ELIMINATION OF GLYCOSYLATION HETEROGENEITY AFFECTING HEPARIN AFFINITYOF RECOMBINANT HUMAN ANTITHROMBIN-III BY EXPRESSION OF A BETA-LIKE VARIANT IN BACULOVIRUS-INFECTED INSECT CELLS

In order to promote homogeneity of recombinant antithrombin III intera ctions with heparin, an asparagine-135 to alanine substitution mutant was expressed in baculovirus-infected insect cells. The N135A variant does not bear an N-linked oligosaccharide on residue 135 and is theref ore similar to the beta isoform of plasma antithrombin. Purified bv.ha t3.N135A is homogeneous with respect to molecular mass, charge and elu tion from immobilized heparin. Second-order rate constants for thrombi n and factor Xa inhibition determined in the absence and presence of h eparin are in good agreement with values established for plasma antith rombin and these enzymes. Based on far- and near-UV CD, bv.hat3.N135A has a high degree of conformational similarity to plasma antithrombin. Near-UV CD, absorption difference and fluorescence spectroscopy studi es indicate that it also undergoes an identical or very similar confor mational change upon heparin binding. The K(d)s of bv.hat3.N135A for h igh-affinity heparin and pentasaccharide were determined and are in go od agreement with those of the plasma beta-antithrombin isoform. The d emonstrated similarity of bv.hat3.N135A. and plasma antithrombin inter actions with target proteinases and heparins suggest that it will be a useful base molecule for investigating the structural basis of antith rombin III heparin cofactor activity.