Herbert König

HERV-K : The biologically most active human endogenous retrovirus family

The human genome contains a wide variety of endogenous retrovirus-like sequences. The human endogenous retrovirus type K (HERV-K) family comprises 30-50 members per haploid genome in humans and is highly conserved in Old World monkeys and apes. Some proviruses are displaying open reading frames (ORF) with coding capacity for viral particles. HERV-K sequences most likely code for the previously described human teratocarcinoma-derived virus (HTDV) and correlated expression of HERV-K Gag has been demonstrated by immunoelectron microscopy studies. Protease, but not yet reverse transcriptase (RT), enzymatic activity was demonstrated for recombinant HERV-K proteins. However, an ultrasensitive RT assay revealed specific polymerase activity associated with the HTDV particles. HERV-K transcription is specifically regulated by viral long terminal repeats and RNA is expressed at low steady-state levels in a variety of human tissues and tumours. In teratocarcinoma cell lines, HERV-K is highly expressed in a complex pattern showing full-length as well as subgenomic envelope (env) and two alternatively spliced small transcripts. The doubly spliced 1.8-kb mRNA codes for cORF protein which resembles Rev of HIV-1 and is located in the nucleolus. In addition, the cORF sequence acts as a leader and is essential for effective expression of glycosylated HERV-K Env protein. Although HERV-K sequences code for all necessary retroviral proteins, infectious particles could not yet be demonstrated. The putative implication of HERV sequences in pathophysiological processes, for example, testicular malignancies, remains to be elucidated.

Detection of a novel plasma serine protease during purification of vitamin K-dependent coagulation factors.

A novel serine protease (PHBSP) was purified from human plasma by two chromatographic steps with a final yield of 1.6 mg/l plasma. The protease consists of two disulfide‐bridged chains of about 50 and 30 kDa with the light chain containing the active site of the enzyme. NH2‐terminal sequence analysis revealed identity to the deduced amino acid sequence of HGFA‐like mRNA. The activity of PHBSP is strongly dependent on Ca2+ ions and is efficiently inhibited by α2‐antiplasmin and aprotinin. Possible functions of PHBSP in the hemostatic system are discussed.

Activation of proPHBSP, the zymogen of a plasma hyaluronan binding serine protease, by an intermolecular autocatalytic mechanism.

Abstract The hyaluronic acid binding serine protease (PHBSP), an enzyme with the ability to activate the coagulation factor FVII and the plasminogen activator precursors and to inactivate factor VIII and factor V, could be isolated from human plasma in the presence of 6m urea as a single-chain zymogen, whereas under native conditions only its activated two-chain form was obtained. The total yield of proenzyme (proPHBSP) was 5–6 mg/l, corresponding to a concentration of at least 80–100nm in plasma. Upon removal of urea, even in the absence of charged surfaces a rapid development of amidolytic activity was observed that correlated with the appearance of the two-chain enzyme. The highest activation rate was observed at pH 6. ProPHBSP processing was concentration-dependent following a second order kinetic and was accelerated by catalytic amounts of active PHBSP, indicating an intermolecular autocatalytic activation. Charged macromolecules like poly-L-lysine, heparin, and dextran sulfate strongly accelerated the autoactivation, suggesting that in vivo proPHBSP activation might be a surface-bound process. The intrinsic activity of the proenzyme was determined to be 0.25–0.3%, most likely due to traces of PHBSP. The presence of physiological concentrations of known plasma inhibitors of PHBSP, like α2 antiplasmin and C1 esterase inhibitor, but not antithrombin III/heparin, slowed down zymogen processing. Our in vitro data suggest that the autoactivation of proPHBSP during plasma fractionation is induced by the removal of inhibitors of PHBSP and is accelerated by charged surfaces of the chromatographic resins.

Azidothymidine triphosphate is an inhibitor of both human immunodeficiency virus type 1 reverse transcriptase and DNA polymerase gamma.

The reverse transcriptase from human immunodeficiency virus type 1 was purified from the virus to near homogeneity. The enzyme was shown to possess both RNA-dependent and DNA-dependent DNA-synthesizing activity. Activated DNA as a heteropolymeric substrate was used as efficiently as was the homopolymeric substrate poly(rA)-oligo(dT). The Michaelis-Menten constants were determined for each of the four nucleotides needed to elongate a natural template primer. Azidothymidine triphosphate, a well-known inhibitor of the enzyme, inhibited the enzyme competitively with respect to dTTP and noncompetitively with respect to the other nucleotides. Azidothymidine triphosphate acted as an efficient inhibitor of cellular DNA polymerase gamma, whereas other enzymes of eucaryotic DNA metabolism, namely, DNA polymerase alpha-primase and DNA polymerase beta, were not inhibited. This finding may explain why some acquired immunodeficiency syndrome patients suffer side effects during azidothymidine therapy. Images

Tissue factor is the only activator of coagulation in cultured human lung cancer cells.

