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Transgenically Produced Human Antithrombin: Structural and Functional
Comparison to Human Plasma-Derived Antithrombin
Tim Edmunds,
Scott M. Van Patten,
Julie Pollock,
Eric Hanson,
Richard Bernasconi,
Elizabeth Higgins,
Partha Manavalan,
Carol Ziomek,
Harry Meade,
John M. McPherson, and
Edward S. Cole
From the Cell and Protein Therapeutics Department, Genzyme Corp, and
Genzyme Transgenics Corp, Framingham, MA.
Recombinant human antithrombin (rhAT) produced in transgenic goat
milk was purified to greater than 99%. The specific activity of the
rhAT was identical to human plasma-derived AT (phAT) in an in vitro
thrombin inhibition assay. However, rhAT had a fourfold higher affinity
for heparin than phAT. The rhAT was analyzed and compared with phAT by
reverse phase high-performance liquid chromatography, circular
dichroism, fluorophore-assisted carbohydrate electrophoresis (FACE),
amino acid sequence, and liquid chromatography/mass spectrography peptide mapping. Based on these analyses, rhAT was determined to be
structurally identical to phAT except for differences in glycosylation.
Oligomannose structures were found on the Asn 155 site of the
transgenic protein, whereas only complex structures were observed on
the plasma protein. RhAT contained a GalNAc for galactose substitution
on some N-linked oligosaccharides, as well as a high degree of
fucosylation. RhAT was less sialylated than phAT and contained both
N-acetylneuraminic and N-glycolylneuraminic acid. We postulate that the
increase in affinity for heparin found with rhAT resulted from the
presence of oligomannose-type structures on the Asn 155 glycosylation
site and differences in sialylation.
Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4561-4571
© 1998 by The American Society of Hematology.
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