REGULATION OF GLYCOSYLATION IN MAMMALIAN CELL LINES: INSIGHTS FROM STRUCTURAL GLYCOMICS, ANALYSIS OF GLYCOSYLATION-RELATED GENES AND FUNCTIONAL RNAI SCREENS
type: CreativeWork
description: This thesis represents a multifaceted approach to elucidate the regulation mechanism of glycosylation in mammalian cell lines. First, N-linked and O-linked glycan structures of five commonly used mammalian cell lines, namely CHO-K1, BHK-21, HEK293, COS-7 and 3T3, were characterized by a combination of mass spectrometric and chromatographic techniques. Such glycomic profiling revealed several immunogenic non-human N-glycan structures in BHK-21 and 3T3 cells. The cell lines were also shown to have significantly different capabilities in N-glycan sialylation, which follows the order: (BHK-21, CHO) > (COS-7, HEK293) > 3T3. Significant difference in O-glycosylation was also detected: CHO-K1 was found to produce only mono- or di-sialyl T antigen structures whereas the other four cell lines were found to produce additional core 2 structures. This glycomic profiling study suggests that glycosylation is differentially regulated in cell lines. Second, regulation of N-glycosylation at pathway level was functionally investigated by focusing on sialylation of recombinant EPO. Thirty one genes that are directly involved in the N-glycosylation pathway were cloned and heterologously expressed in the aforementioned five cell lines as well as NS0 cells. None of these genes could enhance EPO sialylation in BHK-21 and CHO-K1 cells. Overexpression of three sialyltransferases led to significant enhancement of EPO sialylation in HEK293, COS-7, 3T3 and NS0 cells. The other glycogenes involved in upstream reaction steps could not synergize the effect of these sialyltransferases to further enhance sialylation. Transcriptional analysis of endogenous glycogene expression showed a relatively low abundance of sialyltransferases ST3GalIII in HEK293, 3T3 and NS0 and relatively higher abundance of an upstream glycogene, GlcI. The expression level of ST3GalIII in these three cell lines positively correlated to EPO sialylation extent. This study represents a pathway-focused functional analysis of gly…
abstract: This thesis represents a multifaceted approach to elucidate the regulation mechanism of glycosylation in mammalian cell lines. First, N-linked and O-linked glycan structures of five commonly used mammalian cell lines, namely CHO-K1, BHK-21, HEK293, COS-7 and 3T3, were characterized by a combination of mass spectrometric and chromatographic techniques. Such glycomic profiling revealed several immunogenic non-human N-glycan structures in BHK-21 and 3T3 cells. The cell lines were also shown to have significantly different capabilities in N-glycan sialylation, which follows the order: (BHK-21, CHO) > (COS-7, HEK293) > 3T3. Significant difference in O-glycosylation was also detected: CHO-K1 was found to produce only mono- or di-sialyl T antigen structures whereas the other four cell lines were found to produce additional core 2 structures. This glycomic profiling study suggests that glycosylation is differentially regulated in cell lines. Second, regulation of N-glycosylation at pathway level was functionally investigated by focusing on sialylation of recombinant EPO. Thirty one genes that are directly involved in the N-glycosylation pathway were cloned and heterologously expressed in the aforementioned five cell lines as well as NS0 cells. None of these genes could enhance EPO sialylation in BHK-21 and CHO-K1 cells. Overexpression of three sialyltransferases led to significant enhancement of EPO sialylation in HEK293, COS-7, 3T3 and NS0 cells. The other glycogenes involved in upstream reaction steps could not synergize the effect of these sialyltransferases to further enhance sialylation. Transcriptional analysis of endogenous glycogene expression showed a relatively low abundance of sialyltransferases ST3GalIII in HEK293, 3T3 and NS0 and relatively higher abundance of an upstream glycogene, GlcI. The expression level of ST3GalIII in these three cell lines positively correlated to EPO sialylation extent. This study represents a pathway-focused functional analysis of gly…
author: ZHANG PEIQING
keywords: Glycosylation, sialylation, fucosylation, erythropoietin, mass spectrometry, recombinant protein
date published: 2012-01-06
date modified: 2026-07-24T03:31:00Z
provider: National University of Singapore
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: National University of Singapore