Role of O-glycosylation in Animal Development

O-糖基化在动物发育中的作用

• 批准号: 8344134
• 负责人: KELLY G TEN HAGEN
• 金额: $ 127.97万
• 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/8344134
• 关键词: Actins; Address; Affect; Animal Model; Animals; Apoptosis; Architecture; Basement membrane; Binding Proteins; Biological; Biological Models; Carbohydrates; Cell Adhesion; Cell Culture Techniques; Cell Line; Cell Proliferation; Cell physiology; Cells; Cellular Morphology; Cellular Structures; Colon; Communication; Complex; Cytokinesis; Defect; Development; Disease; Disease susceptibility; Drosophila genus; Drosophila melanogaster; Enzymes; Epithelial Cell Proliferation; Epithelial Cells; Essential Genes; Eukaryota; Event; Extracellular Matrix; Extracellular Matrix Proteins; Family; Family Sizes; Family member; Fibroblast Growth Factor; Fishes; Genes; Genetic; Gland; Glean; Goals; Golgi Apparatus; Growth; Human; Insecta; Integrin Binding; Integrin-mediated Cell Adhesion Pathway; Integrins; Intracellular Transport; Laboratories; Laminin; Link; MAP Kinase Gene; Mammals; Mediating; Membrane Proteins; Modification; Mucins; Multigene Family; Mus; Mutation; Organ; Organism; Organogenesis; Phosphorylation; Play; Polypeptide N-acetylgalactosaminyltransferase; Post-Translational Protein Processing; Protein Glycosylation; Protein Secretion; Proteins; Proto-Oncogene Proteins c-akt; RNA Interference; Role; Signal Transduction; Small Intestines; Staging; Stomach; Structure; Submandibular gland; System; Tissues; Transferase; Transferase Gene; Wing; Work; endoplasmic reticulum stress; fly; follow-up; fungus; gland development; glycosylation; glycosyltransferase; in vivo; interest; member; migration; mutant; neuronal cell body; polypeptide; sugar; Actins; Address; Affect; Animal Model; Animals; Apoptosis; Architecture; Basement membrane; Binding Proteins; Biological; Biological Models; Carbohydrates; Cell Adhesion; Cell Culture Techniques; Cell Line; Cell Proliferation; Cell physiology; Cells; Cellular Morphology; Cellular Structures; Colon; Communication; Complex; Cytokinesis; Defect; Development; Disease; Disease susceptibility; Drosophila genus; Drosophila melanogaster; Enzymes; Epithelial Cell Proliferation; Epithelial Cells; Essential Genes; Eukaryota; Event; Extracellular Matrix; Extracellular Matrix Proteins; Family; Family Sizes; Family member; Fibroblast Growth Factor; Fishes; Genes; Genetic; Gland; Glean; Goals; Golgi Apparatus; Growth; Human; Insecta; Integrin Binding; Integrin-mediated Cell Adhesion Pathway; Integrins; Intracellular Transport; Laboratories; Laminin; Link; MAP Kinase Gene; Mammals; Mediating; Membrane Proteins; Modification; Mucins; Multigene Family; Mus; Mutation; Organ; Organism; Organogenesis; Phosphorylation; Play; Polypeptide N-acetylgalactosaminyltransferase; Post-Translational Protein Processing; Protein Glycosylation; Protein Secretion; Proteins; Proto-Oncogene Proteins c-akt; RNA Interference; Role; Signal Transduction; Small Intestines; Staging; Stomach; Structure; Submandibular gland; System; Tissues; Transferase; Transferase Gene; Wing; Work; endoplasmic reticulum stress; fly; follow-up; fungus; gland development; glycosylation; glycosyltransferase; in vivo; interest; member; migration; mutant; neuronal cell body; polypeptide; 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 have taken advantage of a simpler model system (Drosophila melanogaster) to investigate the biological role of glycosylation during development. Previous work from our group demonstrated that there are at least 9 functional transferase genes in Drosophila 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. More recently, we have performed in vivo RNA interference (RNAi) to identify the remaining family members that are also essential for viability. We have discovered that 4 additional family members are required for viability and are essential in specific tissues. Moreover, one of these newly defined essential genes is responsible for proper gut function. Loss of this glycosyltransferase results in reduced secretion of O-glycosylated proteins