InteractiveFly: GeneBrief

Polypeptide N-Acetylgalactosaminyltransferase 4: Biological Overview | References

Gene name - Polypeptide N-Acetylgalactosaminyltransferase 4

Synonyms -

Cytological map position - 23F3-23F3

Function - enzyme

Keywords - O-glycosyltransferase, initiates the formation of mucin-type O-linked glycans, secretion of components of the peritrophic/mucous membrane in the larval digestive tract, mutation can be rescued by expression of conserved cargo receptor Tango1 and partially rescued by supplementation with exogenous mucins or treatment with antibiotics, mutants exhibit disruption of the mucosal barrier

Symbol - Pgant4

FlyBase ID: FBgn0051956

Genetic map position - chr2L:3,472,603-3,475,338

NCBI classification - pp-GalNAc-T: pp-GalNAc-T initiates the formation of mucin-type O-linked glycans

Cellular location - cytoplasmic

NCBI link: EntrezGene, Nucleotide, Protein

The mucous barrier of the digestive tract is the first line of defense against pathogens and damage. Disruptions in this barrier are associated with diseases such as Crohn's disease, colitis and colon cancer, but mechanistic insights into these processes and diseases are limited. Loss of a conserved O-glycosyltransferase (PGANT4) in Drosophila has been shown to result in aberrant secretion of components of the peritrophic/mucous membrane in the larval digestive tract. This study shows that loss of pgant4 disrupts the mucosal barrier, resulting in epithelial expression of the IL-6-like cytokine Upd3, leading to activation of JAK/STAT signaling, differentiation of cells that form the progenitor cell niche and abnormal proliferation of progenitor cells. This niche disruption could be recapitulated by overexpressing upd3 and rescued by deleting upd3, highlighting a crucial role for this cytokine. Moreover, niche integrity and cell proliferation in pgant4-deficient animals could be rescued by overexpression of the conserved cargo receptor Tango1 and partially rescued by supplementation with exogenous mucins or treatment with antibiotics. These findings help 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 (Zhang, 2017).

The mucous barrier that lines the respiratory and digestive tracts is the first line of defense against pathogens and provides hydration and lubrication. This unique membrane separates the delicate epithelia from factors present in the external environment. In mammals, the mucous lining of the intestine allows nutrient penetration while conferring protection from both bacteria and the mechanical damage associated with digestion of solid food. The principal components of mucous membranes are mucins, a diverse family of proteins that are expressed in tissue-specific fashions. Whereas secreted mucins vary greatly in sequence and size, they all share highly O-glycosylated serine- and threonine-rich regions that confer unique structural and rheological properties, allowing the formation of hydrated gels. Deletion of specific components of the mucous membranes present in the lung (Muc5b) or the intestinal tract (Muc2) resulted in defects in mucociliary clearance or an increased incidence colorectal cancer, respectively. Changes in mucus production or the glycosylation status of mucins have also been associated with oral pathology in Sjogren's syndrome, the development of colitis and intestinal tumors in mice, and the progression of ulcerative colitis and colon cancer in humans. However, the detailed mechanisms by which loss/alteration of this protective layer results in epithelial pathology remain unknown (Zhang, 2017).

Many components and factors that confer the unique properties of this protective lining, including the mucins and the enzymes that mediate their dense glycosylation, are conserved across species. Indeed, Drosophila melanogaster contains a similar protective lining known as the peritrophic membrane, consisting of a chitin scaffold that is bound by highly O-glycosylated mucins. During larval development, where animals ingest solid food and undergo massive growth in preparation for metamorphosis, specialized secretory cells (PR cells) of the anterior midgut produce this membrane, which is thought to protect epithelial cells of the digestive tract from mechanical and microbial damage. Previous work in Drosophila has shown that loss of one component of the peritrophic membrane resulted in a thinner and more permeable membrane, where adult flies were viable yet more susceptible to oral infections. However, the exact role of the entirety of this lining in the integrity and protection of cells of the digestive tract remains unknown (Zhang, 2017).

