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Increased expression of vascular cell adhesion molecule 1 (VCAM1) is associated with a variety of chronic inflammatory conditions, making its expression and function a target for therapeutic intervention1,2,3. We have recently identified CAM741, a derivative of a fungus-derived cyclopeptolide that acts as a selective inhibitor of VCAM1 synthesis in endothelial cells. Here we show that the compound represses the biosynthesis of VCAM1 in cells by blocking the process of cotranslational translocation, which is dependent on the signal peptide of VCAM1. CAM741 does not inhibit targeting of the VCAM1 nascent chains to the translocon channel but prevents translocation to the luminal side of the endoplasmic reticulum (ER), through a process that involves the translocon component Sec61β. Consequently, the VCAM1 precursor protein is synthesized towards the cytosolic compartment of the cells, where it is degraded. Our results indicate that the inhibition of cotranslational translocation with low-molecular-mass compounds, using specificity conferred by signal peptides, can modulate the biosynthesis of certain secreted and/or membrane proteins. In addition, they highlight cotranslational translocation at the ER membrane as a potential target for drug discovery.

Interleukin 8 (IL-8) is a member of the CXC subfamily of chemokines which attracts and activates preferentially neutrophilic granulocytes. At nanomolar concentrations monomeric and dimeric forms of the molecule are in equilibrium, with the monomer being the prevalent form. Five amino acids from position 23 to 29 of the 72-amino acid IL-8 sequence form the dimer interface, with Leu25 and Val27 being highly conserved among the CXC chemokines. To investigate the contribution of these amino acids to the dimerization of IL-8, we produced in escherichia coli IL-8 derivatives with phenylalanine substitutions at position 25 or 27, or both 25 and 27. All three recombinant proteins were characterized by a significantly impaired potential to form dimers in solution as seen in chemical crosslinking experiments. IL-8 Val27 also could not be crosslinked as a dimer on its receptors. Receptor affinities and in vitro chemotactic activities, however, were not significantly different between wild-type and IL-8 with single mutations. The dimerization deficient IL-8 analogue had also full inflammatory activity in vivo. Thus, the monomer is the biologically active form of IL-8.

A 72-amino-acid chimeric protein, Chi1, was constructed from the N-terminal part of interleukin 8, IL-8-(1-53), and the C-terminal part of melanoma growth stimulatory activity, MGSA-(54-72). Chi1 protein showed receptor-binding specificity and biological activity similar, but not identical to IL-8 and decidedly different from MGSA. The structure of Chi1 was determined in solution by two-dimensional NMR and molecular-dynamics calculations. The structure resembled the structures of MGSA and IL-8 closely, containing a triple-stranded beta-sheet in the IL-8 part and an amphipathic alpha-helix in the MGSA part. Chi1 formed dimers at millimolar concentrations via the first strand from the N-terminus, analogous to IL-8 and MGSA. In contrast to the latter molecules, however, the alpha-helix of Chi1 did not pack against the beta-sheet part, but was an independent structural element. This structural difference could be explained mainly by the modulation of hydrophobic interactions between the helix and the rest of the protein in Chi1 as compared to IL-8 and MGSA. It is concluded that tight helix packing is not required for receptor binding and biological activity of Chi1.

By chemical cross-linking experiments we show that at physiologically relevant concentrations IL-8 and NAP-2 monomers are in an equilibrium with dimers and even oligomers (Kd 300–800 nM). Oligomerization seems to be more prevalent for IL-8 than for NAP-2. The form in which IL-8 and NAP-2 bind to their specific receptors was analyzed in binding experiments with COS-1 cells expressing IL-8 receptor A or B in recombinant forms. Both receptors were cloned from the human myeloid leukemic cell line AML-193. Type A receptor had high affinity for IL-8 (K d 4 nM) and low affinity for NAP-2 (K d ≥700 nM), whereas the type B receptor was of equally high affinity (K d 2 nM) for both IL-8 and NAP-2. However, IL-8 receptor B could bind specifically three to four times more IL-8 than NAP-2, and NAP-2 was a weak competitor for IL-8 binding to the same receptor. In addition, IL-8, but not NAP-2, could be cross-linked to dimers when bound to IL-8 receptor B. We suggest from these findings that IL-8, but not NAP-2, binds as a dimer and oligomer to IL-8 receptor B. J. Leukoc. Biol. 55: 763-770; 1994.