into the lumen of the gut and affects the structure of the cells responsible for proper gut acidifcation. Mutations in this family member result in improper gut acidification. These studies have implications for the role of this protein modification in proper gut function in higher eukaryotes, as these genes are abundantly expressed in the stomach, small intestine and colon of mice and humans. We also performed RNAi to each pgant in fly cell culture to examine the effects of each gene on specific cellular processes (cell adhesion, division, viability, apoptosis, morphological changes, intracellular transport, subcellular alterations). Using this approach, we obtained evidence for the role of pgants in the proper formation and structure of the secretory apparatus. RNAi to either pgant3 or pgant6 resulted in altered Golgi organization. Disruption of the normal Golgi structure in both cases was accompanied by a reduction in secretion, indicating a functional consequence of the loss of each transferase. Additionally, RNAi to pgant3 also resulted in alteration of the normal actin cytoskeletal architecture, changes in cell morphology and loss of cell adhesion. Other effects observed included multi-nucleated cells seen after RNAi to pgant2 or pgant35A in both cell lines, suggesting a role for these genes in the completion of cytokinesis. These studies provide a new platform for interrogating the cellular effects of mucin-type O-linked glycosylation and evidence for unique subcellular roles of the pgants in secretory apparatus structure and function. By examining the consequences of mutations in pgant family members in the fly, we found that mutations in pgant3 alter integrin-mediated epithelial cell adhesion in the Drosophila wing blade. We discovered that the loss of pgant3 resulted in the improper secretion and localization of the extracellular matrix (ECM) protein Tiggrin. Tiggrin is an integrin-binding protein that is normally O-glycosylated in wild type wing discs. Loss of Tiggrin within the basement membrane region in pgant3 mutants resulted in disruption of integrin-mediated cell adhesion and defects in wing formation. These studies provided the first example of the effects of O-glycosylation on protein secretion, establishment of the basement membrane and modulation of integrin-mediated cell adhesion in vivo. We followed up on these results to ask whether the loss of a mammalian O-glycosyltransferase (Galnt1) has an effect on basement membrane formation and organogenesis using the murine submandibular gland (SMG) as a model system. The basement membrane of the developing SMG is a complex array of components that influence cell signaling, proliferation and differentiation; additionally, it is rich in O-glycosylated proteins. In these studies, we demonstrate that the loss of Galnt1 affects FGF-mediated cell proliferation during mammalian SMG organogenesis by influencing the secretion of basement membrane proteins. Mice deficient for the enzyme Galnt1 (that adds sugars to proteins during early stages of SMG development) resulted in intracellular accumulation of major BM components along with increased endoplasmic reticulum (ER) stress. Along with changes in BM composition, Galnt1 deficient glands displayed decreased FGF signaling, reduced AKT and MAPK phosphorylation, and reduced epithelial cell proliferation. Exogenous addition of BM component laminin to Galnt1 deficient glands rescued FGF signaling and the growth defects in a β1-integrin-dependent manner. Our work demonstrates that O-glycosylation influences the composition of the secreted ECM during mammalian organ development, with resultant effects on cell signaling, proliferation and organ growth. These results highlight a conserved role for O-glycosylation in the establishment of cellular microenvironments and have implications for the role of this protein modification in both development and disease. 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 the mechanisms by which this conserved protein modification operates in both normal development and in disease susceptibility.