Extensive research elucidating the development and function of the many cell types that comprise the Drosophila digestive tract has provided insights into mammalian digestive system formation and function. The Drosophila larval midgut is composed of specialized epithelial cells (enterocytes [ECs] and enteroendocrine cells) for digestion and nutrient absorption, as well as progenitor cells that will eventually form the adult midgut epithelium. These adult midgut progenitor cells (AMPs) reside in a protected niche, formed by peripheral cells (PCs) that wrap and shield them from external signaling. PCs are characterized by a unique crescent shape, with long processes that surround AMPs, restricting proliferation and differentiation until metamorphosis. The larval digestive tract therefore represents an ideal system to interrogate the role of the mucous layer in protection of both the epithelium and the progenitor cell niche at a stage when the mechanical and microbial stresses associated with the ingestion of solid food are abundant (Zhang, 2017).

Previous work has shown that loss of a conserved UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase responsible for initiating O-linked glycosylation (PGANT4) resulted in aberrant secretion of components of the peritrophic membrane in the larval digestive tract (Tran, 2012). This study shows that pgant4 mutants are devoid of a peritrophic membrane, resulting in epithelial cell damage and expression of the IL-6-like inflammatory cytokine, unpaired 3 (Upd3). Upd3 expression resulted in increased JAK/STAT signaling in the progenitor cell niche, causing niche cell differentiation and aberrant progenitor cell proliferation. These effects were dependent on Upd3 and could be rescued by deleting upd3 or partially rescued by feeding animals antibiotics or exogenous mammalian intestinal mucins. Moreover, overexpression of the conserved extracellular matrix cargo receptor, Tango1 (transport and Golgi organization 1), in secretory cells of the digestive tract resulted in restoration of the peritrophic membrane and rescue of niche integrity. These results elucidate new mechanistic details regarding how a compromised mucous lining can influence epithelial integrity and the progenitor cell niche and provide an in vivo screening platform for compounds and strategies that could restore mucosal barrier function (Zhang, 2017).

This study shows the peritrophic membrane is essential to protect the integrity of the epithelial cell layer and maintain an appropriate environment for the progenitor cell niche. Moreover, a dynamic and specific response to the loss of this membrane occurs via the production of the IL-6-like cytokine Upd3 from epithelial cells, which in turn signals to niche cells in a paracrine fashion, causing differentiation and morphological changes. This demonstrates the multipotent nature of PCs, which can respond to specific cytokines to alter their fate. Once the PC morphology and fate were altered, JAK/STAT signaling was activated in AMPs exposed to Upd3, causing aberrant cell proliferation/DNA replication. This represents the first example where loss of the protective mucous lining activates signaling from epithelial cells to alter the fate of niche cells and change the behavior of progenitor cells. These studies highlight the importance of this membrane in both epithelial and progenitor cell biology and elucidate the paracrine signaling cascade that is specifically activated when this barrier is compromised (Zhang, 2017).

Interestingly, the mucinous peritrophic membrane could be restored by overexpression of the conserved cargo receptor Tango1. Tango1 is an essential protein that functions to package large extracellular matrix proteins, such as collagen and mucins, into secretory vesicles. Loss of the mammalian ortholog of Tango1 (Mia3) in a murine model resulted in lethality with global defects in collagen secretion and extracellular matrix composition. Alterations in Tango1/Mia3 expression have also been associated with colon and hepatocellular carcinomas in humans. Previous work in Drosophila demonstrated that PGANT4 glycosylates Tango1, protecting it from Dfur2-mediated proteolysis in the digestive tract. This study shows that Tango1 overexpression specifically in the secretory PR cells of the digestive tract can restore the mucinous membrane throughout the midgut to rescue epithelial viability and niche integrity, further demonstrating the crucial role of the peritrophic membrane in digestive system homeostasis and health. These results suggest the possibility of exogenous Tango1 expression as a potential strategy to restore secretion, mucous membranes, and/or extracellular matrix composition and confer epithelial protection (Zhang, 2017).

