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9-(2-Phosphonylmethoxyethyl)-N6-cyclopropyl-2,6-diaminopurine (cpr-PMEDAP) is an acyclic nucleotide analog of the [9-(2-phosphonylmethoxyethyl)-] (PME) series containing a cyclopropyl substituent on the N6 position of the 2,6-diaminopurine (DAP) base. Growth inhibition assays in a broad range of tumor cell lines demonstrated that this analog had potent antiproliferative activity with IC50 values similar to those of the structurally related guanine analog 9-(2-phosphonylmethoxyethyl)guanine (PMEG). A substantially lower growth inhibitory effect was observed for the 2,6-diaminopurine analog, PMEDAP. To dissect the basis for these varying potencies, the metabolism of the three analogs was examined in a human pancreatic carcinoma cell line, BxPC-3. HPLC analysis of the intracellular metabolites demonstrated that the cpr-PMEDAP was deaminated to PMEG and subsequently phosphorylated to PMEG mono- and diphosphates (PMEGp and PMEGpp). The level of PMEGpp generated from cpr-PMEDAP-treated cells was 50% greater than the level generated from cells incubated with PMEG. The presence of PMEG in the DNA of cells incubated with cpr-PMEDAP confirmed that the cpr-PMEDAP was converted to PMEG. In contrast, PMEDAP was not deaminated to PMEG, but directly phosphorylated to PMEDAPp and PMEDAPpp. The adenylate deaminase inhibitor 2'-deoxycoformycin (dCF) inhibited the conversion of cpr-PMEDAP in a rat liver cytosolic extract and increased the IC50 value for growth inhibition by 40-fold. The antiproliferative activities of PMEG and PMEDAP were unaffected by dCF. Thus, it appears that cpr-PMEDAP, but not PMEDAP, is converted by an adenylate deaminase-like enzyme and functions as a prodrug of PMEG.

PMEG (9-(2-phosphonylmethoxyethyl)guanine) is an acyclic nucleotide analog being evaluated for its anti-proliferative activity. We examined the inhibitory effects of PMEG diphosphate (PMEGpp) toward DNA polymerases (pol) delta and epsilon and found it to be a competitive inhibitor of both these enzymes. The apparent Ki values for PMEGpp were 3-4 times lower than the Km values for dGTP. The analog was shown to function as a substrate and to be incorporated into DNA by both enzymes. Examination of the ability of pol delta and pol epsilon to repair the incorporated PMEG revealed that pol epsilon could elongate PMEG-terminated primers in both matched and mismatched positions with an efficiency equal to 27 and 85% that observed for dGMP-terminated control template-primers. Because PMEG acts as an absolute DNA chain terminator, the elongation of PMEG-terminated primers is possible only by cooperation of the 3'-5'-exonuclease and DNA polymerase activities of the enzyme. In contrast to pol epsilon, pol delta exhibited negligible activity on these template-primers, indicating that pol epsilon, but not pol delta, can repair the incorporated analog.

Previous alanine scanning mutagenesis of thrombin revealed that substitution of residues W50, K52, E229, and R233 (W60d, K60f, E217, and R221 in chymotrypsinogen numbering) with alanine altered the substrate specificity of thrombin to favor the anticoagulant substrate protein C. Saturation mutagenesis, in which residues W50, K52, E229, and R233 were each substituted with all 19 naturally occurring amino acids, resulted in the identification of a single mutation, E229K, that shifted the substrate specificity of thrombin by 130-fold to favor the activation of the anticoagulant substrate protein C over the procoagulant substrate fibrinogen. E229K thrombin was also less effective in activating platelets (18-fold), was resistant to inhibition by antithrombin III (33-fold and 22-fold in the presence and absence of heparin), and displayed a prolonged half-life in plasma in vitro (26-fold). Thus E229K thrombin displayed an optimal phenotype to function as a potent and specific activator of endogenous protein C and as an anticoagulant in vivo. Upon infusion in Cynomolgus monkeys E229K thrombin caused an anticoagulant effect through the activation of endogenous protein C without coincidentally stimulating fibrinogen clotting and platelet activation as observed with wild-type thrombin. In addition, E229K thrombin displayed enhanced potency in vivo relative to the prototype protein C activator E229A thrombin. This enhanced potency may be attributable to decreased clearance by antithrombin III, the principal physiological inhibitor of thrombin.

