Cells of the body are decorated with a variety of carbohydrates (sugars) that serve many diverse functions. These sugars not only act as a protective barrier on the outside of the cell, but have been found to be involved in cell adhesion, migration, communication and signaling events in many organisms. Indeed, many recently described birth defects and syndromes in humans are the result of defects in enzymes responsible for the regulation, synthesis or incorporation of carbohydrates in cells (Congential Disorders of Glycosylation or CDG). While sugars are recognized as being important for embryonic development and adult organ function, we still do not fully understand how they mediate these processes at the molecular level. Our group studies one type of sugar addition to proteins known as mucin-type O-linked glycosylation, which is initiated by the enzyme family known as the polypeptide GalNAc transferases (ppGaNTases or pgants). This sugar addition is seen in most higher organisms including mammals, fish, insects, worms and some types of fungi. The conservation of this protein modification across species suggests that it plays a crucial role(s) during many aspects of development. It is known that there are as many as 20 family members encoding functional ppGaNTases in mammals. Given the size of the family and the complexity it generates, we sought an alternative, simpler model system to aid in investigating the biological role of glycosylation. Analysis of the genome databases from other organisms indicated that the fruit fly (Drosophila melanogaster) had only 12 potential members and may therefore be a more tractable experimental system. Additionally, the fruit fly offers more sophisticated genetic techniques, shorter generation times and a wealth of well-characterized stocks on which to build future studies. Moreover, the fruit fly has been used successfully in the past to decode biological problems and translate what has been learned back into the more complex mammalian systems.? ? We began these studies by cloning and characterizing the genes responsible for O-linked glycosylation in Drosophila. We demonstrated that there are at least 9 functional transferase genes in Drosophila (potentially 12 members total) and that at least one (pgant35A) is required for viability (Ten Hagen and Tran, 2002; Ten Hagen et al., 2003). These studies provided the first example that a member of this multigene family is required for development and viability in any eukaryote. Additionally, we have defined the spatial and temporal patterns of expression of all the pgant family members throughout Drosophila development (Tian and Ten Hagen, 2006). During this past year, we have also elucidated the developmental profile of specific O-glycans using a variety of fluorescent-labeled sugar binding lectins and antibodies that are specific for certain carbohydrate structures (Tian and Ten Hagen, 2007 in press). Using confocal microscopy, we were able to visualize the diverse array of O-glycans present on all developing structures and organs throughout embryogenesis. This information will aid us in determining what tissues and developmental pathways may require O-glycans as well as provide information as to what lectins would be most useful for identifying native proteins that contain O-glycans. This will further allow us to directly interrogate changes in carbohydrate composition in mutant strains during development. All of the studies mentioned above provide us with the background information and tools we will need to decipher the role these enzymes are playing during Drosophila development. ? ? This year, we have demonstrated that one gene is required at multiple distinct times during development for viability. Specifically, pgant35A is required during embryogenesis; homozygous mutants devoid of wild type maternal RNA show abnormal tracheal tube formation and migration of secondary branches in developing embryos (Tian and Ten Hagen, 2007). This is particularly interesting given that the Drosophila tracheal system serves as a model for branching morphogenesis in many mammalian organ systems, including the salivary gland, lung, kidney and vasculature. Specifically, we have found that pgant35A mutants have defects in epithelial cell shape, polarity and diffusion barrier formation within the tracheal system. We observed a decrease in apical staining of certain apical and luminal markers, concomitant with increased staining in cytoplasmic vesicles, suggesting that the phenotypes observed are the result of disruption of transport of proteins destined for the apical and luminal regions. We have also performed lectin staining of embryos and determined that the primary glycan in the tracheal system is GalNAc-Ser/Thr present in the apical and luminal regions; this glycan is severely reduced in pgant35A mutants. We have shown that adding back the functional form of the pgant35A gene will rescue the lethality, conclusively demonstrating that the defects observed are due to the loss of this specific gene.? ? In addition to pgant35A, we are also examining whether other members of this family are required for proper development as well. Like pgant35A, many of these genes have distinct mammalian counterparts and display similar enzymatic activities in vitro, suggesting the results from studies in flies will shed light on the functional role of these genes in mice and humans. To that end, we are also constructing mice deficient in the mammalian counterpart of the fly pgant35A gene to determine its role in mammalian development. In summary, we are using information gleaned from Drosophila to better focus on crucial aspects of development affected by O-glycosylation in more complex mammalian systems. Our hope is that the cumulative results of the studies described above will elucidate why O-linked glycosylation is necessary and what role sugars play in cellular communication and interactions occurring during eukaryotic development.