The larval digestive system offers unique opportunities to investigate the role of the individual components of the mucinous membrane and restorative strategies in epithelial biology. Unlike the adult stage, the larval portion of the life cycle is devoted to continuous feeding and digestion to orchestrate the massive growth of cells and tissues in preparation for metamorphosis. As such, larvae consume many types of solid food and will readily ingest various compounds. Indeed, oral supplementation with an intestinal mucin (Muc2) partially rescued JAK/STAT signaling, suggesting that this could serve as a strategy for epithelial protection. Muc2 is a major component of the protective mucous membrane that lines the small intestine and colon of mammals. Muc2 is thought to confer lubrication for food passage as well as to form a barrier between microbes and epithelial cells of the digestive tract. The current results suggest that Muc2 supplementation could be providing similar properties in the Drosophila digestive tract. Interestingly, supplementation with the gastric mucin (Muc5AC) dramatically exacerbated JAK/STAT signaling, suggesting that the different structural, rheological, or binding properties of each mucin are mediating distinct cellular responses in this system. Current work is focused on deciphering the specific functional regions of various secreted mucins and testing their ability to confer epithelial protection using this in vivo system. Human diseases of the digestive tract are associated with disrupted mucinous linings, and disease severity is often correlated with the severity of barrier disruption. Interestingly, these diseases are also characterized by increased levels of the mammalian ortholog of Upd3 (IL-6), increased JAK/STAT activation, and increased cell proliferation, similar to what is seen in Drosophila, suggesting conserved mechanisms for responding to mucosal disruption/injury. It is widely known that immune cells are one source of IL-6 in mammals, but recent studies have demonstrated that mechanically damaged epithelial and endothelial cells also produce IL-6. This study demonstrated that epithelial expression of Upd3 is both necessary and sufficient for the changes in PC fate and AMP proliferation, as disruption of the niche could be recapitulated by overexpression of upd3 from ECs and rescued by deletion of upd3. How peritrophic membrane loss is signaling to up-regulate upd3 expression in ECs is currently unknown. However, previous studies in the adult Drosophila digestive system have shown up-regulation of upd, upd2, and upd3 in response to enteric infection or damage-inducing agents, such as bleomycin or dextran sulfate sodium, suggesting roles for both microbial insults and physical/mechanical damage to epithelial cells. Indeed, the results also suggest roles for microbial and mechanical damage in the absence of the peritrophic membrane, as both antibiotics and mucin supplementation were able to reduce upd3 expression and cell proliferation. This study demonstrates that the larval midgut can serve as a model system to study how cells/tissues sense and respond to damage as well as to decipher how upd3 is specifically activated in epithelial cells under various conditions (Zhang, 2017).

As a mucous layer is present across most internal epithelial surfaces, understanding the mechanisms by which it confers protection and epithelial homeostasis will be informative in treating various diseases affecting the integrity of this layer. Mucosal healing has been proposed as a treatment option for inflammatory bowel disease and other diseases of the digestive tract that are characterized by destruction of the mucosa and epithelial surfaces. Likewise, mucins are a component in some oral treatments for dry mouth caused by head and neck irradiation or Sjogren's syndrome. Other therapeutics for various autoimmune and inflammatory diseases include JAK inhibitors (Jakinibs) and drugs directed against particular cytokines. This study has shown that genetic restoration of the peritrophic membrane can restore digestive system health and that antibiotic treatment or mucin supplementation can partially rescue damage-induced signaling cascades, suggesting that this Drosophila system may be a viable platform for testing compounds to remediate epithelial damage. Future studies will focus on testing newly emerging mucin mimetics (designed to confer epithelial protection and appropriate rheology/hydration), synthetic mucins (where the extent of glycosylation can be specifically modified), glycan-based hydrogels, and drugs that target conserved steps in the JAK/STAT signaling cascade. Lessons learned in Drosophila may inform future strategies for functional restoration of mucosal protection (Zhang, 2017).

O-glycosylation regulates polarized secretion by modulating Tango1 stability

Polarized secretion is crucial in many tissues. The conserved protein modification, O-glycosylation, plays a role in regulating secretion. However, the mechanisms by which this occurs are unknown. This study demonstrates that an O-glycosyltransferase functions as a novel regulator of secretion and secretory vesicle formation in vivo by glycosylating the essential Golgi/endoplasmic reticulum protein, Tango1 (Transport and Golgi organization 1), and conferring protection from furin-mediated proteolysis. Loss of the O-glycosyltransferase PGANT4 resulted in Tango1 cleavage, loss of secretory granules, and disrupted apical secretion. The secretory defects seen upon loss of pgant4 could be rescued either by overexpression of Tango1 or by knockdown of a specific furin (Dfur2) in vivo. These studies elucidate a novel regulatory mechanism whereby secretion is influenced by the yin/yang of O-glycosylation and proteolytic cleavage. Moreover, the data have broader implications for the potential treatment of diseases resulting from the loss of O-glycosylation by modulating the activity of specific proteases (Zhang, 2014).

