Role of O-glycosylation in Animal Development

身体的细胞装饰有各种各样的碳水化合物（糖），它们具有许多不同的功能。这些糖不仅作为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，通讯和信号事件。我们的小组研究了一种类型的糖添加到蛋白质中，称为粘蛋白型O-连接糖基化，其由多肽GalNAc转移酶（ppGalNAcT或PGANT）酶家族启动。这种糖添加在大多数真核生物中可见，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起着至关重要的作用。已知在哺乳动物中有多达20个家族成员编码功能性ppGalNAcTs。考虑到家族的规模和它产生的复杂性，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。 我们开始我们的研究，克隆和表征的基因负责O-连接糖基化的果蝇。我们证明了果蝇中至少有9个功能性转移酶基因（可能总共有12个成员），并且至少有一个是生存所必需的。这些研究提供了第一个证据，证明这个多基因家族的成员是任何真核生物发育和生存所必需的。此外，我们定义了整个果蝇发育过程中所有pgant家族成员表达的空间和时间模式。我们还使用各种糖结合凝集素和抗体阐明了特定O-聚糖的发育概况，以确定在发育过程中何时何地可能需要O-聚糖。 最近，我们发现，在这个家庭的另一个成员（pgant 3）的突变改变上皮细胞粘附在果蝇翅膀叶片。 pgant 3的转座子插入突变或对pgant 3的RNA干扰（RNAi）导致了红翅，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pgant 3的表达和翅膀的完整性。 形成稳定但无活性的高尔基体定位酶的突变也导致翅膀起泡，表明适当的细胞粘附依赖于糖基转移酶活性。 野生型pgant 3在突变体背景中的表达挽救了翅起泡表型，而另一个家族成员的表达没有，揭示了对pgant 3活性的独特要求。 最近在pgant 3中分离的点突变显示了与整合素突变体的遗传相互作用，证明了O-糖基化和整合素介导的细胞粘附之间的遗传联系。 我们已经确定了一个主要的O-糖基化的蛋白质在翼盘（tiggrin）使用亲和纯化，生物化学和生物信息学的组合。Tiggrin是一种整合素结合的细胞外基质（ECM）蛋白，其在野生型翼盘中特异性O-糖基化，但在pgant 3突变体中不特异性O-糖基化。 我们认为pgant 3通过ECM蛋白的糖基化调节整合素介导的细胞粘附。 我们目前正在研究O-聚糖在这一过程中的机制作用。 我们也在研究每种转移酶在体内和细胞培养中使用RNAi的作用。 今年，我们已经检查了每个pgant家族成员含有诱导型dsRNA的转基因果蝇。 dsRNA的表达重现了先前对pgant 35 A和pgant 3描述的表型，验证了RNAi在体内特异性敲低这些基因的用途。通过这种方法对其他同种型的研究表明，需要额外的pgant来维持活力。 我们正在继续这项研究，通过在各种组织特异性启动子的控制下诱导RNAi到每个家族成员，以解决每个亚型在某些发育组织中的作用。 我们正在对这些RNAi实验进行后续研究，对感兴趣的家族成员进行额外的生化、细胞和遗传分析。 最后，我们正在研究O-聚糖在哺乳动物器官系统发育中的作用。 使用唾液腺作为我们的模型系统，我们已经评估了18 ppGalNAcTs中的每一个在腺体发育的各个阶段的表达水平，以确定其表达的时间模式。 我们的分析表明，某些异构体在腺体发育的特定阶段表达。 此外，存在许多在间充质或上皮中组织特异性表达的同种型。 为了研究O-糖基化的功能后果，我们已经检查了缺乏ppGalNAcT家族的一个成员的小鼠胚胎唾液腺的生长和发育，该家族在发育的早期阶段（E12-E13）表达最丰富。 虽然这种转移酶缺陷的小鼠是可行的和可生育的，但它们的唾液腺在生长中显示出阶段特异性发育延迟。 这种阶段特异性延迟可能是腺体发育早期特异性蛋白糖基化丧失的结果。 我们目前正在努力确定在早期发育过程中表达的糖蛋白，以确定它们如何影响腺体生长和形态发生。 总之，我们正在使用从果蝇中收集的信息，以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。我们的希望是，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，以及糖在真核生物发育过程中发生的细胞通讯和相互作用中起什么作用。

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. We began our 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 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. We have also elucidated the developmental profile of specific O-glycans using a variety of sugar binding lectins and antibodies to determine where and when O-glycans may be required during development. Recently, we have found that mutations in another member of this family (pgant3) alter epithelial cell adhesion in the Drosophila wing blade. A transposon insertion mutation in pgant3 or RNA interference (RNAi) to pgant3 resulted in blistered wings, a phenotype characteristic of genes involved in integrin-mediated cell interactions. Precise excision of the transposon restored pgant3 expression and wing integrity. Mutations that form a stable yet inactive Golgi-localized enzyme also resulted in wing blistering, indicating that proper cell adhesion is dependent upon glycosyltransferase activity. Expression of wild type pgant3 in the mutant background rescued the wing blistering phenotype, whereas expression of another family member did not, revealing a unique requirement for pgant3 activity. Recently isolated point mutations in pgant3 show genetic interactions with an integrin mutant, demonstrating a genetic link between O-glycosylation and integrin-mediated cell adhesion. We have identified one of the main O-glycosylated proteins in the wing disc (tiggrin) using a combination of affinity purification, biochemistry and bioinformatics. Tiggrin is an integrin-binding extracellular matrix (ECM) protein that is specifically O-glycosylated in wild type wing discs but not in pgant3 mutants. We believe that pgant3 modulates integrin-mediated cell adhesion through glycosylation of this ECM protein. We are currently investigating the mechanistic role of O-glycans in this process. We are also investigating the role of each transferase using RNAi in vivo and in cell culture. This year, we have examined transgenic flies containing inducible dsRNA for each pgant family member. Expression of dsRNA has recapitulated the phenotypes described previously for pgant35A and pgant3, verifying the use of RNAi to specifically knockdown these genes in vivo. Interrogation of other isoforms by this approach indicates that additional pgants are required for viability. We are continuing this study by inducing RNAi to each family member under the control of various tissue-specific promoters to address the role of each isoform in certain developing tissues. We are following up these RNAi experiments with additional biochemical, cellular and genetic analyses of family members of interest. Finally, we are investigating the role of O-glycans in mammalian organ system development. Using salivary glands as our model system, we have assessed the expression levels of each of the 18 ppGalNAcTs during various stages of glandular development to determine their temporal patterns of expression. Our analyses demonstrate that certain isoforms are expressed at specific stages of glandular development. Additionally, there are a number of isoforms that are expressed tissue-specifically, in either the mesenchyme or the epithelium. To investigate the functional consequences of O-glycosylation, we have examined the growth and development of embryonic salivary glands from mice lacking one member of the ppGalNAcT family that is expressed most abundantly at early stages of development (E12-E13). While mice deficient in this transferase are viable and fertile, their salivary glands show a stage-specific developmental delay in growth. This stage-specific delay is likely the result of loss of specific protein glycosylation early in gland development. We are currently working to identify the glycoproteins that are expressed during early development to determine how they influence gland growth and morphogenesis. 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.