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：
9555618
负责人：
KELLY G TEN HAGEN
金额：
$ 233.2万
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/9555618
关键词：
Ablation Acetylgalactosamine Address Affect Alternative Splicing Animal Model Animals Appearance Biogenesis Biological Bone Density Calcinosis Cell Adhesion Cell Differentiation process Cell Proliferation Cell membrane Colitis Colon Carcinoma Communication Complex Congenital Heart Defects Crohn&apos s disease Cytoplasmic Granules Defect Development Disease Disease Progression Disease susceptibility Drosophila genus Drosophila melanogaster Enzymes Epithelial Epithelium Eukaryota Event Extracellular Matrix Proteins Familial disease Family Family member Fibroblast Growth Factor Gastrointestinal tract structure Genes Genetic Glean Goals High Density Lipoprotein Cholesterol Hormones Integrin-mediated Cell Adhesion Pathway Integrins Interleukin-6 Investigation Laboratories Link Mammals Membrane Modification Morphology Mouth Diseases Movement Mucins Mucous Membrane Mucous body substance Neoplasm Metastasis Oral mucous membrane structure Organ Paracrine Communication Play Polysaccharides Post-Translational Protein Processing Protein Glycosylation Protein Isoforms Proteins RNA Splicing Research Role Salivary Glands Secretory Vesicles Serine Signal Transduction Stem cells Substrate Specificity Supplementation Syndrome System Threonine Tissues Triglycerides Variant Vesicle Work body system cytokine ex vivo imaging genome wide association study glycosylation human disease hydroxyl group imaging platform in vivo interest member mimetics novel organ growth overexpression pathogen receptor screening sugar tumor tumor progression

Ablation Acetylgalactosamine Address Affect Alternative Splicing Animal Model Animals Appearance Biogenesis Biological Bone Density Calcinosis Cell Adhesion Cell Differentiation process Cell Proliferation Cell membrane Colitis Colon Carcinoma Communication Complex Congenital Heart Defects Crohn&apos s disease Cytoplasmic Granules Defect Development Disease Disease Progression Disease susceptibility Drosophila genus Drosophila melanogaster Enzymes Epithelial Epithelium Eukaryota Event Extracellular Matrix Proteins Familial disease Family Family member Fibroblast Growth Factor Gastrointestinal tract structure Genes Genetic Glean Goals High Density Lipoprotein Cholesterol Hormones Integrin-mediated Cell Adhesion Pathway Integrins Interleukin-6 Investigation Laboratories Link Mammals Membrane Modification Morphology Mouth Diseases Movement Mucins Mucous Membrane Mucous body substance Neoplasm Metastasis Oral mucous membrane structure Organ Paracrine Communication Play Polysaccharides Post-Translational Protein Processing Protein Glycosylation Protein Isoforms Proteins RNA Splicing Research Role Salivary Glands Secretory Vesicles Serine Signal Transduction Stem cells Substrate Specificity Supplementation Syndrome System Threonine Tissues Triglycerides Variant Vesicle Work body system cytokine ex vivo imaging genome wide association study glycosylation human disease hydroxyl group imaging platform in vivo interest member mimetics novel organ growth overexpression pathogen receptor screening sugar tumor tumor progression

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项目摘要

Mucin-type O-linked glycosylation is a widespread and evolutionarily conserved protein modification catalyzed by a family of enzymes (PGANTs in Drosophila or ppGalNAcTs in mammals) that transfer the sugar N-acetylgalactosamine (GalNAc) to the hydroxyl group of serines and threonines in proteins that are destined to be membrane-bound or secreted. Defects in this type of glycosylation are responsible for the human diseases familial tumoral calcinosis and Tn syndrome. Additionally, changes in O-glycosylation have been associated with tumor progression and metastasis. More recently, genome-wide association studies have identified the genes encoding the enzymes that are responsible for initiating O-glycosylation among those associated with HDL-cholesterol levels, triglyceride levels, congenital heart defects, colon cancer and bone mineral density. From these studies, it is apparent that this conserved protein modification has a multitude of biological roles. The focus of our research group is to elucidate the mechanistic roles of O-glycans during development in order to understand how