This study demonstrates that O-glycosylation regulates polarized secretion and secretory vesicle formation in vivo by modulating the stability of an essential component of the secretory apparatus. While previous studies have documented the effects of loss of O-glycosylation on constitutive secretion and secretion of extracellular matrix (ECM) proteins in vivo, the current studies elucidate a mechanism by which these effects may occur. This study shows that Tango1 stability is modulated by the presence of O-glycans, which serve to protect it from furin-mediated cleavage. This represents the first example of O-glycans modulating secretion by stabilizing an essential component of the secretory apparatus (Zhang, 2014).

Roles for Tango1 in constitutive secretion, Golgi structure, and COPII vesicle formation have been identified previously, but the factors that regulate its activity and stability are not completely understood. This study demonstrates that Tango1 stability is modulated by the competing activities of a specific O-glycosyltransferase (PGANT4) and a specific furin (Dfur2) in secretory cells of the Drosophila digestive tract. Tango1 is a ubiquitously expressed protein that regulates not only constitutive secretion, but also the formation of large secretory vesicles that transport bulky ECM proteins in certain cells. Tango1/Mia3 is proposed to bind secretory cargo via its luminal domain and COPII coat subunits via its cytoplasmic domain, thereby coordinating the size of secretory vesicles to accommodate large ECM proteins. However, not all cells have the same secretory demands nor produce large vesicular carriers. This study demonstrates that the expression of PGANT4 specifically in the secretory PR cells of the digestive tract confers increased stability to Tango1 by protecting it from Dfur2-mediated cleavage, thereby allowing the formation of large mucin-containing secretory vesicles within these cells. As the enzymes controlling the initiation of O-glycosylation are typically abundantly expressed in cells under high secretory burden, it raises the possibility that O-glycosylation may modulate the stability of Tango1 in other tissues, ensuring that Tango1 activity is commensurate with the secretory demands of the cell (Zhang, 2014).

How are O-glycans specifically influencing Tango1 stability? Based on in vitro studies, O-glycans may influence protease sensitivity by blocking access to vicinal protease cleavage sites. Additionally, O-glycans may affect protein conformation, thus altering protease sensitivity at more distant sites. Finally, O-glycosylation may also influence the binding of partner or cargo proteins, thereby affecting protease access. Although the current data suggest that O-glycans added by PGANT4 may influence vicinal protease cleavage, the possibility cannot be ruled out that O-glycans may influence Tango1 conformation and/or cargo binding. Future studies mapping all sites of glycosylation of Tango1 by PGANT4 will aid in determining the mechanisms involved (Zhang, 2014).

PGANT4 is one member of a large family of enzymes (PGANTs in Drosophila, GALNTs in humans, and ppGalNAcTs in mice) that control the initiation of O-glycosylation. These family members are expressed in unique spatial and temporal patterns during development and in adult tissues. Additionally, these enzymes display unique substrate specificities, with some members preferring to add the initial GalNAc (peptide transferases) and others preferring to add GalNAc to previously glycosylated substrates (glycopeptide transferases). The concerted activity of these many members is thought to result in the elaborate glycosylation patterns typically seen in mucin-like molecules. As members of this family are abundantly expressed in many secretory cells and tissues, it raises the possibility that the yin/yang provided by the opposing forces of O-glycosylation and proteolytic cleavage may serve as a more widespread, dynamic system to regulate the stability and bioactivity of many proteins. In support of this theory, recent glycoproteomic studies performed in mammalian cell culture have mapped sites of O-glycosylation to be in close proximity to potential furin cleavage sites on many proteins. Additionally, these studies also identified O-glycans on mammalian Tango1/Mia3, suggesting that O-glycans may perform similar protective functions to control secretion in mammalian systems. Indeed, alterations in both Tango1/Mia3 and O-glycosylation have been associated with diseases of the mammalian gastrointestinal tract, which are typically characterized by loss of secretion, loss of mucous membrane formation, and increased diffusion barrier permeability. Interestingly, PGANT4 is most similar to the mammalian ppGalNAc-T10, which is abundantly expressed in the digestive system. It will be interesting to determine if mammalian Tango1/Mia3 is similarly regulated by O-glycosylation and proteolytic cleavage (Zhang, 2014).

