{"id":{"repo_id":"nus","oai_identifier":"oai:scholarbank.nus.edu.sg:10635/32965"},"canonical_url":"https://search.dev.ndltd.org/etd/nus/oai:scholarbank.nus.edu.sg:10635/32965","repository":{"repo_id":"nus","name":"National University of Singapore","base_url":"https://scholarbank.nus.edu.sg/oai/request"},"display":{"title":"REGULATION OF GLYCOSYLATION IN MAMMALIAN CELL LINES: INSIGHTS FROM STRUCTURAL GLYCOMICS, ANALYSIS OF GLYCOSYLATION-RELATED GENES AND FUNCTIONAL RNAI SCREENS","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 glycogenes which supports the notion that glycan structural diversity is mainly a consequence of different expressions of glycogenes that control terminal modifications. Third, structure-function relationship of glycogene-encoded proteins, especially nucleotide-sugar transporters, represents a major interest in understanding the regulation of glycogene-encoded proteins and possible targets for controlling glycosylation. On the basis of a CHO glycosylation mutant of GDP-fucose transporter (GFT), a set of GFT variants were functionally analyzed. Through these analyses, we identified a conserved Glu-Met motif and a cluster of Lys residues in the C-terminal tail of GFT having a critical impact on the transport activity of GFT. In addition, three conserved Gly residues located in different transmembrane helices are also essential for GFT activity. These elements could represent viable targets for inactivating GFT as a therapeutic approach. Finally, functional genomic screening of the kinases and phosphotases in the human genome was performed aiming to identify novel genes that control the cell surface glycosylation. Using ConA and AAL lectins to specifically label oligomannose-type and fucosylated glycans at the cell surface, the primary screening results suggested a significant number of genes may play a role in the transition of oligomannose/hybrid-type glycans to complex-type glycans as well as in fucosylation of glycans. On this basis, it is tempting to speculate that multiple kinases and phosphotases can regulate the glycosylation process upstream of the glycosylation pathway in response to extracellular or intracellular signals. In summary, this multi-layered investigation provides a set of essential information for selection of cell lines for biotech application as well as promising molecular targets for controlling the glycosylation for enhancing drug efficacy and disease intervention.","abstract_html":"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 glycogenes which supports the notion that glycan structural diversity is mainly a consequence of different expressions of glycogenes that control terminal modifications. Third, structure-function relationship of glycogene-encoded proteins, especially nucleotide-sugar transporters, represents a major interest in understanding the regulation of glycogene-encoded proteins and possible targets for controlling glycosylation. On the basis of a CHO glycosylation mutant of GDP-fucose transporter (GFT), a set of GFT variants were functionally analyzed. Through these analyses, we identified a conserved Glu-Met motif and a cluster of Lys residues in the C-terminal tail of GFT having a critical impact on the transport activity of GFT. In addition, three conserved Gly residues located in different transmembrane helices are also essential for GFT activity. These elements could represent viable targets for inactivating GFT as a therapeutic approach. Finally, functional genomic screening of the kinases and phosphotases in the human genome was performed aiming to identify novel genes that control the cell surface glycosylation. Using ConA and AAL lectins to specifically label oligomannose-type and fucosylated glycans at the cell surface, the primary screening results suggested a significant number of genes may play a role in the transition of oligomannose/hybrid-type glycans to complex-type glycans as well as in fucosylation of glycans. On this basis, it is tempting to speculate that multiple kinases and phosphotases can regulate the glycosylation process upstream of the glycosylation pathway in response to extracellular or intracellular signals. In summary, this multi-layered investigation provides a set of essential information for selection of cell lines for biotech application as well as promising molecular targets for controlling the glycosylation for enhancing drug efficacy and disease intervention.","abstract_has_math":false,"creators":["ZHANG PEIQING"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2012,"date_issued":"2012-01-06","date_published":"2012-01-06","updated_at":"2026-07-24T03:31:00Z","subjects":["Glycosylation, sialylation, fucosylation, erythropoietin, mass spectrometry, recombinant protein"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":null,"outbound_label":null,"outbound_source":null},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["ZHANG PEIQING"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2012-01-06"]},{"key":"dc:relation.isreferencedby","label":"Dc Relation Isreferencedby","values":["https://scholarbank.nus.edu.sg/handle/10635/32965"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Glycosylation, sialylation, fucosylation, erythropoietin, mass