Improvement of Erythropoietin N-glycan Branching and Sialylation by Overexpression of Glycosyltransferases
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Date
2015-07-24
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Johns Hopkins University
Abstract
The emergence of recombinant therapeutic proteins whose indications range from anti-cancer to rheumatoid arthritis treatments represented a significant revolution for modern pharmaceutical industry. When introduced into patients, the immunogenicity, bioactivity, and serum half-life of the therapeutic proteins are greatly impacted by their glycosylation patterns, especially the antennarity of the N-glycan and the level of terminal sialylation. To introduce α2,6-sialic acids and elevate overall sialylation level, a human α2,6-sialytransferase (ST6GAL1) was expressed in Chinese hamster ovary (CHO-K1) cells which produce recombinant human erythropoietin (EPO). In addition, both UDP-N-acetylglucosamine: α-1,3-D-mannoside β1,4-N-acetylglucosaminyltransferase (GNTIV/Mgat4 ) and UDP-N-acetylglucosamine:α-1,6-D-mannoside β1,6-N-acetylglucosaminyltransferase (GNTV/Mgat5) were further incorporated into the CHO-K1 cell line expressing EPO and ST6GAL1.
Firstly, stable clones of CHO-K1 cells expressing glycosyltransferases were established. CHO-K1 cell line expressing recombinant EPO (ChEPO) was transfected with ST6GAL1. The stable pool was created by drug selection and stable clone (ChEPO-S) was picked by lectin blot. Similarly, GNTIV and GNTV were simultaneously transfected into ChEPO-S cells. Once the stable pool was constructed, the stable clone (ChEPO-SG) was selected using western blot and lectin blot analysis.
The second goal of this thesis was to examine the expression and function of ST6GAL1, GNTIV and GNTV in ChEPO-S and ChEPO-SG cell lines. The expression of the transfected genes at both transcription and translation levels were confirmed by RT-PCR and western blot, respectively. The impact of these glycosyltransferases on the level of terminal sialylation as well as the degree of branching of intracellular proteins and purified EPO from the various CHO cell lines was evaluated. Lectin blot analysis indicated that transfection of ST6GAL1 indeed introduced α2,6-sialic acids and increased overall sialylation level. Higher extent of β1,4 and β1,6 branching at the N-glycan trimannosyl core was also observed. HPLC analysis quantified the sialic acid content of recombinant human EPO purified from each cell line. Results demonstrated that recombinant human EPO produced by ChEPO-S and ChEPO-SG cells respectively contained ~26% and ~45% more sialic acids compared to EPO from ChEPO cells.
In this study, for the first time three glycosyltransferases, ST6GAL1, GNTIV and GNTV, were overexpressed in CHO-K1 cells in a simultaneous and coordinated manner. This strategy allows robust enhancement of the N-glycan complexity and terminal sialylation of recombinant therapeutic proteins produced by CHO cells.
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Keywords
GNTIV, GNTV, sialic acid, CHO, erythropoietin, N-glycosylation