More broadly, the current results provide in vivo evidence for models where competing activities of O-glycosyltransferases and proteases may modulate protein stability, with imbalances in these activities contributing to disease. Genome-wide association studies demonstrating a link between O-glycosylation and blood lipid levels have led to the proposal that O-glycans on proteins involved in lipid metabolism may confer protection from proteolysis, thereby influencing HDL-cholesterol and triglyceride levels, and thus cardiovascular disease risk. Similarly, in the case of familial tumoral calcinosis, it is proposed that mutations in an O-glycosyltransferase (GALNT3 in humans and Galnt3 in mice) may lead to decreased FGF23 glycosylation, increased FGF23 cleavage, abnormal phosphate levels, and calcified tumor development in patients. However, although cell culture and in vitro assays have shown that FGF23 can be glycosylated by Galnt3, the glycosylation status of endogenous FGF23 in the presence or absence of Galnt3 has not been examined (in mice or humans). Similarly, although O-glycans can confer protection from protease cleavage of peptides in vitro, a demonstration that cleavage of endogenous FGF23 can be rescued by altering levels of an endogenous protease in vivo has not been previously shown. The current results provide in vivo evidence for the interplay between O-glycosylation and protease sensitivity, supporting the model of how loss of O-glycosylation may contribute to human disease and disease associations. More importantly, the ability to rescue the effects of loss of O-glycosylation by altering the levels of a specific furin in vivo suggests that inhibition/modulation of specific proteases may be a viable treatment option for certain diseases associated with aberrant O-glycosylation (Zhang, 2014).

Multiple members of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase family are essential for viability in Drosophila

Mucin-type O-glycosylation represents a major form of post-translational modification that is conserved across most eukaryotic species. This type of glycosylation is initiated by a family of enzymes (GalNAc-Ts in mammals and PGANTs in Drosophila) whose members are expressed in distinct spatial and temporal patterns during development. Previous work has group demonstrated that one member of this family is essential for viability and another member modulates extracellular matrix composition and integrin-mediated cell adhesion during development (Tian, 2007). To investigate whether other members of this family are essential, RNA interference (RNAi) was employed to each gene in vivo. Using this approach, four additional pgant genes were identified that are required for viability. Ubiquitous RNAi to pgant4, pgant5, pgant7, or the putative glycosyltransferase CG30463 resulted in lethality. Tissue-specific RNAi was also used to define the specific organ systems and tissues in which each essential family member is required. Interestingly, each essential pgant had a unique complement of tissues in which it was required. Additionally, certain tissues (mesoderm, digestive system, and tracheal system) required more than one pgant, suggesting unique functions for specific enzymes in these tissues. Expanding upon the RNAi results, it was found that conventional mutations in pgant5 resulted in lethality and specific defects in specialized cells of the digestive tract, resulting in loss of proper digestive system acidification. In summary, these results highlight essential roles for O-glycosylation and specific members of the pgant family in many aspects of development and organogenesis (Tran, 2012).

This study identified 4 additional members of this multigene glycosyltransferase family that are essential for viability by taking advantage of the highly efficient and specific in vivo RNAi system in Drosophila. This work serves to highlight the biological importance of O-linked glycosylation and of specific family members responsible for initiating this conserved post-translational modification. Additionally, these studies indicate that at least 5 family members in the fly are serving unique and nonredundant functions during development. Thus, whereas these members all catalyze the transfer of GalNAc to serine or threonine, they are doing so on specific substrates and/or at specific positions within these substrates. Previous enzymatic studies demonstrated that many mammalian GalNAc-Ts and Drosophila PGANTs have unique substrate preferences and sites of GalNAc addition in vitro, suggesting that certain substrates can only be glycosylated by certain enzymes. The current in vivo results support this hypothesis and suggest a highly complex system for proper O-glycosylation of proteins (Tran, 2012).

The in vivo RNAi system also offers the unique advantage of allowing the knockdown of genes in specific tissues to determine exactly where a particular gene is required. Gal4 driver lines were used that induced RNAi in most major tissue types and developing organ systems to determine where each essential pgant was required. In agreement with previous studies demonstrating that conventional pgant35A mutants affect respiratory system development, this study found that RNAi to pgant35A in the tracheal system resulted in significantly reduced viability. Interestingly, each essential gene was found to be required in a specific subset of tissues and organ systems, suggesting unique functional roles for each. Developing organs known for the abundant production of O-glycoproteins (digestive and respiratory systems)) were most significantly affected by the knockdown of pgant family members. Knockdown of pgant4, pgant5, pgant35A, or the putative transferase CG30463 in the digestive system resulted in significant losses of viability. In addition to pgant35A, knockdown of pgant5, pgant7, or CG30463 in the respiratory system also affected viability. CG30463 was also found to be required in the amnioserosa, an embryonic tissue involved in developmentally regulated cell migration events. Finally, 3 of the 5 essential pgants were required in the mesoderm. The results presented here will form the basis for future studies investigating the mechanistic role of these essential pgants in diverse tissues and developmental stages with the hope of gaining insight into the role of their mammalian orthologues (Tran, 2012).