they contribute to disease susceptibility and progression. Previous work from our group demonstrated that O-linked glycosylation is essential for viability in Drosophila. Our studies have demonstrated roles for this protein modification in the secretion of extracellular matrix (ECM) proteins. Specifically, we found that loss of one PGANT family member alters secretion of ECM proteins, disrupting integrin-mediated cell adhesion during Drosophila development and influencing integrin and FGF signaling during mammalian organ development. Mechanistically, we have shown that O-glycosylation of a conserved cargo receptor modulates its stability and ability to form secretory vesicles. These results highlight a conserved role for O-glycosylation in secretion and in the establishment of cellular microenvironments. Recent studies by our group have focused on further investigation of the role of O-glycosylation in secretion and secretory vesicle formation. Additionally, we are interested in investigating how the synthesis, packaging and secretion of large, highly O-glycosylated proteins, such as mucins, are regulated. We have therefore developed a powerful imaging platform using Drosophila salivary glands, allowing us to visualize the initial formation of mucin-containing secretory vesicles, their maturation as they undergo homotypic fusion and regulated secretion after hormone stimulation. The powerful combination of Drosophila genetics with in vivo and ex vivo imaging allows one to rapidly interrogate the role of factors in many aspects of secretion, including vesicle biogenesis, vesicle movement, fusion with the plasma membrane, release of granule cargo and expansion of cargo once in the lumen. Given that many genes and mechanisms involved in secretion are conserved across species, this system can provide detailed mechanistic information regarding the function of many components involved in mammalian secretion. We have recently discovered a role for one member of the pgant family in secretory vesicle formation. We identified a novel member of the pgant family that undergoes tissue-specific differential splicing within the salivary gland of Drosophila. This splicing event generates 2 isoforms that differ within a subdomain of this enzyme and differentially modulate secretory granule morphology. Interestingly, splicing of this subdomain confers unique substrate specificity, allowing the complete glycosylation of a cargo mucin. In the absence of one splice variant, the cargo mucin is not fully glycosylated and secretory granules take on an irregular, shard-like appearance. Our study provides the first evidence that alternative splicing of a member of the pgant family can alter substrate specificity and that the glycosylation status of mucin cargo can influence secretory granule morphology. We are also examining the function of the mucous membrane that lines the internal epithelia of various organ systems of our bodies. The mucous barrier of our digestive tract is the first line of defense against pathogens and damage. Disruptions in this barrier are associated with diseases, such as Crohns disease, colitis and colon cancer. In our research, we show that genetic ablation of the mucosal barrier in Drosophila causes epithelial expression of the IL-6-like cytokine Upd3, leading to differentiation of cells that form the progenitor cell niche. Disruption of the niche leads to abnormal proliferation of progenitor cells. Niche disruption could be recapitulated by overexpressing upd3 and rescued by deleting upd3, highlighting a crucial role for this cytokine. Additionally, niche integrity and cell proliferation could be rescued by overexpression of the conserved cargo receptor Tango1 or by supplementation with exogenous mucins. Our studies elucidate the paracrine signaling events activated by a compromised mucosal barrier and provide a novel in vivo screening platform for mucin mimetics and other strategies to treat diseases of the oral mucosa and digestive tract. In summary, we are using information gleaned from Drosophila to better focus on crucial aspects of development and organ function affected by O-glycosylation in more complex mammalian systems. Our hope is that the cumulative results of our research will elucidate the mechanisms by which this conserved protein modification operates in both normal development and disease susceptibility.

