A rapid method for efficiently generating site-directed mutations on a clean sequence background is described. This modification of the megaprimer PCR mutagenesis approach can be performed in one tube in less than 4.5 hours, and does not require purification of intermediate products. High fidelity of DNA sequence replication is obtained by employing Pfu DNA polymerase and limiting the total number of amplification cycles to 30. The mutagenesis efficiency of the procedure is high enough to allow rapid, direct identification of mutants by restriction digest or sequencing techniques.

Two antithrombin III (ATIII) isoforms occur naturally in human plasma. The alpha-ATIII isoform has four N-linked oligosaccharides attached to asparagines 96, 135, 155, and 192. The beta-ATIII isoform lacks carbohydrate on asparagine-135 (N135), which is near the heparin binding site, and binds heparin with higher affinity than does alpha-ATIII. Two isoforms are also produced when the normal human ATIII cDNA sequence is expressed in baculovirus-infected insect cells, and the recombinant beta' isoform similarly binds heparin with higher affinity than the recombinant alpha' isoform. Consensus sequences (CSs) of the ATIII N-glycosylation sites are N-X-S for 135 and N-X-T for 96, 155, and 192. On the basis of database and in vitro glycosylation studies suggesting that N-X-S CSs are utilized less efficiently than N-X-T CSs, we hypothesized that the beta-ATIII isoform might result from inefficient core glycosylation of the N135 N-X-S CS due to the presence of a serine, rather than a threonine, in the third position. ATIIIs with N-X-S, N-X-T, and N-X-A consensus sequences were expressed in baculovirus-infected insect cells. In contrast to the N-X-S sequence, which expressed a mixture of alpha' and beta' molecules, the N-X-T variant produced alpha' exclusively, while the N-X-A variant produced beta' exclusively. Thus, serine in the third position of the N135 CS is responsible for its "partial" glycosylation and leads to production of beta-ATIII.(ABSTRACT TRUNCATED AT 250 WORDS)

The heparin binding site of the anticoagulant protein antithrombin III (ATIII) has been defined at high resolution by alanine scanning mutagenesis of 17 basic residues previously thought to interact with the cofactor based on chemical modification experiments, analysis of naturally occurring dysfunctional antithrombins, and proximity to helix D. The baculovirus expression system employed for this study produces antithrombin which is highly similar to plasma ATIII in its inhibition of thrombin and factor Xa and which resembles the naturally occurring ␤-ATIII isoform in its interactions with high affinity heparin and pentasaccharide (Ersdal-Badju, E., Lu, A., Peng, X., Picard, V., Zendehrouh, P., Turk, B., Bjö rk, I., Olson, S. T., and Bock, S. C. (1995) Biochem. J. 310, 323-330). Relative heparin affinities of basic-to-Ala substitution mutants were determined by NaCl gradient elution from heparin columns. The data show that only a subset of the previously implicated basic residues are critical for binding to heparin. The key heparin binding residues, Lys-11, Arg-13, Arg-24, Arg-47, Lys-125, Arg-129, and Arg-145, line a 50-Å long channel on the surface of ATIII. Comparisons of binding residue positions in the structure of P14-inserted ATIII and models of native antithrombin, derived from the structures of native ovalbumin and native antichymotrypsin, suggest that heparin may activate antithrombin by breaking salt bridges that stabilize its native conformation. Specifically, heparin release of intramolecular helix D-sheet B salt bridges may facilitate s123AhDEF movement and generation of an activated species that is conformationally primed for reactive loop uptake by central ␤-sheet A and for inhibitory complex formation. In addition to providing a structural explanation for the conformational change observed upon heparin binding to antithrombin III, differences in the affinities of native, heparin-bound, complexed, and cleaved ATIII molecules for heparin can be explained based on the identified binding site and suggest why heparin functions catalytically and is released from antithrombin upon inhibitory complex formation.

In order to promote homogeneity of recombinant antithrombin III interactions 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 therefore similar to the beta isoform of plasma antithrombin. Purified bv.hat3.N135A is homogeneous with respect to molecular mass, charge and elution from immobilized heparin. Second-order rate constants for thrombin and factor Xa inhibition determined in the absence and presence of heparin are in good agreement with values established for plasma antithrombin 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 studies indicate that it also undergoes an identical or very similar conformational change upon heparin binding. The Kds of bv.hat3.N135A for high-affinity heparin and pentasaccharide were determined and are in good agreement with those of the plasma beta-antithrombin isoform. The demonstrated similarity of bv.hat3.N135A and plasma antithrombin interactions with target proteinases and heparins suggest that it will be a useful base molecule for investigating the structural basis of antithrombin III heparin cofactor activity.