To begin to address the specific role of one of the novel essential genes identified in this screen, the phenotypes associated with the loss of pgant5 were further characterized. Interestingly, loss of pgant5 resulted in the disruption of the proper structure and function of specialized cells of the digestive tract responsible for gut acidification. These studies revealed that the loss of pgant5 caused loss of O-glycoproteins along the apical and luminal surfaces of copper cells of the midgut, along with an irregular apical actin-based microvillar structure in pgant5c03193/Df(2L)BSC109. Restoration of pgant5 expression resulted in restoration of O-glycosylation, actin-based microvilli, and proper gut acidification. Western blots revealed that the loss of pgant5 affected the glycosylation of many proteins, making it difficult to discern what the primary target(s) might be. It remains possible that PGANT5 could be glycosylating subunits of the ion transporters responsible for gut acidification. Alternatively, PGANT5 could be glycosylating other proteins involved in the localization of ion transporters or the establishment of proper apical polarity within the copper cells. In support of this, previous work on PGANT35A in the respiratory tract demonstrated that it also glycosylates apical and luminal proteins and affects apical-basal polarity when mutated (Tian, 2007). As O-glycoproteins are abundant along the apical regions of many developing tissues, it is possible that they are performing functions related to maintenance of the unique characteristics of apical surfaces (Tran, 2012).

Previous work on the mammalian orthologue of pgant5 (Galnt1) did not examine the digestive system but did find lymphocyte homing defects (due to decreased presence of selectin ligands on lymphocytes and endothelial cells) and bleeding disorders (due to decreased plasma levels of blood coagulation factors) in Galnt1-/- mice. However, the specific substrates involved and the mechanism(s) by which these phenotypes occur remain unknown. Nonetheless, the results presented in this study highlight a previously unknown role for pgant5 in digestive tract function and provide a basis for examining the role of Galnt1 in mammalian digestive system function. There is precedent for mucins and mucin-type O-glycosylation having functional roles in the digestive system as the loss of a major O-glycosylated mucin (Muc2) or the disruption of certain O-glycan structures results in digestive tract abnormalities, including increased rates of infection, decreased barrier function, and increased susceptibility to colitis and colon cancer. Future studies on PGANT5 in the Drosophila gut will identify direct in vivo targets of this enzyme and define how O-glycosylation regulates their function (Tran, 2012).

As mentioned above, the putative glycosyltransferase CG30463 was found to be essential for viability and required in multiple developing tissues. Based on sequence conservation, CG30463 is assumed to be a member of the pgant family. However, in vitro biochemical assays have failed to detect glycosyltransferase activity for the purified recombinant CG30463 protein, thus its biological relevance has been unclear. The data shown here, indicating that knockdown of CG30463 in specific tissues results in lethality, demonstrates a crucial in vivo role for this gene. It is possible that the CG30463 protein has very specific substrate preferences and thus its in vitro enzymatic activity may not have been detectable with the panel of substrate peptides used. Future experiments will use this RNAi system to investigate in vivo changes in O-glycosylation upon CG30463 knockdown (Tran, 2012).

Finally, the in vivo RNAi system was used to address the role of each family member in a specific developmental process, integrin-mediated cell adhesion. This study demonstrated that pgant3 has a unique role in integrin-mediated wing blade adhesion, as RNAi to other family members had no effect on wing blistering. The results, demonstrating a unique function for pgant3 in the wing, are also supported by genetic data indicating that expression of another family member in the wing could not compensate for loss of pgant3. These studies indicate that PGANT3 may be uniquely responsible for glycosylating the extracellular matrix protein Tiggrin in the wing disc, further supporting the notion of unique substrate preferences in vivo. The role for other pgants in specific cell adhesion events in other tissues will be investigated using this methodology (Tran, 2012).

In summary, this study has identified four additional members of this glycosyltransferase family that are essential for viability in Drosophila, highlighting the importance of this protein modification and unique requirements for individual family members. Future work will define the specific functions and substrates of each member in Drosophila to gain insight into the roles of O-glycosylation in development and disease (Tran, 2012).

