Cells within 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 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). It is termed ?mucin-type? glycosylation because it is present in great abundance on proteins known as mucins, which dominate the salivary secretions, respiratory, digestive and reproductive tracts. 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 24 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 14 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 by characterizing the genes responsible for O-linked glycosylation in Drosophila melanogaster. We have demonstrated that there are at least 9 functional members in Drosophila (potentially 14 total) and that at least one gene (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 a eukaryotic organism. In an effort to elucidate the biological role of O-glycosylation, we have focused on identifying when and where each family member is required during development as well as defining the consequences of the loss of a member of this family. To begin to address these issues, we performed RNA in situ hybridization experiments to define when during embryonic development each gene is expressed. This information will aid us in determining where to look for defects and developmental pathways influenced by mucin-type O-glycosylation. Thus far, we are seeing unique temporal and spatial expression patterns for each member of this family, with some being expressed in many regions throughout embryogenesis while others are expressed very specifically in certain regions at specific times (Tian and Ten Hagen, submitted). A number of family members are highly expressed in the developing salivary glands and specific portions of the digestive tract, suggesting a role for O-glycosylation in these developing tissues. Additionally, we are defining the variety of sugars present during fly development by employing fluorescent-labeled sugar binding proteins known as lectins. These lectin studies will corroborate our data on the expression of the pgant genes as well as tell us what other sugars are also present on the surface of cells at each stage of development. To address the consequences of the loss of a family member, we have examined the development of flies containing a defective pgant35A gene. We have established that this gene is required at multiple distinct times during development for viability. Additionally, we have shown that adding back the functional form of the pgant35A gene will rescue the lethality observed, conclusively demonstrating that the defects observed are due to the loss of this specific gene. We are continuing to do a careful analysis of the various stages of embryogenesis to identify the exact biological processes affected in these mutants. The results we find here will be translated back into the mammalian system, as a distinct mammalian counterpart to the pgant35A gene exists. In addition to the pgant35A studies, we are also examining mutations in other members of this family to determine whether or not they 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. 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. We are currently constructing mice deficient in the mammalian counterpart of the fly pgant35A gene to determine its role in mammalian development. 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.
