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 have taken advantage of a simpler model system (Drosophila melanogaster) to investigate the biological role of glycosylation during development. Previous work from our group demonstrated that there are at least 9 functional transferase genes in Drosophila 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. More recently, we have performed in vivo RNA interference (RNAi) to identify the remaining family members that are also essential for viability. We have discovered that 4 additional family members are required for viability and are essential in specific tissues. Moreover, one of these newly defined essential genes is responsible for proper gut function. Loss of this glycosyltransferase results in reduced secretion of O-glycosylated proteins into the lumen of the gut and affects the structure of the cells responsible for proper gut acidifcation. Mutations in this family member result in improper gut acidification. These studies have implications for the role of this protein modification in proper gut function in higher eukaryotes, as these genes are abundantly expressed in the stomach, small intestine and colon of mice and humans. We also performed 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). Using this approach, we obtained 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. 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. 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. These studies provide a new platform for interrogating the cellular effects of mucin-type O-linked glycosylation and evidence for unique subcellular roles of the pgants in secretory apparatus structure and function. By examining the consequences of mutations in pgant family members in the fly, we found that mutations in pgant3 alter integrin-mediated epithelial cell adhesion in the Drosophila wing blade. We discovered that the loss of pgant3 resulted in the improper secretion and localization of the extracellular matrix (ECM) protein Tiggrin. Tiggrin is an integrin-binding protein that is normally O-glycosylated in wild type wing discs. Loss of Tiggrin within the basement membrane region in pgant3 mutants resulted in disruption of integrin-mediated cell adhesion and defects in wing formation. These studies provided the first example of the effects of O-glycosylation on protein secretion, establishment of the basement membrane and modulation of integrin-mediated cell adhesion in vivo. We followed up on these results to ask whether the loss of a mammalian O-glycosyltransferase (Galnt1) has an effect on basement membrane formation and organogenesis using the murine submandibular gland (SMG) as a model system. The basement membrane of the developing SMG is a complex array of components that influence cell signaling, proliferation and differentiation;additionally, it is rich in O-glycosylated proteins. In these studies, we demonstrate that the loss of Galnt1 affects FGF-mediated cell proliferation during mammalian SMG organogenesis by influencing the secretion of basement membrane proteins. Mice deficient for the enzyme Galnt1 (that adds sugars to proteins during early stages of SMG development) resulted in intracellular accumulation of major BM components along with increased endoplasmic reticulum (ER) stress. Along with changes in BM composition, Galnt1 deficient glands displayed decreased FGF signaling, reduced AKT and MAPK phosphorylation, and reduced epithelial cell proliferation. Exogenous addition of BM component laminin to Galnt1 deficient glands rescued FGF signaling and the growth defects in a β1-integrin-dependent manner. Our work demonstrates that O-glycosylation influences the composition of the secreted ECM during mammalian organ development, with resultant effects on cell signaling, proliferation and organ growth. These results highlight a conserved role for O-glycosylation in the establishment of cellular microenvironments and have implications for the role of this protein modification in both development and disease. 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 the mechanisms by which this conserved protein modification operates in both normal development and in disease susceptibility.

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National Institute of Dental & Craniofacial Research
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Ji, Suena; Samara, Nadine L; Revoredo, Leslie et al. (2018) A molecular switch orchestrates enzyme specificity and secretory granule morphology. Nat Commun 9:3508
Zhang, Liping; Ten Hagen, Kelly G (2018) Pleiotropic effects of O-glycosylation in colon cancer. J Biol Chem 293:1315-1316
Zhang, Liping; Ten Hagen, Kelly G (2017) Enzymatic insights into an inherited genetic disorder. Elife 6:
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Revoredo, Leslie; Wang, Shengjun; Bennett, Eric Paul et al. (2016) Mucin-type O-glycosylation is controlled by short- and long-range glycopeptide substrate recognition that varies among members of the polypeptide GalNAc transferase family. Glycobiology 26:360-76
Tian, E; Stevens, Sharon R; Guan, Yu et al. (2015) Galnt1 is required for normal heart valve development and cardiac function. PLoS One 10:e0115861
Tran, Duy T; Masedunskas, Andrius; Weigert, Roberto et al. (2015) Arp2/3-mediated F-actin formation controls regulated exocytosis in vivo. Nat Commun 6:10098

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