Dissecting the biological role of mucin-type O-glycosylation using RNA interference in Drosophila cell culture

Mucin type O-glycosylation is a highly conserved form of post-translational modification initiated by the family of enzymes known as the polypeptide alpha-N-acetylgalactosaminyltransferases (ppGalNAcTs in mammals and PGANTs in Drosophila). To address the cellular functions of the many PGANT family members, RNA interference (RNAi) to each pgant gene was performed in two independent Drosophila cell culture lines. RNAi to individual pgant genes results in specific reduction in gene expression without affecting the expression of other family members. Cells with reduced expression of individual pgant genes were then examined for changes in viability, morphology, adhesion, and secretion to assess the contribution of each family member to these cellular functions. This study found that RNAi to pgant3, pgant6, or pgant7 resulted in reduced secretion, further supporting a role for O-glycosylation in proper secretion. Additionally, RNAi to pgant3 or pgant6 resulted in altered Golgi organization, suggesting a role for each in establishing or maintaining proper secretory apparatus structure. Other subcellular effects observed included multinucleated cells seen after RNAi to either pgant2 or pgant35A, suggesting a role for these genes in the completion of cytokinesis. These studies demonstrate the efficient and specific knockdown of pgant gene expression in two Drosophila cell culture systems, resulting in specific morphological and functional effects. This work provides new information regarding the biological roles of O-glycosylation and illustrates a new platform for interrogating the cellular and subcellular effects of this form of post-translational modification (Zhang, 2010).

Expression of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase family is spatially and temporally regulated during Drosophila development

The UDP-GalNAc : polypeptide N-acetylgalactosaminyltransferase (ppGaNTase or ppGalNAcT or pgant) enzyme family is responsible for the first committed step in the synthesis of mucin-type O-glycans on protein substrates. Previous work has demonstrated both sequence and functional conservation between members of this family in mammals and the fruit fly, Drosophila melanogaster. One member of this family in Drosophila has been shown to be essential for viability and development. In an effort to understand the developmental stages and processes in which O-glycosylation is involved, this study determined the expression pattern of each functional family member as well as putative isoforms during Drosophila development. These studies indicate that isoforms are expressed in discrete spatial and temporal fashions during development, with some isoforms being found uniquely in restricted areas of the developing embryo (brain, trachea, pharynx, esophagus, proventriculus, and amnioserosa), whereas others are found in multiple regions and overlap with the expression of other isoforms (salivary glands, posterior midgut, anterior midgut, and the fore-/hindgut) during embryogenesis. Additionally, expression patterns were examined in imaginal discs from third instar larvae, which will become the adult structures. Most isoforms are also expressed in the imaginal discs, with some showing unique transcript localization and spatial regulatory control. Thus, this report provides insight into the specific regions during Drosophila development that may require O-linked glycosylation in vivo as well as which isoforms may act cooperatively in certain tissues and which may be uniquely responsible for glycosylation in others (Tian, 2006).


Search PubMed for articles about Drosophila Pgant4

Tian, E. and Ten Hagen, K. G. (2006). Expression of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase family is spatially and temporally regulated during Drosophila development. Glycobiology 16(2): 83-95. PubMed ID: 16251381

Tran, D. T., Zhang, L., Zhang, Y., Tian, E., Earl, L. A. and Ten Hagen, K. G. (2012). Multiple members of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase family are essential for viability in Drosophila. J Biol Chem 287(8): 5243-5252. PubMed ID: 22157008

Zhang, L. and Ten Hagen, K. G. (2010). Dissecting the biological role of mucin-type O-glycosylation using RNA interference in Drosophila cell culture. J Biol Chem 285(45): 34477-34484. PubMed ID: 20807760

Zhang, L., Syed, Z. A., van Dijk Hard, I., Lim, J. M., Wells, L. and Ten Hagen, K. G. (2014). O-glycosylation regulates polarized secretion by modulating Tango1 stability. Proc Natl Acad Sci U S A 111(20): 7296-7301. PubMed ID: 24799692

Zhang, L., Turner, B., Ribbeck, K. and Ten Hagen, K. G. (2017). Loss of the mucosal barrier alters the progenitor cell niche via Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling. J Biol Chem 292(52): 21231-21242. PubMed ID: 29127201

Biological Overview

date revised: 3 March, 2018

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