Biomedical subjects

Ralf David

Publications and source records attributed to Ralf David.

4 recordsLinked to original sources

PubMed2006

Identification of the dimerisation interface of human interleukin-8 by IL-8-variants containing the photoactivatable amino acid benzoyl-phenylalanine.

The three-dimensional structure of human interleukin-8 (hIL-8) was determined by the use of NMR and X-ray methodology. At high concentrations interleukin-8 and many other chemokines form a non-covalent homodimer. Several studies have been performed to investigate the relevance of the dimer on receptor activation and led to contradictory results. In order to obtain a better understanding of the dimerisation process, covalently linked homo- and heterodimers were produced by photo-induced dimerisation of hIL-8 analogues that contain the photo-activatable amino acid p-benzoyl-phenylalanine (Bpa) at different positions. Whereas the N-terminal fragment (1-54) was expressed as recombinant thioester, the C-terminal fragments (55-77) that contain Bpa either at position 65 or 74 were obtained by solid-phase peptide synthesis. The segments were combined by expressed protein ligation and led to full length IL-8 variants containing the non-proteinogenic amino acid Bpa at single positions. IP(3) activity tests showed high biological activity for the CXCR1-GFP receptor for both variants comparable to that of the native ligand. The refolded and purified ligation-products were used for dimer formation by UV-irradiation. The analysis of the reaction mixture was performed by gel-electrophoresis and mass spectrometry and showed that dimer formation of IL-8 occurred in a position dependent manner. [Bpa(74)]hIL-8 has a high tendency to form covalent dimers whereas no dimer formation was observed for the variant with Bpa at position 65. Accordingly one residue of the dimerisation interface could be identified.

Amino Acid Substitution↗

PubMed2006

Conformational constraining of inactive and active States of a seven transmembrane receptor by metal ion site engineering in the extracellular end of transmembrane segment V.

The extracellular part of transmembrane segment V (TM-V) is expected to be involved in the activation process of 7TM receptors, but its role is far from clear. Here, we study the highly constitutively active CXC-chemokine receptor encoded by human herpesvirus 8 (ORF74-HHV8), in which a metal ion site was introduced at the extracellular end of TM-V by substitution of two arginines at positions V:01 and V:05 with histidines [R208H; R212H]. The metal ion site conferred high-potency inverse agonist properties (EC(50), 1.7 microM) to Zn(II) in addition to agonist and allosteric enhancing properties at concentrations >10 microM. The chemokine interaction with [R208H;R212H]-ORF74 was altered compared with wild-type ORF74-HHV8 with decreased agonist (CXCL1/GROalpha) potency (84-fold), affinity (5.8- and 136-fold in competition against agonist and inverse agonist, respectively), and binding capacity (B(max); 25-fold). Zn(II) in activating concentrations (100 microM) acted as an allosteric enhancer as it increased the B(max) (7.1-fold), the potency (9.9-fold), the affinity (1.7- and 6.1-fold in competition against agonist and inverse agonist, respectively), and the efficacy (2.5-fold) of CXCL1/GROalpha. The activating properties of Zn(II) were not due to a metal ion site between the ligand and the receptor because CXCL1/GROalpha analogs in which the putative metal-ion binding residues had been substituted-[H19A] and [H34A]-acted like wild-type CXCL1/GROalpha. Based on the complex action of Zn(II) and on the chemokine interaction for [R208H;R212H]-ORF74, we conclude that the extracellular end of TM-V is important for the activation of this CXC-chemokine receptor.

Amino Acid Sequence↗

PubMed2004

Expressed protein ligation. Method and applications.

The introduction of noncanonical amino acids and biophysical probes into peptides and proteins, and total or segmental isotopic labelling has the potential to greatly aid the determination of protein structure, function and protein-protein interactions. To obtain a peptide as large as possible by solid-phase peptide synthesis, native chemical ligation was introduced to enable synthesis of proteins of up to 120 amino acids in length. After the discovery of inteins, with their self-splicing properties and their application in protein synthesis, the semisynthetic methodology, expressed protein ligation, was developed to circumvent size limitation problems. Today, diverse expression vectors are available that allow the production of N- and C-terminal fragments that are needed for ligation to produce large amounts and high purity protein(s) (protein alpha-thioesters and peptides or proteins with N-terminal Cys). Unfortunately, expressed protein ligation is still limited mainly by the requirement of a Cys residue. Of course, additional Cys residues can be introduced into the sequence by site directed mutagenesis or synthesis, however, those mutations may disturb protein structure and function. Recently, alternative ligation approaches have been developed that do not require Cys residues. Accordingly, it is theoretically possible to obtain each modified protein using ligation strategies.

Animals↗

PubMed2003

Semisynthesis and application of carboxyfluorescein-labelled biologically active human interleukin-8.

Human interleukin 8 (hIL-8), a neutrophil-activating and chemotactic cytokine, is known to play an important role in the pathogenesis of a large number of neutrophil-driven inflammatory diseases. This cytokine belongs to the family of CXC chemokines, mediating the response through binding to the seven-transmembrane helical G protein-coupled receptors CXCR1 and CXCR2. For the first time, we employed the expressed protein ligation (EPL) strategy to chemokine synthesis and subsequent modification. The ligation site was chosen with respect to the position of four cysteine residues within the hIL-8 sequence. Ligation with synthetic peptides that carry cysteine at their N-termini resulted in full-length hIL-8 and the specifically carboxyfluorescein-labelled analogue [K69(CF)]hIL-8(1-77). [K69(CF)]hIL-8(1-77) was fully active as shown by inhibition of cAMP production. Furthermore, this analogue was used to study receptor internalisation in human promyelotic HL60 cells that express CXCR1 and CXCR2 receptors. Binding and quenching studies were performed on HL60 membranes and suggest that the C-terminus of IL-8 is accessible to solvent in the receptor-bound state. Thus, we introduce here a powerful approach that allows the site-specific incorporation of chemical modifications into the sequence of chemokines, which opens new avenues for studying IL-8 function.

Amino Acid Sequence↗