Role of O-glycosylation in Animal Development

National Institute Of Dental & Craniofacial Research

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Abstract

Cells are decorated with a variety of carbohydrates that serve many diverse functions. These glycans not only act as a protective barrier on the external surface of the cell, but are also involved in cell adhesion, migration, communication and signaling events (Tran and Ten Hagen, 2013; Zhang and Ten Hagen, 2015). While roles for diverse glycans in many developmental processes continue to be elucidated, we still lack a clear understanding of the roles of mucin-type O-glycans during development and organ function. Mucin-type O-linked glycosylation is a widespread and evolutionarily conserved modification that involves the addition of the sugar N-acetylgalactosamine (GalNAc) to the hydroxyl group of serines and threonines in proteins that are destined to be membrane-bound or secreted (Tian and Ten Hagen, 2009; Ten Hagen et al., 2009; Zhang and Ten Hagen, 2011; Tran et al., 2012a; Tran and Ten Hagen, 2013; Zhang et al., 2014a; Ten Hagen, 2015; Zhang and Ten Hagen, 2019). Our initial interest in mucin-type O-glycans stemmed from their abundance in salivary secretions, where they lubricate the soft and hard tissues of the oral cavity. We and others determined that this type of protein modification is initiated by a large family of enzymes known as the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs or Galnts in mammals and PGANTs in Drosophila). There are 19-20 Galnt family members in mammals. Mutations in some family members are now known to cause certain human diseases, including hyperphosphatemic familial tumoral calcinosis (HFTC), and changes in O-glycans have historically been associated with tumor development and progression (Tran and Ten Hagen, 2013). Our group cloned and characterized the genes responsible for O-linked glycosylation in Drosophila and demonstrated that multiple members are essential for viability in Drosophila (Ten Hagen and Tran, 2002; Tran et al., 2012a). Moreover, we have elucidated key roles for certain members, including proper formation of the Drosophila respiratory system (Tian and Ten Hagen, 2007b); proper cell adhesion during development (Zhang et al., 2008; Zhang et al., 2010); and protection of a cargo receptor required for secretory granule formation (Zhang et al., 2014b). Our studies in Drosophila provided the basis for hypotheses regarding the role of O-glycans and Galnts during mammalian development and organ function. Factors involved in biosynthesis and packaging of mucins Mucins are large extracellular matrix proteins that protect epithelial surfaces throughout the body. Changes in mucin production and secretion are associated with colon cancer, inflammatory bowel disease and cystic fibrosis. We have previously shown that the proper packaging of one mucin is dependent on a small cysteine-rich adaptor protein. This adaptor acts via cysteine bonding between itself and the C-terminal cysteine-rich domain of the mucin. Loss of this adaptor protein disrupts mucin packaging in secretory granules, alters the mobile fraction within granules and results in granules that are larger, more circular and more fragile. We are continuing to examine other factors essential for proper mucin production. Understanding mucin biosynthesis, packaging and secretion may provide insight into novel treatment strategies for diseases of the respiratory and digestive tracts. Quantitative mapping of the in vivo glycoproteome of Galnt family members In collaborative work with the Tabak lab, we continue to define the in vivo substrates of each Galnt in various mouse tissues using our Galnt-deficient lines. We have previously mapped the Galnt2 glycoproteome in liver, to highlight the substrates that may be involved in the complex metabolic phenotypes and congenital disorder of glycosylation associated with this glycosyltransferase. In another recent collaborative study, additional substrates of Galnt3 have been identified that are involved in bone development and remodeling, mineral homeostasis, calcium ion binding and skeletal morphology, highlighting a potential network of proteins that may be involved in disease presentation beyond the previously identified FGF23. We are continuing to map and examine the glycoproteome of other members of this glycosyltransferase family. Single cell RNAseq of salivary glands to identify factors controlling regulated secretion We are continuing to examine the role of other factors in secretory granule formation and secretion. To that end, we are analyzing single cell RNA data on salivary glands that are at various stages of secretory granule biogenesis. This will provide information about the genes present in cells that have near-mature secretory granules as well as cells that are just beginning to form secretory granules. By analyzing differential gene expression across these diverse cell stages, we are identifying genes that are involved in each step of the secretory program. We have performed RNA interference to identified genes to demonstrate their roles in this process. We are currently preparing this manuscript for submission. Enzymatic characterization of HFTC mutations in GALNT3 The human disease HFTC is caused by mutations in GALNT3. However, the effect of these mutations on GALNT3 activity or substrate specificity has not been examined. We have therefore cloned and expressed GALNT3 mutations to assess their enzymatic activity in vitro. We have now finished characterization of these mutations and are preparing a manuscript describing them. Interestingly, some mutations were unable to be stably expressed, suggesting that they result in structural changes that induce instability. Using structural modeling, we demonstrate which bonds are disrupted and provide explanations for why certain mutations likely result in an unstable enzyme. Mutations that could be stably expressed were analyzed across a variety of substrates. While some mutations abrogated all activity, others displayed low but detectable activity against a variety of substrates.