spectrometry, recombinant protein"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://scholarbank.nus.edu.sg/bitstreams/89cdf81f-1566-46c9-8249-c23a55fa536c/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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 glycogenes which supports the notion that glycan structural diversity is mainly a consequence of different expressions of glycogenes that control terminal modifications. Third, structure-function relationship of glycogene-encoded proteins, especially nucleotide-sugar transporters, represents a major interest in understanding the regulation of glycogene-encoded proteins and possible targets for controlling glycosylation. On the basis of a CHO glycosylation mutant of GDP-fucose transporter (GFT), a set of GFT variants were functionally analyzed. Through these analyses, we identified a conserved Glu-Met motif and a cluster of Lys residues in the C-terminal tail of GFT having a critical impact on the transport activity of GFT. In addition, three conserved Gly residues located in different transmembrane helices are also essential for GFT activity. These elements could represent viable targets for inactivating GFT as a therapeutic approach. Finally, functional genomic screening of the kinases and phosphotases in the human genome was performed aiming to identify novel genes that control the cell surface glycosylation. Using ConA and AAL lectins to specifically label oligomannose-type and fucosylated glycans at the cell surface, the primary screening results suggested a significant number of genes may play a role in the transition of oligomannose/hybrid-type glycans to complex-type glycans as well as in fucosylation of glycans. On this basis, it is tempting to speculate that multiple kinases and phosphotases can regulate the glycosylation process upstream of the glycosylation pathway in response to extracellular or intracellular signals. In summary, this multi-layered investigation provides a set of essential information for selection of cell lines for biotech application as well as promising molecular targets for controlling the glycosylation for enhancing drug efficacy and disease intervention."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["357d7b8da3cee1bebf80aa56784e3532","d43f4b687b163c51efb31042ebe0f8c1"]},{"key":"dc:title","label":"Title","values":["REGULATION OF GLYCOSYLATION IN MAMMALIAN CELL LINES: INSIGHTS FROM STRUCTURAL GLYCOMICS, ANALYSIS OF GLYCOSYLATION-RELATED GENES AND FUNCTIONAL RNAI SCREENS"]}]}],"canonical_facts":{"dc:creator":["ZHANG PEIQING"],"dc:date.issued":["2012-01-06"],"dc:description.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 glycogenes which supports the notion that glycan structural diversity is mainly a consequence of different expressions of glycogenes that control terminal modifications. Third, structure-function relationship of glycogene-encoded proteins, especially nucleotide-sugar transporters, represents a major interest in understanding the regulation of glycogene-encoded proteins and possible targets for controlling glycosylation. On the basis of a CHO glycosylation mutant of GDP-fucose transporter (GFT), a set of GFT variants were functionally analyzed. Through these analyses, we identified a conserved Glu-Met motif and a cluster of Lys residues in the C-terminal tail of GFT having a critical impact on the transport activity of GFT. In addition, three conserved Gly residues located in different transmembrane helices are also essential for GFT activity. These elements could represent viable targets for inactivating GFT as a therapeutic approach. Finally, functional genomic screening of the kinases and phosphotases in the human genome was performed aiming to identify novel genes that control the cell surface glycosylation. Using ConA and AAL lectins to specifically label oligomannose-type and fucosylated glycans at the cell surface, the primary screening results suggested a significant number of genes may play a role in the transition of oligomannose/hybrid-type glycans to complex-type glycans as well as in fucosylation of glycans. On this basis, it is tempting to speculate that multiple kinases and phosphotases can regulate the glycosylation process upstream of the glycosylation pathway in response to extracellular or intracellular signals. In summary, this multi-layered investigation provides a set of essential information for selection of cell lines for biotech application as well as promising molecular targets for controlling the glycosylation for enhancing drug efficacy and disease intervention."],"dc:format.checksum.md5":["357d7b8da3cee1bebf80aa56784e3532","d43f4b687b163c51efb31042ebe0f8c1"],"dc:identifier.uri":["https://scholarbank.nus.edu.sg/bitstreams/89cdf81f-1566-46c9-8249-c23a55fa536c/download"],"dc:relation.isreferencedby":["https://scholarbank.nus.edu.sg/handle/10635/32965"],"dc:subject":["Glycosylation, sialylation, fucosylation, erythropoietin, mass spectrometry, recombinant protein"],"dc:title":["REGULATION OF GLYCOSYLATION IN MAMMALIAN CELL LINES: INSIGHTS FROM STRUCTURAL GLYCOMICS, ANALYSIS OF GLYCOSYLATION-RELATED GENES AND FUNCTIONAL RNAI SCREENS"],"dc:type":["Thesis"]},"updated_at":"2026-07-24T03:31:00Z"}