Role of O-glycosylation in Animal Development

O-糖基化在动物发育中的作用

• 批准号: 10003743
• 负责人: KELLY G TEN HAGEN
• 金额: $ 191.9万
• 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/10003743
• 关键词: Acetylgalactosamine; Active Sites; Address; Affect; Alternative Splicing; Animal Model; Animals; Apical; Appearance; Biogenesis; Biological; Biological Process; Biology; Bone Density; Catalytic Domain; Cell Adhesion; Cell Communication; Cell membrane; Charge; Chronic Kidney Failure; Collaborations; Colon Carcinoma; Complex; Congenital Heart Defects; Crystallization; Cytoplasmic Granules; Defect; Development; Disease Progression; Disease susceptibility; Drosophila genus; Drosophila melanogaster; Enzymes; Eukaryota; Event; Extracellular Matrix Proteins; Failure; Familial tumoral calcinosis; Family; Fibroblast Growth Factor; Focus Groups; Genes; Genetic; Glean; Glycopeptides; Goals; High Density Lipoprotein Cholesterol; Hormones; Integrin-mediated Cell Adhesion Pathway; Integrins; Investigation; Kidney; LDL-Receptor Related Protein 2; Laboratories; Lectin; Link; Mammals; Mediating; Membrane; Modification; Molecular Weight; Morphology; Movement; Mucins; Mus; Neoplasm Metastasis; Organ; Orthologous Gene; Peptides; Phenotype; Polysaccharides; Post-Translational Protein Processing; Process; Protein Glycosylation; Protein Isoforms; Proteins; Proteinuria; RNA Splicing; Renal function; Research; Role; Salivary Glands; Secretory Vesicles; Serine; Signal Transduction; Structure; Substrate Specificity; Syndrome; System; Threonine; Tissues; Triglycerides; Tubular formation; Variant; Vesicle; Work; base; ex vivo imaging; genome wide association study; glycosylation; glycosyltransferase; human disease; hydroxyl group; imaging platform; in vivo; interest; member; novel; organ growth; preference; receptor; sugar; tumor progression