Protein purification has been made significantly easier by the use of affinity tags that can be genetically engineered at either the amino- or carboxyl-terminus of recombinant proteins. One of the most widely used tags is six consecutive histidine residues or 6His tag. These residues bind with high affinity to metal ions immobilized on chelating resins even in the presence of denaturing agents and can be mildly eluted with imidazole. We report the methodology for the immobilization of six histidine-containing proteins onto microtiter plates. A derivative of nitrilotriacetic acid (NTA) was prepared. This derivative, N,N-bis[carboxymethyl]lysine (BCML), was easily coupled to a maleic anhydride-activated polystyrene microtiter plate. The plate was then charged with Ni2+ for the capture of the 6His-tagged proteins. Using two different recombinant proteins with the 6His tag at either the N- or C-terminus, we demonstrated that the binding to the Ni(2+)-NTA plate was specific for six histidine-containing proteins. Proteins lacking the 6His tags did not bind to the plate. The plate was used in a modified enzyme-linked immunoabsorbent assay format to quantitate protein concentrations and determine the affinity of protein-ligand interactions. The technology can also be extended to include high-throughput screening assays for antagonists of protein-protein interactions.

Tissue factor (TF) is a transmembrane protein that functions in the initiation of blood coagulation in vivo. At sites of vascular injury, TF serves as a cell-surface receptor for the serine protease factor VIIa (FVIIa), forming an enzyme--cofactor complex and enhancing the catalytic activity of FVIIa. Tissue factor, along with the receptors for alpha- and gamma-interferons, is a member of the class 2 cytokine receptor superfamily. Crystallographic analysis demonstrated that the extracellular domain of TF consists of two immunoglobulin-like domains joined by a linker region. Each domain is comprised of two antiparallel beta-sheets containing seven conserved beta-strands separated by more variable loop regions. Extensive mutagenesis has been performed in order to map the FVIIa binding site on TF. Results indicated that the discontinuous binding site for FVIIa lies at the domain--domain interface and includes residues from extended loops and beta-strands within both the N- and C-terminal domains. Our previous study provided evidence that three consecutive residues (D44, W45, K46) within the TF loop region between beta-strands C and C' of the N-terminal domain were important for interactions with FVIIa. We have presently extended our alanine-scanning mutagenesis to include the residues within the flanking beta-strands. Thirteen sTF mutants were screened for their ability to enhance FVIIa activity. Three residues within strand C (Y34, Q37, I38) and two residues within C' (K48, Y51) were shown to be important for TF cofactor function.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue factor (TF) is a membrane-bound glycoprotein that functions as a cofactor for coagulation factor VIIa (VIIa) and initiates blood coagulation at sites of vascular injury. On the basis of sequence alignments, TF was predicted to be a member of the cytokine receptor superfamily. Utilizing the structural information available for the cytokine receptor superfamily, we have used site-directed mutagenesis to identify the binding site on TF for VIIa. The predicted loop regions in TF were systematically replaced with the homologous loops from the gamma-interferon receptor (gamma-IFN-R), the protein most related to TF in the superfamily of cytokine receptors. Six discontinuous regions (residues 16-20, 40-46, 60-69, 101-111, 129-151, 193-207) were identified that are required for interaction with VIIa and enhancement of activity. Individual substitution of 68 residues within these loops with alanine revealed eight residues (K20, D44, W45, K46, Q110, R135, F140, V207) that are required for cofactor activity. These residues fall into two groups, those that are required only for interactions with VIIa (K46, Q110, R135, F140, V207) and those that are also required to induce the conformational change in VIIa required for enhanced activity (K20, D44, W45). The discontinuous regions of TF required for interactions with VIIa form a single binding surface for VIIa that is analogous to the interface defined by the crystal structure of the complex between growth hormone and its receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