The human neutrophil activating peptides-1 and -2 (NAP-1/IL-8, NAP-2) are two structurally and functionally related members of the chemokine cytokine family. They are chemoattractants and activators of neutrophils and exert their effects by binding to specific receptors which are expressed on responsive cells. Two closely related IL-8 receptors of neutrophils have been characterized recently by molecular cloning. We show here that NAP-1/IL-8 and NAP-2 can be cross-linked to at least four protein bands from human neutrophil surfaces with apparent molecular masses of 55, 65, 71 and 81 kDa. The two cross-linked proteins with lower masses were associated with high, the two with the higher masses with low affinity binding of NAP-2, NAP-1/IL-8 was bound to all bands with high affinity. NAP-1/IL-8 and NAP-2 could also be cross-linked to form dimers when bound to cells and in solution. Our results show that more than two NAP-1/IL-8 receptors, or more than two forms of the known receptors exist. Alternatively, the four protein bands can be explained by cross-linking of ligand monomers and dimers, respectively, to the known receptors of neutrophils.

The majority of patients suffering from myelodysplastic syndromes (MDS) die of complications due to cytopenia. Clinical trials have demonstrated that in an essential number of MDS patients cytopenia can be ameliorated by exogenously supplied growth factors. We investigated endogenous serum levels of GM-CSF and IL-3 in 15 healthy individuals and 34 patients suffering from MDS. No circulating growth factors were detected in the serum of healthy controls, nor was IL-3 measurable in MDS patients. GM-CSF serum levels, however, were elevated in a significant number of patients (26.5%). Levels did not correlate with FAB classification, leukocyte count or chromosomal abnormalities. No significant differences in GM-CSF or IL-3 receptor expression were detected between healthy individuals and MDS patients. One patient with increased endogenous GM-CSF serum level and normal surface receptor expression responded to exogenously applied GM-CSF. In the light of these results, a funetional alteration of growth factor receptors or disturbances of signal transduction pathways must be discussed for MDS.

Summary Neutrophil receptor(s) for neutrophil activating peptides 1 and 2 were studied by competition binding experiments with radiolabeled NAP-1 and NAP-2 preparations. NAP-1 bound with one affinity, NAP-2 with two quite different affinities, to common receptor(s) on neutrophils. Concentrations of NAP-2 needed to induce exocytosis of β-glucosaminidase corresponded to the higher dissociation constant of the two binding equilibria. Thus, the binding of NAP-2 to PMN with high affinity does not activate the cells.

After its identification in 1987 as a novel neutrophil-activating cytokine, interleukin-8 (IL-8) was the center of interest of a large variety of interdisciplinary scientists for study of its action in vitro and in vivo (1,2,3,4). Soon it became clear that interleukin-8 plays a critical role in a series of diseases which are all characterized by strong neutrophil involvement. Rheumatoid artrithis, psoriasis, polytrauma, septicemia, adult respiratory distress syndrome (ARDS), asthma, emphysema, myocardial infarction, nephritis, inflammatory bowel disease, and gout are diseases which affect millions of people, and IL-8 is implicated in all these disease states. Therefore, inhibition of interleukin-8 itself, or blocking of the interaction with its seven transmembrane domain cellular receptor represents an attractive and logical approach for treatment of these inflammatory states. Possibilities for interference include neutralising antibodies, small molecules binding to the chemokine at critical positions, (e.g. domains involved in dimerization, heparin binding or receptor binding) or molecules which bind to the receptor. An additional approach would be the use of peptides based on the IL-8 structure or mutant IL-8 molecules to act as antagonists. As an alternative to screening for inhibitors in a series of biological assays, a rational way of finding an inhibitory principle involves the elucidation of structure/function relationships. This is usually done by production of mutant proteins of the target molecule and subsequent determination of biological activities. As only the primary structures of the mutants are known this might be called a “one-dimensional structure activity relationship”. We are attempting to go one step further and actually determine the three-dimensional structure of mutant IL-8 derivatives with interesting and clear changes in biological activities and, based on structure/function relationships in three dimensions, get a more detailed insight into structural requirements for specific biological activities.

