后果。鉴于该家族在进化上是保守的，并且哺乳动物和果蝇直系同源物在体外表现出相似的酶活性和底物偏好，我们还在研究 O-聚糖在哺乳动物器官系统发育中的作用。作为 NIDCR 唾液腺计划的一部分，我们目前正在使用 Matthew Hoffman 博士生成的微阵列数据集来探究胚胎唾液腺发育过程中 ppGaNAcT 的表达模式。我们正在评估 18 种小鼠 ppGalNAcT 中每种基因在腺体发育各个阶段的表达水平，以便对每个基因的表达时间进行分类。我们还在检查每个基因是否在上皮和/或间质组织中表达，以评估每种酶在该器官的发育中可能发挥的独特作用。初步分析表明某些亚型在腺体发育的关键阶段表达。此外，还有许多在间充质或上皮细胞中特异性表达的亚型。从这里开始，将在下颌下器官培养中使用 siRNA 来探究异构体的功能作用。此外，缺乏许多 ppGalNAcT 家族成员的小鼠已经存在。因为这些品系是纯合的，我们正在使用器官培养系统来检查这些基因敲除小鼠唾液腺的发育。总之，我们正在利用从果蝇收集的信息来更好地关注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。我们希望上述研究的累积结果将阐明为什么 O-连接糖基化是必要的，以及糖在真核发育过程中发生的细胞通讯和相互作用中发挥什么作用。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Characterization of mucin-type core-1 beta1-3 galactosyltransferase homologous enzymes in Drosophila melanogaster.

果蝇粘蛋白型 core-1 beta1-3 半乳糖基转移酶同源酶的表征。

DOI：
发表时间：
2005-09
期刊：
影响因子：
0
作者：
Muller, Reto;Hulsmeier, Andreas J;Altmann, Friedrich;Ten Hagen, Kelly;Tiemeyer, Michael;Hennet, Thierry
通讯作者：
Hennet, Thierry

A UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase is required for epithelial tube formation.

UDP-GalNAc：多肽N-乙酰半乳糖氨基转移酶是上皮管形成所必需的。