A new class of thrombin inhibitors based on sequence-specific single-stranded DNA oligonucleotides (thrombin aptamer) has recently been identified. The aptamer-binding site on thrombin was examined by a solid-phase plate binding assay and by chemical modification. Binding assay results demonstrated that the thrombin aptamer bound specifically to alpha-thrombin but not to gamma-thrombin and that hirudin competed with aptamer binding, suggesting that thrombin's anion-binding exosite was important for aptamer-thrombin interactions. To identify lysine residues of thrombin that participated in the binding of the thrombin aptamer, thrombin was modified with fluorescein 5'-isothiocyanate in the presence or absence of the thrombin aptamer, reduced, carboxymethylated, and digested with endoproteinase Arg-C. The digestion products were analyzed by reversed-phase high performance liquid chromatography and the peptide maps compared. Four peptides with significantly decreased modification in the presence of the aptamer were identified and subjected to N-terminal sequence analysis. Results indicated that B chain Lys-21 and Lys-65, both located within the anion-binding exosite, are situated within or in close proximity to the aptamer-binding site of human alpha-thrombin. The thrombin aptamer binds to the anion-binding exosite and inhibits thrombin's function by competing with exosite binding substrates fibrinogen and the platelet thrombin receptor.

The biochemical behavior and peptide binding properties of a soluble form of the human class II DR4Dw4 molecule (PI-DR4Dw4) were compared to DR4Dw4 molecules containing the transmembrane and cytoplasmic domains that were purified both from B and transfected chinese hamster ovary cells. Recombinant and B cell-derived DR4Dw4 molecules bound monoclonal anti-DR4Dw4 antibodies with different affinities and varied in their stability in the presence of sodium dodecyl sulfate. The three forms of DR4Dw4 bound peptides with a similar apparent affinity constant, but soluble class II molecules bound up to ten times more peptide than DR4Dw4 containing a transmembrane region. Peptide binding kinetics for soluble DR4Dw4 molecules were 10-20 times faster than for the other two forms of DR4Dw4 molecules. Finally, soluble PI-DR4Dw4/peptide complexes were shown to stimulate T cell proliferation.

Full-length tissue factor (263 rTF) and three truncated forms have been expressed in human kidney 293 cells; 1) 243 rTF, which lacks the cytoplasmic tail, is fully functional in the chromogenic assay and has a specific activity comparable with that of the full-length molecule, 263 rTF; 2) 219 rTF, which lacks both the transmembrane and cytoplasmic domains, is not functional; 3) the third variant, referred to as TF-PI, is a fusion protein containing the extracellular domain of TF (amino acids 1-219) fused to the last 37 amino acids of decay-accelerating factor which contain a signal for attachment of a phosphatidylinositol membrane anchor (PI). TF-PI is a membrane-bound protein expressed on the cell surface. The PI anchor restores TF activity lost when the transmembrane domain is deleted from the 219 rTF variant. The ability of the PI anchor to restore activity to 219 rTF clearly demonstrates that while the transmembrane domain is not required for TF activity, lipid association is required.

The possible self-association of tissue factor molecules was investigated by treating cells expressing tissue factor with bifunctional cross-linking agents. The two reagents chosen were 3,3'-dithiobis(sulfosuccinimidylpropionate) and sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate, both of which are membrane-impermeable and thiol-cleavable. A human bladder carcinoma cell line, J82, and a transfected human kidney cell line expressing high amounts of recombinant tissue factor were used in these studies. Exposure of the intact cells to the crosslinking reagents was found to result in the formation of multimeric tissue factor-containing complexes, the extent of which appeared to be dependent upon the amount of tissue factor expressed by the cell. The self-association of tissue factor was prevented in a variant tissue factor molecule harboring a non-homologous transmembrane domain.

