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 are also involved in cell adhesion, migration, communication and signaling events in many organisms. Our group studies one type of sugar addition to proteins, known as mucin-type O-linked glycosylation, which is initiated by the polypeptide GalNAc transferase (ppGalNAcT or PGANT) enzyme family. This sugar addition is seen in most eukaryotic organisms including mammals, fish, insects, worms and some types of fungi. The conservation of this protein modification across species suggests that it plays crucial roles during many aspects of development. It is known that there are as many as 20 family members encoding functional ppGalNAcTs in mammals. Given the size of the family and the complexity it generates, we sought an alternative, simpler model system to investigate 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. We began our 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 is required for viability. These studies provided the first evidence that a member of this multigene family is required for development and viability in any eukaryote. Additionally, we defined the spatial and temporal patterns of expression of all the pgant family members throughout Drosophila development. We have also elucidated the developmental profile of specific O-glycans using a variety of sugar binding lectins and antibodies to determine where and when O-glycans may be required during development. Recently, we have found that mutations in another member of this family (pgant3) alter epithelial cell adhesion in the Drosophila wing blade. A transposon insertion mutation in pgant3 or RNA interference (RNAi) to pgant3 resulted in blistered wings, a phenotype characteristic of genes involved in integrin-mediated cell interactions. Precise excision of the transposon restored pgant3 expression and wing integrity. Mutations in PGANT3 that form a stable yet inactive Golgi-localized enzyme also resulted in wing blistering, indicating that proper cell adhesion is dependent upon glycosyltransferase activity. Expression of wild type pgant3 in the mutant background rescued the wing blistering phenotype, whereas expression of another family member did not, revealing a unique requirement for PGANT3 activity. Recently isolated point mutations in pgant3 show genetic interactions with an integrin mutant, demonstrating a genetic link between O-glycosylation and integrin-mediated cell adhesion. We have identified one of the main O-glycosylated proteins in the wing disc (Tiggrin) using a combination of affinity purification, biochemistry and bioinformatics. Tiggrin is an integrin-binding extracellular matrix (ECM) protein that is specifically O-glycosylated in wild type wing discs but not in pgant3 mutants. Our recent results demonstrate that Tiggrin is normally specifically localized at the interface between the dorsal and ventral cell layers of the developing wild type wing. However, Tiggrin fails to be localized to this interface in pgant3 mutant wings. Indeed, when comparing the location of Tiggrin in wing cells from wild type and pgant3 mutants, we found that Tiggrin accumulates intracellularly in pgant3 mutants, demonstrating that Tiggrin secretion is affected when O-glycosylation is disrupted. Our studies provide the first in vivo example of the effects of O-glycosylation on protein secretion, establishment of the basal matrix and modulation of integrin-mediated cell adhesion in vivo. We are also performing RNAi to each pgant in fly cell culture to examine the effects of each gene on specific cellular processes (cell adhesion, division, viability, apoptosis, morphological changes, intracellular transport, subcellular alterations). We used Drosophila S2R+ cells, a line of embryonic origin that is adherent via integrin-dependent mechanisms, and S2 cells, a non-adherent embryonic line that can be induced to adhere via integrin independent mechanisms when plated on concanavalin A (ConA). Using this approach, we obtained specific knockdown of each individual isoform in each cell line. Interestingly, we obtained additional evidence for the role of pgants in the proper formation and structure of the secretory apparatus. RNAi to either pgant3 or pgant6 resulted in altered Golgi organization. In each instance, the Golgi lost its normal punctate appearance and was either more diffuse (after pgant3 RNAi) or was comprised of smaller, fainter puncta (after pgant6 RNAi). Disruption of the normal Golgi structure in both cases was accompanied by a reduction in secretion, indicating a functional consequence of the loss of each transferase. Additionally, RNAi to pgant3 also resulted in alteration of the normal actin cytoskeletal architecture, changes in cell morphology and loss of cell adhesion. Furthermore, the cell adhesion defects seen after pgant3 RNAi were specific to the cells that adhere via integrins, lending further support to a role for pgant3 in integrin-mediated adhesion. Other effects observed included multi-nucleated cells seen after RNAi to pgant2 or pgant35A in both cell lines, suggesting a role for these genes in the completion of cytokinesis. No effects on endocytosis were observed after RNAi to any pgant. No evidence of apoptosis was observed after RNAi to any of the pgants. These studies provide evidence for efficient and specific knockdown of pgant transcripts and enzyme activity in Drosophila cell culture, providing a new platform for interrogating the cellular and sub-cellular effects of mucin-type O-linked glycosylation. These data also provide additional evidence for unique subcellular roles for the pgants in secretory apparatus structure and function. In summary, we are using information gleaned from Drosophila to better focus on crucial aspects of development affected by O-glycosylation in more complex 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.
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