Role of O-glycosylation in Animal Development

粘蛋白型o -连接糖基化是一种广泛的、进化上保守的蛋白质修饰，由一系列酶（果蝇中的pants或哺乳动物中的ppGalNAcTs）催化，这些酶将糖n-乙酰半乳糖胺（GalNAc）转移到注定是膜结合或分泌的蛋白质中的丝氨酸和苏氨酸的羟基上。这种糖基化的缺陷是人类家族性肿瘤钙质沉着症和Tn综合征的原因。此外，o -糖基化的改变与肿瘤的进展和转移有关。全基因组关联研究已经确定了编码酶的基因，这些酶在与高密度脂蛋白胆固醇水平、甘油三酯水平、先天性心脏缺陷、结肠癌和骨矿物质密度相关的疾病中负责启动o糖基化。从这些研究中，很明显，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制作用，以了解这种糖基化的变化如何促进疾病的易感性和进展。

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.