Relationship of N-glycans to Neuronal Dysfunction
Whitman, Austin A
N-glycosylation is important in regulating protein activity and serves several vital roles contributing to protein folding, protein assembly, stability, and interactions. Changes in branching of N-glycans are linked to development and maintenance of multicellular organisms. All N-glycans share a common core sugar sequence and are classified as three major types: oligomannose, complex, and hybrid. Here the aim is to determine the roles N-glycans have in cell behavior, as well as, vertebrae development. To characterize the contribution of complex N-glycans to neuroblastoma (NB), CRISPR-Cas9 technology was employed to silence the Mgat2 gene in a NB cell line. Mgat2 encodes for GlcNAcT-II, N-acetylglucosaminyltransferase-II, responsible for converting hybrid type N-glycans to complex type. Lectin binding studies were conducted to support the successful knockout of Mgat2. Electrophoretic mobility shifts of Kv3.1, a voltage-gated K+ channel, supports that N220 and N229, possess complex N-glycans in our wild type cell line and hybrid N-glycans in our N-glycosylation mutant cell line. Wound healing, anchorage-independent growth, and MMP-2 expression studies found that the loss of Mgat2 resulted in decreased tumorigenicity. Mgat1b was silenced in zebrafish to study how increased oligomannose N-glycans affect vertebrae development. Magt1b encodes for GlcNAct-I, responsible for converting oligomannose N-glycans to hybrid type, which in turn form complex N-glycans. DNA sequencing and various genotyping of zebrafish generations support the knockout of Mgat1b. The loss of Mgat1b resulted in decreased embryo viability but also increased the number of eggs spawned. Overall, this data reveals that complex N-glycans contribute to an increase in NB tumorigenicity, and also, increased levels of oligomannose N-glycans impede development of embryos in vertebrae.
Whitman, Austin A. (June 2019). Relationship of N-glycans to Neuronal Dysfunction (Master's Thesis, East Carolina University). Retrieved from the Scholarship. (http://hdl.handle.net/10342/7465.)
Whitman, Austin A. Relationship of N-glycans to Neuronal Dysfunction. Master's Thesis. East Carolina University, June 2019. The Scholarship. http://hdl.handle.net/10342/7465. September 27, 2022.
Whitman, Austin A, “Relationship of N-glycans to Neuronal Dysfunction” (Master's Thesis., East Carolina University, June 2019).
Whitman, Austin A. Relationship of N-glycans to Neuronal Dysfunction [Master's Thesis]. Greenville, NC: East Carolina University; June 2019.
East Carolina University