Heparin (HP), a highly-sulfated heparan sulfate (HS), is widely utilized as anticoagulant drug. HP from animal source has been associated with health hazards, leading to an interest in the development of HP from non-animal source. N-deacetylase/N-sulfotransferase (NDST2) and HS-3-O-sulfotransferase (HS3ST1) are critical for anticoagulant HP biosynthesis. We previously demonstrated CHO cell-line stably expressing NDST2 and HS3ST1, produced highly-sulfated-bioengineered HS with insufficient anticoagulant activity. The results led us to investigate HP biosynthetic enzymes, specifically, NDST2 and HS3ST1 localization and activity. Preliminary results from immunoflourescence and immunoblotting showed NDST2 is localized in the Golgi; however, HS3ST1 is more broadly localized. In parallel, the N-sulfation pattern in fractionated HP from stable-clones demonstrate a mixture of heparosan and highly-sulfated HS/HP, suggesting incomplete activity of HS3ST1 and/or NDST1. We are currently comparatively profiling HS/HP biosynthetic enzyme, their effect on HS/HP composition and HS/HP biosynthetic activity in CHO stable-clones and other cell-lines, including mastocytoma cell-line.

Synthesis of bioengineered heparin in mammalian cell lines

244th National Meeting (2012)

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