The structure of a complex of bovine alpha-thrombin and recombinant hirudin at 2.8-A resolution.

Crystals of the complex of bovine alpha-thrombin with recombinant hirudin variant 1 have space group C222(1) with cell constants a = 59.11, b = 102.62, and c = 143.26 A. The orientation and position of the thrombin component was determined by molecular replacement and the hirudin molecule was fit in 2 magnitude of Fo - magnitude of Fc electron density maps. The structure was refined by restrained least squares and simulated annealing to R = 0.161 at 2.8-A resolution. The binding of hirudin to thrombin is generally similar to that observed in the crystals of human thrombin-hirudin. Several differences in the interactions of the COOH-terminal polypeptide of hirudin, specifically of residues Asp-55h, Phe-56h, Glu-57h, and Glu-58h, and a few differences in the interactions of the hirudin core, specifically of residues Asp-5h, Ser-19h, and Asn-20h, with thrombin from human thrombin-hirudin suggest that there is some flexibility in the binding of these 2 molecules. Most of the residues in the 9 subsites that bind fibrinopeptide A7-16 to thrombin also interact with the NH2-terminal domain of hirudin. The S1 subsite is a notable exception in that only 1 of its 6 residues, namely Ser-214, interacts with hirudin. The only difference between human and bovine thrombins that appears to influence the binding of hirudin is the replacement of Lys-149E by an acidic glutamate in the bovine enzyme.

Chemical modifications and amino acid substitutions in recombinant hirudin that increase hirudin-thrombin affinity.

Recombinant hirudin (r-hirudin), unlike the naturally occurring leech protein, lacks a sulfate ester on Tyr-63 which reduces its binding affinity to thrombin by 3-10-fold. We demonstrate that nitration or iodination of Tyr-63 restores hirudin-thrombin affinity to levels similar to or exceeding that of the natural inhibitor. In contrast, nitration of Tyr-3 reduces the affinity of hirudin for thrombin. These chemical modifications results in multiple reaction products that are readily separated by reverse-phase HPLC. The mechanism of the observed changes in thrombin affinity may involve a reduction in the pK of the hydroxyl group of tyrosine due to substitution of the electrophilic iodo or nitro group on the phenyl ring, resulting in an increased negative charge at neutral pH. For Tyr-63, this effect mimics the sulfatotyrosine of natural hirudin, leading to an increased thrombin affinity at the anion-binding exosite. For Tyr-3, the increased polarity may destabilize its interaction within the apolar-binding site of thrombin. Substitution of the highly conserved Tyr-3 residue with Phe or Trp not only enables specific and quantitative chemical modification at Tyr-63 but also independently increases hirudin-thrombin affinity. Kinetic analysis of thrombin inhibition showed that enhanced binding by r-hirudin(nitro-Tyr-63) is due to an increase in the association rate between hirudin and thrombin whereas the reduced binding of r-hirudin(nitro-Tyr-3) results from a large increase in the dissociation rate. These observations indicate that specific segments within both the amino- and carboxy-terminal regions of hirudin interact with thrombin.

Hirudin: amino-terminal residues play a major role in the interaction with thrombin.

Amino acid substitutions within the amino-terminal 5 residues of the thrombin-specific inhibitor hirudin dramatically alter its ability to inhibit the thrombin-catalyzed hydrolysis of both a chromogenic substrate and fibrinogen. Replacing the highly conserved Tyr-3 residue with Trp or Phe increases hirudin's affinity for thrombin 3-6-fold (decreases the inhibition constant, Ki) whereas Thr results in a 450-fold increase in Ki. A more extensive modification involving deletion of the amino-terminal Val, and Tyr-3----Val, Thr-4----Gln, and Asp-5----Ile replacement, results in a large reduction in thrombin inhibitory activity corresponding to greater than a 10(7)-fold increase in Ki and a 10(3)-fold increase in IC50, using D-Phe-L-pipecolyl-Arg-p-nitroanilide (S-2238) and fibrinogen, respectively, as substrates. Kinetic analysis of these mutant proteins and synthetic peptide fragments and available structural information on thrombin and hirudin derived from protein crystallography and two-dimensional NMR studies indicate that the amino-terminal region of hirudin binds at the apolar binding/active site region of thrombin, with Tyr-3 occupying the S3 specificity site. The large effect of these modifications on hirudin activity suggests that alteration of the amino-terminal segment can destabilize the interaction of other regions of hirudin with thrombin.

Structure-function and refolding studies of the thrombin-specific inhibitor hirudin.