Three derivatives of mutant ga10P-308::IS2-I of Escherichia coli were characterized by DNA sequence analysis. Deletions and DNA sequence rearrangements were observed which apparently were initiated at short A-T rich inverted repeats within IS2. Two of the mutants carried newly synthesized DNA sequences which were inverted copies of already existing IS2 sequences. Thus long stretches with twofold symmetry were formed. It is discussed whether these inverted repeats were formed by DNA repair synthesis which was initiated at the A-T rich palindromes of IS2.

Phenotypic revertants of galOP::IS1 and galOP::IS2 mutations have been isolated after mutagenesis with nitrosoguanidine, they are probably caused by mutations in gene suA. The polarity suppressor mutations described in this study and a known mutation in gene suA isolated by D. Morse (Morse and Guertin, 1972) suppress polarity caused by IS1 more effectively than that caused by IS2 or IS4. Furthermore, suppressibility is influenced by the site and orientation of IS integration. The synthesis of the three enzymes in galOP::IS suA double mutants is constitutive and the ratio of the three enzymes is altered in comparison to the wild type. The reasons for constitutive synthesis of the galactose enzymes and for the altered ratio of enzyme synthesis are discussed.

The isolation and characterization of three unstable and constitutive revertants of mutant galOP-308 of E. coli is described. In this mutant an IS2 element is integrated between the promoter and the first structural gene of the galactose operon, and exerts a strong polar effect on the expression of the three galactose genes. In the three revertants under investigation it was observed that relief of polarity and constitutive expression of the gal-operon were accompanied by the deletion of 90% of the IS2 sequence and of various lengths of the adjacent sequences including the gal-promoter. We conclude from this result that the transcription termination signals causing strong polarity were located on the deleted part of IS2, and that in our revertants the galactose genes are now under the control of a new promoter which is apparently unstable.

DNA of the IS-elements IS1 and IS2 was prepared by digestion of appropriate heteroduplex molecules with endonuclease S1, followed by sucrose gradient centrifugation or gel electrophoresis. The material obtained is homogeneous with regard to size. The length of IS1 DNA is 820 +/- 65 nucleotides, the length of IS2 DNA is 1,350 +/- 70 nucleotides. IS1 DNA is not cleaved by the restriction endonucleases Eco R1, Hind II or Hind III. IS2 DNA is cleaved once by each of the two latter enzymes. The buoyand density determined by equilibrium centrifugation of Hg-complexes in Cs2so4 corresponds to a GC content of approximately 50%. Labelling with polynucleotide kinase indicates that both IS DNA's have a guanosyl residue at both of their 5'-termini.

phi80dgal transducing bacteriophages have been isolated by the F-fusion technique of Press et al. (1971) and gal-operator-promoter insertion mutations have been introduced by homogenate formation. Five different phi80dgal isolates have been studied in more detail. One of the phi80 phages transduces the gal operon and gene aroG as well as at least part of the trp-operon; the gal operon of another phi80dgal transducing phage is inverted with respect to the phi80dgal sequences. Heteroduplex DNA mapping indicates that one of the phi80dgal isolates in addition to the gal operon and a portion of the adjacent chromosomal region carries an IS2-element which is derived from the F'gal episome. The isolated phi80dgal phages may be utilized for preparing pure gal mRNA and insertion-RNA as well as pure gal operon DNA.

Galactose negative mutations are described which reduce the maximum expression of all three gal genes about 100-fold. The residual enzyme synthesis is not or only slightly inducible.