Role of O-glycosylation in Animal Development

National Institute Of Dental & Craniofacial Research

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Abstract

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.