We have developed a novel expression and purification system that yields recombinant desulfo-hirudin (HV-1) with high specific activity (10,000 antithrombin units/mg) and an inhibition constant (Ki) for human alpha-thrombin of 0.2 pM. Reduced and denatured hirudin rapidly refolds to the native, fully active conformation at high concentration (greater than 50 mg/ml) by incubation at pH 10. Analytical gel filtration studies at neutral pH suggest that hirudin is a multimer. Initial binding of hirudin to thrombin appears to be followed by dissociation of the hirudin multimer to give a tight-binding 1:1 hirudin:thrombin complex. Thrombin inhibition studies showed that hirudin synthetic peptide fragments 42-65 and 51-65 [but not (Ala22)-6-28, containing two of the three disulfide bonds formed in native hirudin] were similarly effective in inhibiting thrombin cleavage of fibrinogen (IC50 = 4.9 and 6.0 microM, respectively, at a thrombin concentration of 1 microM). We conclude that hirudin has unusual structural and refolding properties and that its mechanism of inhibition involves noncovalent interaction with multiple sites on thrombin. The interaction of hirudin (specifically the region of Lys-47) with the basic specificity pocket of thrombin may contribute to the binding but is not essential for its inhibitory activity.

Characterization and immunoassay of human tumor-associated galactosyltransferase isoenzyme II.

Galactosyltransferase (GT) (EC 2.4.1.38) was purified to homogeneity from human ovarian tumor effusion fluid and normal human serum by chromatography on alpha-lactalbumin and anti-human immunoglobulin affinity (to selectively absorb contaminating IgG) columns. Both preparations showed a single, broad band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis centered at a molecular weight of 48,000, but nondenaturing polyacrylamide gel electrophoresis of GT isolated from tumor effusion fluid revealed the presence of a series of oligomeric proteins possessing GT activity, which were barely detectable in normal human serum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of N-glycanase- and O-glycanase-treated GT revealed that each endoglycanase removed carbohydrate with an approximate molecular weight of 3,000, revealing the presence of both N-linked and O-linked oligosaccharide substitutions on GT. Purified GT (containing a mixture of GT isoenzymes) was used to immunize BALB/c mice for monoclonal antibody (MAb) preparation. Four of the MAb isolated reacted with GT. MAb 3872 (patent pending; an IgG1) was determined to be specific for a cancer-associated GT isoenzyme (GT-II) by immunostaining of Western blots and nondenaturing polyacrylamide gel electrophoresis of GT specifically eluted from a MAb 3872 affinity column. Two 125I-labeled cyanogen bromide peptides (Mr 8,400 and 7,400) prepared from 125I-GT were specifically bound and eluted from a MAb 3872 affinity column, demonstrating that the MAb 3872 GT-II-specific antigenic epitope resides on these peptides. MAb 3872 was immobilized on 1,1'-carbonyldiimidazole-activated trisacryl GF-2000 and used to specifically assay serum GT-II levels in 29 individual normal human serum samples and 77 serum samples from 38 patients with advanced ovarian tumors. The normal serum GT-II level was found to be 85.3 +/- 30.9 milliunits/ml, with a range of 17 to 160 milliunits/ml. Of the 38 tumor patients, 33 showed GT-II values in excess of 200 milliunits/ml, with a range of 216 to 8,469 milliunits/ml. Serial samples obtained from the ovarian tumor patients suggested that the serum GT-II level reflected the tumor burden of the patient.

Immunoassay of serum galactosyltransferase isoenzyme II in cancer patients and control subjects.

Serum galactosyltransferase isoenzyme II (GT-II) was assayed in 409 coded serum samples obtained from the National Cancer Institute Tumor Serum Bank using a monoclonal antibody-based immunoassay. The serum panel consisted of samples from patients with confirmed, metastatic ovarian, breast, stomach, esophageal, pancreatic, lung, colorectal, bladder, prostate, and cervical cancer, as well as benign disease controls corresponding to each cancer type, and confirmed healthy normal controls. The serum panel was matched for age and sex; 176 of 179 cancer patients had metastatic disease, and many had undergone previous therapy. GT-II was significantly elevated (P less than 0.01) in all pairwise tests (Wilcoxon) comparing cancer cases with normals and cancer cases with benign disease cases of the same site. A cutpoint of 200 milliunits of GT-II activity/ml of serum was selected, and only one of 50 normal control sera was elevated above this value, yielding a specificity of 98%. The overall sensitivity of the GT-II assay was 55.3%, with higher sensitivity shown by pancreatic (77%), prostate (65%), esophageal (64%), cervical (59%), and bladder cancer (58%).