Transgenically produced human antithrombin: structural and functional comparison to human plasma-derived antithrombin.
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.
Edmunds
T
T
Cell and Protein Therapeutics Department, Genzyme Corp, and Genzyme Transgenics Corp, Framingham, MA 01701-9322, USA.
Van Patten
S M
SM
Pollock
J
J
Hanson
E
E
Bernasconi
R
R
Higgins
E
E
Manavalan
P
P
Ziomek
C
C
Meade
H
H
McPherson
J M
JM
Cole
E S
ES
D000818:Animals; D030801:Animals, Genetically Modified; D000990:Antithrombin III / Q000032:analysis* / Q000235:genetics* / Q000302:isolation & purification*; D005260:Female; D006041:Goats; D006801:Humans; D008894:Milk Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification
D008894:Milk Proteins; D011994:Recombinant Proteins; D000990:Antithrombin III