The COG complex is essential for retrograde trafficking and glycosylation at the Golgi. The COG complex performs this function through interaction with vesicle docking and fusion machinery at the Golgi; however, its exact mechanism of action is unknown. Previous studies of the COG complex were limited to the use of COG-CDG patient fibroblasts, siRNA mediated knockdowns, or protein relocalization approaches. In this work, I have generated a new cellular model for COG deficiency using CRISPR/Cas9 mediated gene editing to create a full set of HEK293T knock-out (KO) cell lines missing each individual COG subunit. A combination of molecular, cell, biochemical and microscopy techniques was used to characterize COG KO cells for glycosylation and trafficking defects, cell proliferation rates, stability of COG subunits, localization of Golgi markers, changes in Golgi structure, and N-glycan profiling. All KO cell lines were uniformly deficient in Golgi glycosylation and showed defects in Golgi morphology, retrograde trafficking and sorting, and abnormal accumulation of enlarged endolysosomal structures.
To elucidate how much COG KO induced misglycosylation was contributing to mutant phenotypes three additional knockouts of components of glycosylation machinery were created. Side-by-side analysis of glycosylation deficient cells with COG KO cells led to the discovery that many COG knock-out phenotypes were glycosylation independent including: Golgi and lysosomal morphology changes, trafficking abnormalities, and enhanced secretion. Importantly, these COG deficiency specific phenotypes were also apparent in COG7-CDG patient fibroblasts proving the human disease relevance of this new COG knockout model. The knowledge gained from this study has important implications, both for understanding the physiological role of COG complex in Golgi homeostasis, and for better understanding human diseases associated with COG/Golgi impairment.
|Advisor:||Lupashin, Vladimir V.|
|Commitee:||Baldini, Guilia, Gaddy, Dana, Morello, Roy, Ware, Jerry|
|School:||University of Arkansas for Medical Sciences|
|Department:||Physiology and Biophysics|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 79/10(E), Dissertation Abstracts International|
|Keywords:||Congenital disorders of glycosylation, Conserved oligomeric golgi complex, Crispr, Glycosylation, Golgi, Membrane trafficking|
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