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. 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 and organ function in order to understand how changes in this type of glycosylation contribute to disease susceptibility and progression. Previous work from our group demonstrated that disruption of O-linked glycosylation alters secretion of extracellular matrix 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. To further investigate the role of O-glycosylation in secretion, we developed a powerful imaging platform using Drosophila salivary glands, allowing us to visualize the initial formation of secretory vesicles, secretory granule maturation, 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 components involved in mammalian secretion. Recent work by our group using this system has elucidated a novel in vivo role for O-glycosylation in secretory vesicle morphology as well as identified a novel regulatory mechanism for controlling the substrate specificity of the enzymes that initiate O-glycosylation. We identified a new member of the PGANT family PGANT9 that undergoes tissue-specific differential splicing within the salivary gland of Drosophila. This splicing event generates 2 isoforms (PGANT9A and PGANT9B) that differ within a non-catalytic subdomain (lectin domain) 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 protein (a 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, providing the first evidence that cargo glycosylation can influence secretory vesicle morphology. Additionally, in collaboration with Nadine Samara and Lawrence Tabak, the crystal structure of each splice variant was solved to demonstrate how the differential splicing affects substrate preferences at the atomic level. In short, the splicing event creates either a positively or negatively charged loop that lies in proximity to the active site of the catalytic domain. This charged loop appears to control access of charged peptide substrates to the active site, thus determining which substrates will be glycosylated. This represents the first example of a splicing event within this enzyme family altering substrate specificity. It is also the first example of changes within the lectin domain (which was previously thought to only affect interactions with glycopeptide substrates) altering peptide preferences. In summary, our results elucidate a novel mechanism for altering the substrate preferences of members of this O-glycosyltransferase enzyme family through alternative splicing within subregions of the lectin domain. Additionally, we provide evidence that the glycosylation status of cargo influences secretory vesicle morphology. We are also finishing a mouse study based on our prior work in Drosophila that demonstrated that one member of this glycosyltransferase family (pgant35A) was essential for viability and affected the apical composition of tubular organs. We have made and characterized a mouse deficient for the ortholog of pgant35A (Galnt11). Galnt11-deficient mice are viable yet suffer from kidney defects, characterized by proteinuria and the specific failure to resorb low molecular weight proteins. After extensive phenotyping, we have determined that the conserved endocytic receptor megalin is no longer appropriately glycosylated in Galnt11-deficient mice. Interestingly, Galnt11 was previously identified by GWAS as being associated with chronic kidney disease. Our studies demonstrate how the loss of Galnt11 may affect kidney function and elucidate another role for O-glycosylation in mammalian biology (that was informed by studies in Drosophila). Ongoing studies by our group are focused on further investigation of the factors involved 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 continue to use the Drosophila salivary gland system to identify components that influence the formation and/or morphology of the secretory vesicles. We are currently deciphering the mechanisms by which these proteins work. 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。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-聚糖在发育和器官功能中的机械作用，以了解这种类型的糖基化的变化如何有助于疾病的敏感性和进展。 我们小组的先前工作表明，O连锁的糖基化的破坏会改变细胞外基质蛋白的分泌，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。 为了进一步研究O-糖基化在分泌中的作用，我们使用果蝇唾液腺开发了一个强大的成像平台，使我们能够可视化分泌囊泡的初始形成，分泌颗粒成熟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。鉴于分泌物中涉及的许多基因和机制在整个物种之间都是保守的，因此该系统可以提供有关哺乳动物分泌所涉及的成分功能的详细机械信息。 我们小组使用该系统的最新工作阐明了在分泌囊泡形态中O-糖基化的新型体内作用，并确定了一种用于控制启动O-糖基化酶底物特异性的新型调节机制。 我们确定了PGANT家族PGANT9的新成员，该成员在果蝇的唾液腺内经历组织特异性差异剪接。 该剪接事件会产生2种同工型（PGANT9A和PGANT9B），这些同工型在该酶的非催化子域（凝集素结构域）中有所不同，并且对分泌颗粒形态进行了差异调节。 有趣的是，该亚域的剪接赋予了独特的底物特异性，从而使货物蛋白（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，这提供了第一个证据，证明货物糖基化会影响分泌的囊泡形态。 此外，与Nadine Samara和Lawrence Tabak合作，解决了每个剪接变体的晶体结构，以证明差异剪接如何影响原子水平的底物偏好。 简而言之，剪接事件会产生一个阳性或负电荷的环，该环路与催化域的活跃位点相邻。 该带电的回路似乎控制了带电肽底物对活性位点的访问，因此确定将糖基化哪些底物。 这代表了此酶家族中改变底物特异性的剪接事件的第一个示例。 它也是凝集素结构域内变化（以前被认为仅影响与糖肽底物的相互作用）的第一个示例改变了肽偏爱。 总而言之，我们的结果阐明了一种新的机制，该机制是通过静脉蛋白结构域子区域内的替代剪接来改变该O-糖基转移酶家族的底物偏好。 此外，我们提供的证据表明货物的糖基化状态会影响分泌囊泡的形态。 我们还根据我们在果蝇的先前工作完成了一项小鼠研究，该研究表明该糖基转移酶家族的一个成员（PGANT35A）对于生存能力至关重要，并影响了管状器官的顶端组成。 我们已经制作并表征了缺乏pgant35a直系同源物（GALNT11）的小鼠。 GALNT11缺陷型小鼠可行，但患有肾脏缺陷，其特征是蛋白尿和吸收低分子量蛋白的特定失败。 经过广泛的表型，我们确定了保守的内吞受体梅加林不再在GALNT11缺陷型小鼠中适当地糖基化。 有趣的是，GALNT11先前被GWAS鉴定为与慢性肾脏疾病有关。 我们的研究表明，GALNT11的丧失如何影响肾脏功能，并阐明哺乳动物生物学中O-糖基化的另一种作用（这是由果蝇的研究告知的。 我们小组正在进行的研究集中于进一步研究分泌和分泌囊泡形成的因素。 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。 我们继续使用果蝇唾液腺系统来识别影响分泌囊泡形成和/或形态的组成部分。 我们目前正在解密这些蛋白质起作用的机制。 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。