粘蛋白型O连接糖基化是一种广泛而进化的蛋白质修饰，由酶（果蝇中的PGANT或PPGALNACTS中的PGANTS）催化，可将糖N-乙酰基乳糖苷（galnac）转移到糖N-乙酰乳糖苷（galnac）中，将蛋白质素和THREN REANINE SERENINE SERON INROON构成，以下膜结合或分泌。这种类型的糖基化缺陷导致人类疾病家族性肿瘤钙化和TN综合征。另外，O-糖基化的变化与肿瘤进展和转移有关。最近，全基因组关联研究已经确定了编码与HDL-胆固醇水平，甘油三酸酯水平，先天性心脏缺陷，结肠癌和骨矿物质密度相关的酶的编码基因。从这些研究中可以明显看出，这种保守的蛋白质修饰具有多种生物学作用。我们研究小组的重点是阐明O-Glycans在开发过程中的机械作用，以了解它们如何促进疾病的敏感性和进展。我们小组的先前工作表明，O连接的糖基化对于果蝇的生存能力至关重要。我们的研究表明，这种蛋白质修饰在细胞外基质（ECM）蛋白的分泌中的作用。具体而言，我们发现一个PGANT家族成员的丧失会改变ECM蛋白的分泌，从而破坏果蝇发育过程中整联蛋白介导的细胞粘附，并影响哺乳动物器官发育过程中整联蛋白和FGF信号传导。从机械上讲，我们已经表明，保守的货物受体的O-糖基化调节其稳定性和形成分泌囊泡的能力。这些结果强调了O-糖基化在分泌和细胞微环境中的保守作用。我们小组的最新研究集中在进一步研究O-糖基化在分泌和分泌囊泡形成中的作用。此外，我们有兴趣研究如何调节大型，高糖基化蛋白的合成，包装和分泌。因此，我们已经使用果蝇唾液腺开发了功能强大的成像平台，使我们能够可视化含粘蛋白的分泌囊泡的初始形成，它们的成熟，因为它们经过同型融合和激素刺激后的调节分泌。果蝇遗传学与体内和体内成像的强大组合使人们可以迅速质疑因子在分泌的许多方面的作用，包括囊泡生物发生，囊泡运动，与质膜的融合，释放颗粒货物和货物在Lumen中的扩张。鉴于在分泌物中涉及的许多基因和机制在各种物种之间都是保守的，因此该系统可以提供有关许多参与哺乳动物分泌的成分功能的详细机械信息。我们最近在分泌囊泡形成中发现了Pgant家族的一个成员的角色。我们确定了PGANT家族的一个新成员，该家族在果蝇的唾液腺内经历组织特异性差异剪接。该剪接事件产生的2种同工型在该酶的亚域内不同，并差异调节了分泌颗粒的形态。有趣的是，该子域的剪接赋予了独特的底物特异性，从而使货物粘蛋白完全糖基化。在没有一个剪接变体的情况下，货物粘蛋白没有完全糖基化，分泌颗粒具有不规则的，类似碎片的外观。我们的研究提供了第一个证据，表明PGANT家族成员的替代剪接可以改变底物特异性，并且粘蛋白货物的糖基化状态可以影响分泌的颗粒形态。我们还在研究粘膜的功能，该粘膜在身体各种器官系统的内部上皮上排列。我们消化道的粘液屏障是针对病原体和损害的第一道防线。该障碍的破坏与疾病有关，例如克罗恩斯疾病，结肠炎和结肠癌。在我们的研究中，我们表明果蝇中粘膜屏障的遗传消融会导致IL-6样细胞因子UPD3的上皮表达，从而导致形成祖细胞细胞小裂细胞的分化。利基的破坏会导致祖细胞的异常增殖。可以通过过度表达upd3并通过删除upd3来挽救利基破坏，从而突出了这种细胞因子的关键作用。另外，可以通过过度表达保守的货物受体探戈1或补充外源粘蛋白来挽救利基完整性和细胞增殖。我们的研究阐明了通过受损的粘膜屏障激活的旁分泌信号事件，并为粘蛋白模拟物和其他治疗口腔粘膜和消化道疾病的策略提供了一种新颖的体内筛选平台。总而言之，我们正在使用从果蝇收集的信息，以更好地关注更复杂的哺乳动物系统中O-糖基化影响的发育和器官功能的关键方面。我们的希望是，我们研究的累积结果将阐明这种保守的蛋白质修饰在正常发育和疾病易感性中起作用的机制。