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。鉴于家庭的大小及其产生的复杂性，我们利用了更简单的模型系统（果蝇Melanogaster）来研究开发过程中糖基化的生物学作用。 我们小组的先前工作表明，果蝇中至少有9个功能转移酶基因，并且至少需要一个可行性。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。最近，我们进行了体内RNA干扰（RNAI），以识别其余的家庭成员，这些家庭成员也是可行性所必需的。 我们已经发现，可行性需要其他4个家庭成员，并且在特定组织中至关重要。 此外，这些新定义的基本基因之一负责适当的肠道功能。该糖基转移酶的丧失导致O-糖基化蛋白的分泌减少到肠道内腔中，并影响负责适当肠酸裂料的细胞结构。 该家庭成员的突变导致肠道酸化不当。这些研究对这种蛋白质修饰在适当的肠道功能中的作用具有影响，因为这些基因在小鼠和人类的胃，小肠和结肠中大量表达。 我们还向蝇培养中的每个PGANT进行了RNAI，以检查每个基因对特定细胞过程的影响（细胞粘附，分裂，生存力，凋亡，形态变化，细胞内转运，细胞下细胞的变化）。使用这种方法，我们获得了PGANT在分泌仪的正确形成和结构中的作用的证据。 RNAi到PGANT3或PGANT6导致高尔基组织改变。在两种情况下，正常高尔基体结构的破坏都伴随着分泌的减少，表明每个转移酶损失的功能后果。 此外，RNAi至PGANT3还导致正常肌动蛋白细胞骨架结构的改变，细胞形态的变化和细胞粘附的丧失。 观察到的其他效果包括两种细胞系中RNAi在RNAi到PGANT2或PGANT35A之后看到的多核细胞，这表明这些基因在胞质分裂完成中的作用。这些研究提供了一个新的平台，用于询问粘蛋白型O-连接糖基化的细胞效应，并证明了PGANTS在分泌设备结构和功能中的独特亚细胞作用的证据。 通过检查PGANT家族成员的突变的后果，我们发现PGANT3中的突变改变了整联蛋白介导的果蝇翼叶片中的上皮细胞粘附。我们发现PGANT3的损失导致细胞外基质（ECM）蛋白质Tiggrin的分泌不当和定位。 Tiggrin是一种整联蛋白结合蛋白，通常在野生型翼盘中被O-糖基化。 pgant3突变体中基底膜区域内提格格林的丧失导致整联蛋白介导的细胞粘附和机翼形成中的缺陷。这些研究提供了O-糖基化对蛋白质分泌，基底膜的建立以及体内整联蛋白介导的细胞粘附的调节的第一个例子。 我们跟进了这些结果，询问哺乳动物O-糖基转移酶（GALNT1）是否丧失使用鼠类下颌腺（SMG）作为模型系统对地下膜形成和器官发生有效。发育中的SMG的地下膜是影响细胞信号传导，增殖和分化的复杂成分阵列。另外，它富含O-糖基化蛋白。在这些研究中，我们证明了GALNT1的丧失会影响哺乳动物SMG器官发生过程中FGF介导的细胞增殖，从而影响基底膜蛋白的分泌。 缺乏酶Galnt1的小鼠（在SMG发育的早期阶段将糖添加到蛋白质中）导致主要BM成分的细胞内积累，并且内质网应激（ER）胁迫增加。 随着BM组成的变化，GALNT1缺乏腺体显示出降低的FGF信号传导，降低AKT和MAPK磷酸化以及上皮细胞增殖的降低。 在GALNT1缺乏腺体中，外源添加BM成分层粘连蛋白以β1-整合素依赖性方式营救了FGF信号传导和生长缺陷。我们的工作表明，O-糖基化会影响哺乳动物器官发育过程中分泌的ECM的组成，从而对细胞信号，增殖和器官生长产生影响。 这些结果强调了O-糖基化在建立细胞微环境中的保守作用，并对该蛋白质修饰在发育和疾病中的作用具有影响。 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这种保守蛋白质修饰在正常发育和疾病易感性中起作用的机制。