Annear, Nicholas; (2013) Does Glycosylation Play a Role in Adaptation to Altered Oxygenation? Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Hypoxia, defined as low oxygen tension, is a common characteristic of several human diseases, including ischaemia, anaemia and certain cancers. Hypoxia results in major adaptive changes in cell phenotype, metabolism and intercellular signalling. Hypoxia activates the transcription factor complex, hypoxia inducible factor (HIF), which plays a central role in regulating the expression of many genes leading to cellular adaptive responses. Cells with constitutive HIF activation are a powerful system for investigating how HIF is regulated; previously leading to the recognition of the central role of VHL protein in HIF ubiquitination. The chinese hamster ovary (CHO) cell line M6.19, was identified from a mutagenesis programme designed to identify novel pathways that regulate Hif. Alongside constitutive activation of Hif-1α, M6.19 cells displayed enhanced electrophoretic mobility of the Hif-1α target Glut-1, and altered lectin binding. These data together suggested that M6.19 cells harbour a major glycosylation defect. Through lectin resistance assays and genetic sequencing, M6.19 cells were found to harbour a mutation in the UDP-galactose transporter (Slc35a2). This study aims to identify whether glycosylation plays a role in modulating cellular adaptation to altered oxygenation. Slc35a2 was shown to regulate cell-surface lectin binding, Glut-1 electrophoretic mobility and glucose uptake in CHO cells. In addition, Slc35a2 was found to regulate basal Hif-1α protein levels in CHO cells. Finally, transient knockdown of SLC35A2 was shown to enhance HIF-2α protein levels and HIF-2 target gene expression in several human cancer cell lines. By demonstrating that SLC35A2 plays a role in modulating key components of the hypoxic response, this thesis provides insight into novel pathways that regulate hypoxia signalling and HIF in human cells.

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