Recombinant human tissue factor (rTF) purified from transfected mammalian cells is a glycoprotein that contains N-linked, but not O-linked oligosaccharides. Two of the three potential N-linked sites in the extracellular portion are fully glycosylated, while one site is approximately 90% utilized. These sites have complex-type oligosaccharides attached. The potential N-linked site in the cytoplasmic domain near the C-terminus is not glycosylated. Characterization of the tryptic map of rTF confirmed most of the proposed amino acid sequence. In addition, the disulfide bonds (between Cys-49 and Cys-57 and between Cys-186 and Cys-209) were demonstrated by FAB-MS analysis of cysteine-containing fragments obtained from the tryptic map.

We describe a mammalian cell expression system used to rapidly produce microgram quantities of a membrane protein used as an immunogen. A fusion protein expression vector was constructed which contained the signal sequence and 27 amino acids of the Herpes simplex virus glycoprotein D (gD), followed by a factor VIII (fVIII) thrombin cleavage site and the mature tissue factor (TF) sequence. This fusion protein was transiently expressed and then purified using an antibody to gD. The purified fusion protein, gDTF, was incubated with thrombin to remove the gD-fVIII moiety and the resulting rTF served as antigen for the generation of TF-specific antibodies. The antibodies produced were then used for a comparison of the turnover rates of the constitutively and transiently produced fusion protein. In addition, sensitivity to glycosidases indicated that the transiently and constitutively produced recombinant proteins do not contain identical carbohydrate structures.

Tissue factor (TF) is a 263 amino acid membrane-bound procoagulant protein that serves as a cofactor for the serine protease factor VII (fVII). Recombinant human TF (rTF) produced in both human kidney 293 cells and Escherichia coli has been immunoaffinity purified by using a TF-specific monoclonal antibody. Recombinant TF produced in 293 cells is glycosylated and migrates on reducing SDS-PAGE with an apparent molecular weight (Mr) of 45K. Some interchain disulfide-bonded rTF dimers are observed under nonreducing conditions. The E. coli produced rTF has a molecular weight of 33K and 35K, with the 33K band missing nine amino acids at the carboxy terminus. Although the E. coli produced rTF does not contain any carbohydrate, it is fully functional in both a chromogenic assay and a one-stage prothrombin time assay. A variant has been constructed wherein the cytoplasmic cysteine (residue 245) has been mutagenized to a serine residue. The amount of disulfide-linked aggregates is dramatically reduced following immunoaffinity purification of this four-cysteine variant (C2455), which is active in the chromogenic and prothrombin time assays.

We describe the polypeptide structure and some of the catalytic properties of a DNA polymerase alpha.DNA primase complex that can be prepared from KB cells by immunoaffinity purification. The procedure is based on monoclonal antibodies that were raised against a biochemically purified, catalytically active core protomer of the polymerase. In all respects tested, the basic mechanism of substrate recognition and binding by the immunoaffinity-purified polymerase is qualitatively identical to that of the core protomer. The immunoaffinity-purified KB cell polymerase alpha X DNA primase is structurally complex. On the basis of extensive immunochemical analyses with five independent monoclonal antibodies, three of which are potent neutralizers of polymerase alpha activity, peptide mapping studies, and the application of a sensitive immunoassay that permits detection of polymerase alpha antigens in crude cell lysates, we have established that the principal form of catalytically active DNA polymerase alpha in KB cells is a phosphoprotein with a molecular mass of 180 kilodaltons. This protein is stable in vivo, with an estimated half-life of greater than or equal to 15 h. In contrast, the polypeptide is extremely fragile in vitro and generates partial degradation products of p165, p140, and p125 that explain the "microheterogeneity" typically exhibited by polymerase alpha peptides in denaturing polyacrylamide gels. In addition to the catalytically active polymerase alpha polypeptide(s), the immunopurified enzyme fraction typically contains three other proteins, p77, p55, and p49, the functions of which have not yet been established. These proteins do not display polymerase alpha epitopes and have been shown by peptide mapping to be independent species that are unrelated either to the large polymerase peptides or to one another. The polypeptide p77 is also a phosphoprotein, and in both p180 and p77 the phosphorylated amino acids are exclusively serine and threonine.