It is a long-known principle that tumour cells tend to exploit the hosts physiologic systems in order to get support in terms of, for example, nutrition, growth or metastasis. One of these physiologic systems is the blood coagulation cascade, which has been found activated in many tumour patients. The mechanisms of the activation of coagulation have been assessed in numerous animal and in vitro experiments, and the results appeared to point to several distinct activators. The present study used a large panel of different cultivated human lung cancer cell lines and experimental systems involving normal plasma, plasmas deficient of factors V, VII or X, purified coagulation factors II and X, recombinant tissue factor (TF), and specific inhibitory antibodies against factor VII and TF. The results provide strong evidence that there is no activator of coagulation besides TF in the wide array of lung cancer cells examined. However, this work reveals a striking variability of TF content among the cell lines. This might explain ambiguous results of clinical trials of anticoagulation as an adjunct to antineoplastic therapy in lung cancer. By sensitive diagnostic tools like the plasma thrombin-antithrombin complex levels it might be possible to select patients with activated coagulation, who might benefit from anticoagulation.

A secreted metallo protease from Aeromonas hydrophila exhibits prothrombin activator activity

Detection, purification, and partial characterization of a protease from Aeromonas hydrophila capable of cleaving prothrombin into active thrombin is described. The protease has been characterized with respect to enzymatic characteristics such as optimum reaction conditions for prothrombin activation, usage of additional substrates, as well as sensitivity against inhibitors. The protease activity can reversibly be inhibited by Me2+ chelating agents like ethylenediamine tetraacetic acid. The enzyme exhibits a pI value of 4.4 and can withstand temperatures up to 55°C without loss of activity. With respect to prothrombin the enzyme exhibits a KM value of 1.47 μmol/l and a vmax value of 1.66 mol/min per mol enzyme. Amino terminal sequence analysis as well as mass spectrometry of fragments obtained by trypsin digest showed identity to a recently described elastase type protease from the same organism and homology to known proteases from other procaryotes (e.g. Aeromonas caviae, Vibrio proteolytica, Pseudomonas aeruginosa).

Calibrating Thrombin Generation in Different Samples: Less Effort with a Less Efficient Substrate

The technique of thrombin generation has been established as a promising tool for the diagnosis, risk estima- tion, and perhaps prognosis of coagulation insufficiencies. Without further experimentation an indication can be obtained if a sample donor carries the risk for haemophilia or thrombophilia, either congenital or acquired. A comparison of two different thrombin generation assays is presented, demonstrating that a currently not commercially available test allows an easier and cheaper calibration than its commercial counterpart. This is achieved by employment of a less efficient but highly specific peptide substrate for thrombin and the involvement of low amounts of analyte (plasma samples).

A novel assay for factor XIII based on cross-linking of synthetic peptides : analysis of different substrates

A novel assay for factor XIII is described that utilizes exclusively small synthetic peptides as substrates for the cross-linking reaction catalyzed by activated factor XIII (FXIIIa). The acyl donor substrate (selection peptide) is immobilized on a microplate via biotin while the acyl acceptor substrate (detection peptide) is labeled with the fluorochrome Oregon green to allow sensitive detection without the need for secondary enzyme systems for signal amplification. Starting with an amino acid sequence from the fibrin γ-chain (GQQHHLGGAKQAGDV) as a prototype peptide, the influence of amino acid exchanges were investigated with respect to their impact on the FXIIIa-catalyzed reaction. It was found that FXIIIa readily accepts a broad range of substrate peptides, with a proline neighboring the essential lysine having the most detrimental effect. The assay appears to be valuable for the molecular characterization of factor XIII and may be used for a deeper investigation into the substrate requirements of this final enzyme of wound repair, and eventually also for the characterization of other transglutaminases.

Factor VIII determination in patient's plasma and concentrates: a novel test equally suited for both matrices

A novel assay for the determination of factor VIII (FVIII) is described. The assay uses a fluorescence-based detection system comparable with the common chromogenic test. At the same time, the assay is analogous to the clotting test as it does not involve pre-activation of FVIII. The assay was adjusted to perform equally well with patients plasma and FVIII concentrates as samples. The combined employment of two different FVIII-deficient plasmas turned out to be of crucial importance in order to render the matrices as similar as possible to patients plasma and, simultaneously, to obtain maximum sensitivity. Samples with a FVIII content down to 0.01 IU/ml are readily measured as are samples with a FVIII content of 1 IU/ml or somewhere in between. Upon dilution of samples, concentrates and plasma exhibited the same dose–response characteristics.

ATP and the processivity of Xenopus laevis DNA polymerase α

We use specific restriction fragments as defined primers for DNA synthesis on single-stranded circular phage fd DNA. These structures are relatively poor templates for a highly purified DNA polymerase alpha from Xenopus laevis eggs. However, DNA synthesis is stimulated about 5-fold by addition of ATP to the reaction mixture. We show that the deoxynucleotide polymers, synthesized in the presence of ATP, are significantly longer than those produced in the absence of ATP. We also show that this effect is due to a more tenacious binding of DNA polymerase alpha to DNA and conclude that ATP increases the processivity of the enzyme.