Decrease in antithrombin III fucosylation by expressing GDP-fucose transporter siRNA in Chinese hamster ovary cells

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• GDP-fucose transporter (GFT) is crucial for fucose incorporation into glycoproteins.
• Defucosylation of recombinant proteins can impact their efficacy and immunogenicity.
• Chinese hamster ovary (CHO) cells are widely used for recombinant protein production.

Purpose of the Study:

• To clone and characterize the GDP-fucose transporter (GFT) from Chinese hamster ovary (CHO) cells.
• To investigate the effect of GFT inhibition on the fucosylation of recombinant human antithrombin III (AT-III).

Main Methods:

• Cloning of full-length GFT cDNA from CHO cells.
• Synthesis and application of small interfering RNA (siRNA) targeting CHO GFT.
• Construction of a GFT siRNA expression plasmid for cell transfection.
• Analysis of defucosylated AT-III levels and Golgi-GDP fucose reduction.

Main Results:

• The cloned GFT exhibited high similarity to human and mouse GFT.
• siRNA-mediated GFT inhibition in recombinant CHO cells led to a 30-40% increase in defucosylated AT-III.
• Transfection with a GFT siRNA expression plasmid reduced Golgi-GDP fucose by approximately 75% and increased defucosylated AT-III by 10-40%.

Conclusions:

• The cloned GFT is a functional transporter in CHO cells.
• Targeting GFT effectively reduces protein fucosylation in recombinant CHO cells.
• This approach offers a strategy to enhance the production of defucosylated recombinant proteins like AT-III.