Mucin-type O-linked glycosylation is a widespread and evolutionarily conserved protein modification catalyzed by a family of enzymes (PGANTs in Drosophila or pGalNAcTs in mammals) that transfer the sugar N-acetylgalactosamine (GalNAc) to the hydroxyl group of serines and threonines in proteins that are destined to be membrane-bound or secreted. Defects in this type of glycosylation are responsible for the human diseases familial tumoral calcinosis and Tn syndrome. Additionally, changes in O-glycosylation have been associated with tumor progression and metastasis. More recently, genome-wide association studies have identified the genes encoding the enzymes that are responsible for initiating O-glycosylation among those associated with HDL-cholesterol levels, triglyceride levels, congenital heart defects, colon cancer and bone mineral density. From these studies, it is apparent that this conserved protein modification has a multitude of biological roles. The focus of our research group is to elucidate the mechanistic roles of O-glycans during development in order to understand how they contribute to disease susceptibility and progression. Previous work from our group demonstrated that O-linked glycosylation is essential for viability in Drosophila. Our studies have demonstrated roles for this protein modification in the secretion of extracellular matrix (ECM) proteins. Specifically, we found that loss of one PGANT family member alters secretion of an ECM protein, thereby influencing basement membrane composition and disrupting integrin-mediated cell adhesion during Drosophila wing development. Likewise, we demonstrated that O-glycosylation also modulates the composition of the ECM during mammalian organ development, influencing integrin and FGF signaling, thereby affecting cell proliferation and growth of the developing salivary glands. These results highlight a conserved role for O-glycosylation in secretion and in the establishment of cellular microenvironments. Recent studies by our group elucidated the mechanism by which O-glycans influence secretion in the Drosophila digestive tract. We found that one member of this family (PGANT4) modulates secretion by glycosylating an essential component of the secretory apparatus (Tango1), conferring protection from furin-mediated proteolysis. Tango1 is an ER/Golgi transmembrane protein that coordinates packaging of large cargo into secretory vesicles. In the absence of PGANT4, Tango1 is cleaved, resulting in loss of secretory apparatus polarization, loss of secretory vesicle formation and disrupted secretion of proteins that line and protect the digestive tract. 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. Additionally, we have developed a system for real-time imaging of secretory vesicle formation and polarized secretion in a living organ, to define how O-glycosylation is involved in these processes. Using this system, we have defined the order of events that occur as vesicles are formed and eventually fuse with the apical membrane to secrete their contents into the extracellular space. Through 3D time-lapse imaging, we show that F-actin recruitment to fused vesicles occurs after fusion pore formation but before myosin recruitment. Moreover, we show essential roles for the branched actin nucleators, Arp2/3 and WASp in specific aspects of regulated secretion. Taking advantage of facile gene disruption in vivo, we are further investigating how the PGANTs are mediating effects on secretion and secretory apparatus structure through real-time imaging in organs where certain family members have been deleted. Finally, in collaboration with the Tabak laboratory, we are investigating the effects of loss of O-glycosylation on other aspects of mammalian development. Specifically, we have found that loss of Galnt1 affects cardiac function in mice, resulting in aortic and pulmonary valve stenosis, regurgitation, altered ejection fraction and cardiac dilation. The primary defect resulting in compromised function stems from increased cell proliferation during valve development. We found that loss of Galnt1 resulted in the loss of O-glycans in developing valves at embryonic day 11.5 (E11.5). Moreover, the loss of O-glycans was accompanied by loss of the proteases ADAMTS1 and ADAMTS5, along with decreased cleavage of the proteoglycan versican and increased BMP and MAPK signaling. Increased cell proliferation within the developing valves was observed as early as E11.5. Taken together, our study demonstrates that loss of a member of the Galnt family can influence organ development and function by affecting the formation/remodeling of the extracellular matrix during development, with resultant effects on cell proliferation. Furthermore, this study provides the first evidence for the role of this protein modification in heart valve development and may represent a new model for idiopathic valve 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. We are also using real-time imaging within living organs to define the specific processes by which O-glycosylation influences secretion. Our hope is that the cumulative results of our research will elucidate the mechanisms by which this conserved protein modification operates in both normal development and disease susceptibility.

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11
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2015
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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
Tran, Duy T; Ten Hagen, Kelly G (2017) Real-time insights into regulated exocytosis. J Cell Sci 130:1355-1363
Zhang, Liping; Ten Hagen, Kelly G (2017) Enzymatic insights into an inherited genetic disorder. Elife 6:
Milberg, Oleg; Shitara, Akiko; Ebrahim, Seham et al. (2017) Concerted actions of distinct nonmuscle myosin II isoforms drive intracellular membrane remodeling in live animals. J Cell Biol 216:1925-1936
Zhang, Liping; Turner, Bradley; Ribbeck, Katharina et al. (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 292:21231-21242
Ten Hagen, Kelly G (2016) Novel or reproducible: That is the question. Glycobiology 26:429
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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