Mistletoe lectin I (MLI) is the major active constituent of mistletoe extracts, which are widely used for adjuvant tumour therapy. The 66-kDa heterodimeric disulphide-linked glycoprotein is classified as type II ribosome-inactivating protein (RIP) due to the rRNA-cleaving enzyme activity of the A-subunit, also referred to as toxic entity. MLI and the close relative ricin both belong to the family of the two-chain plant type II RIP proteins. Isolation of the glycosylated proteins from plant material yield inhomogeneous material probably due to post-translational modifications. The aim of this study was to prepare pure and homogeneous protein as a prerequisite for structural and mechanistic studies in order to gain insight into the mode of action of this cytotoxic plant protein on tumour and immune cells. Of particular interest was to explain whether the differences in toxicity of ML and ricin are the result of variations of their enzymatic activities. By investigating the sequence homologies between the active sites of different RIPs we were able to deduce a set of primers which were suitable for specific amplification of the mistletoe lectin gene. Applying this PCR strategy the full-length 1923 nucleotide DNA sequence coding for the prepro-protein was obtained showing the existence of a single intron-free gene. In order to elucidate the molecular basis for the observed differences in cytotoxicity within the family of RIP the enzymatic A-subunit was expressed in a heterologous system. Expression of the A-chain in E. coli BL21/pT7 resulted in production of insoluble inclusion bodies constituting 20-30% of total protein. Refolding led to a pure and homogeneous protein species with an apparent molecular mass of 27 kDa and a pI value of 6.4. The ribosome-inactivating activity of the unglycosylated recombinant A-chain (IC50 20.5 pM) protein was in the same range as that of the glycosylated plant-derived ML A-chain (IC50 3.7 pM), which was very similar to that of ricin A-chain (IC50 4.9 pM). Thus, the higher cytotoxicity of ricin cannot be accountable for differences in the enzymatic activities of the type II RIP A-chains.

The kinetic mechanism of the inhibition of a-thrombin by hirudin was analyzed using the hirudin-derived fragments hirudin(1-47) and hirudin(45 -65). Previously, these fragments have been shown to interact with athrombin at distinct sites inhibiting thrombin-mediated clot formation. Binding to the active site the N-terminal fragment hirudin(1-47) competitively inhibits hydrolysis of the substrates Tos-Gly-Pro-Arg-NH-Mec (Tos, tosyl; NH-Mec, 4-methylcoumaryl-7-amide) and fibrinogen with Ki values of 420 f 18 nM and 460 f 25 nM, respectively. Interacting with the anion-binding site of a-thrombin the C-terminal fragment competitively inhibits the hydrolysis of fibrinogen with a Ki of 760 & 40 nM. It was found, however, that this fragment acts as a hyperbolic uncompetitive inhibitor with respect to the hydrolysis of the peptide-NH-Mec substrate. According to the BottsMorales scheme for enzyme inhibition, the parameters Ki = 710 38 nM, Ri = 348 f 22 nM, as well as a = a = 0.49 of thrombin inhibition by the C-terminal fragment hirudin(45 -65), were obtained. The results are discussed in terms of the interaction of hirudin and thrombin.The thrombin-specific inhibitor hirudin is a polypeptide of 65 amino acid residues isolated from the leech Hirudo medicinalis [I -41. Recombinant DNA technology revealed the production of recombinant hirudin and recombinant hirudin mutants as tools for analyzing thrombin-hirudin complex formation [5, 61. This set of probes was completed by synthetic peptides comprising the C-terminal part of the hirudin molecule [7, 81. It has been shown previously that the recognition between thrombin and hirudin resides in a number of contact regions on each molecule [9 -111. Areas of thrombin which interact with hirudin have been identified using proteolyzed and active-site-modified thrombin forms [I 21, as well as peptidespecific antibodies to the anion-binding site of the enzyme [13]. Chemical modification of the complex revealed distinct lysine residues as part of the contact region [14]. With respect to the inhibitor, the importance of an intact C-terminus for thrombin-hirudin complex formation became evident from Cterminal truncated hirudin forms [ 15, 161. Synthesis of hirudin-related C-terminal peptides confirmed their binding to a non-catalytic site of a-thrombin causing inhibition of athrombin-mediated clotting [7,8]. In addition, the importance of the nature of the two N-terminal amino acid residues for complex formation [17], and the lack of a reactive site peptide bond which interacts with the specificity pocket of the target protease [5,6] comprising the disulfide-bonded N-terminus and the negatively charged C-terminus were used to localize regions of thrombin and hirudin that interact with each other [9, 181. These studies have revealed competitive binding of the Nterminal part of hirudin to the active site and direct interactions of C-terminal fragments with the anion-binding site. The conformation of C-terminal fragments of hirudin in solution has been examined using NMR spectroscopy ...

Timely and reliable detection of acute primary human cytomegalovirus (HCMV) infection is important in

The open reading frames ORF 52, ORF 65, K12, and K8.1 of the human herpesvirus 8 (HHV8) were expressed as glutathione-S-transferase (GST) fusion proteins and analysed by Western blotting (WB) and enzyme-linked immunosorbent assay (ELISA). The open reading frame (ORF) 65 and K8.1 antigens gave the highest reactivity (71%) in sera from HIV-dependent Kaposi's sarcoma (KS) patients. Therefore both antigens appear to be essential for HHV8 diagnostics, whereas ORF K12 and ORF 52 were of minor importance. Using polymerase chain reaction (PCR) out of the peripheral blood of these KS patients, 48% were detected as positive. By testing an N-terminal-deleted construct (amino acid 80-171) of ORF 65, we could show that the N-terminal region of this protein is essential to mediate full immunogenic reactivity. By analysing different deletion mutants of ORF K8.1, the major epitope was found to be located between aa 29 and 101. The prevalence of antibodies directed against the different antigens was determined for healthy blood donors to be 3-6%. The different antibody patterns obtained in HIV-patients with and without KS support the hypothesis that different antibody profiles develop during the course of KS.

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