René Devos

Identification and Expression Cloning of a Leptin Receptor, OB-R

The ob gene product, leptin, is an important circulating signal for the regulation of body weight. To identify high affinity leptin-binding sites, we generated a series of leptin-alkaline phosphatase (AP) fusion proteins as well as [125I]leptin. After a binding survey of cell lines and tissues, we identified leptin-binding sites in the mouse choroid plexus. A cDNA expression library was prepared from mouse choroid plexus and screened with a leptin-AP fusion protein to identify a leptin receptor (OB-R). OB-R is a single membrane-spanning receptor most related to the gp130 signal-transducing component of the IL-6 receptor, the G-CSF receptor, and the LIF receptor. OB-R mRNA is expressed not only in choroid plexus, but also in several other tissues, including hypothalamus. Genetic mapping of the gene encoding OB-R shows that it is within the 5.1 cM interval of mouse chromosome 4 that contains the db locus.

A human high affinity interleukin-5 receptor (IL5R) is composed of an IL5-specific α chain and a β chain shared with the receptor for GM-CSF

Abstract cDNA clones encoding two receptor proteins involved in the binding of human interleukin 5 (hIL5) have been isolated. A first class codes for an IL5-specific chain (hIL5Rα). The major transcript of this receptor gene, as analyzed in both HL-60 eosinophilic cells and eosinophilic myelocytes grown from cord blood, encodes a secreted form of this receptor. This soluble hIL5Rα has antagonistic properties. A second component of the hIL5R is found to be identical to the β chain of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) high affinity receptor. The finding that IL5 and GM-CSF share a receptor subunit provides a molecular basis for the observation that these cytokines can partially interfere with each others binding and have highly overlapping biological activities on eosinophils.

Molecular cloning of human interleukin 2 cDNA and its expression in E. coli.

A recombinant plasmid containing human interleukin 2 (IL2) cDNA was identified in a cDNA library constructed from mRNA derived from PHA-TPA induced splenocytes. Using this cDNA as a hybridization probe, a DNA fragment containing the IL2 gene was isolated from a collection of hybrid phages derived from human genomic DNA. A unique reading frame was identified from the nucleotide sequence derived from these plasmids coding for a polypeptide of 153 amino acids and containing a putative signal sequence of 20 amino acids. A mature polypeptide starting with either Met-Ala-Pro or Met-Pro was expressed in E. coli under control of the E. coli trp promoter or using a combination of the phage lambda PL promoter and a ribosome binding site derived from phage Mu. The bacterial IL2 polypeptide had a molecular weight of 15,000 daltons and accounted for more than 10% of the total E. coli proteins in fully induced cells; it was biologically active in the T-cell specific DNA synthesis assay, even after recovery from a SDS-containing polyacrylamide gel.

Leptin Receptor (OB-R) Signaling CYTOPLASMIC DOMAIN MUTATIONAL ANALYSIS AND EVIDENCE FOR RECEPTOR HOMO-OLIGOMERIZATION

The leptin receptor (OB-R) mediates the weight regulatory effects of the adipocyte secreted hormone leptin (OB). Previously we have shown that the long form of OB-R, expressed predominantly in the hypothalamus, can mediate ligand-induced activation of signal transducer and activator of transcription factors 1, 3, and 5 and stimulate transcription via interleukin-6 and hematopoietin receptor responsive gene elements. Here we report that deletion and tyrosine substitution mutagenesis of OB-R identifies two distinct regions of the intracellular domain important for signaling. In addition, granulocyte-colony stimulatory factor receptor/OB-R and OB-R/granulocyte-colony stimulatory factor receptor chimeras are signaling competent and provide evidence that aggregation of two OB-R intracellular domains is sufficient for ligand-induced receptor activation. However, signaling by full-length OB-R appears to be relatively resistant to dominant negative repression by signaling-incompetent OB-R, suggesting that mechanisms exist to permit signaling by the long form of OB-R even in the pretence of excess naturally occurring short form of OB-R.

ESM-1 Is a Novel Human Endothelial Cell-specific Molecule Expressed in Lung and Regulated by Cytokines

We here report the identification of a novel human endothelial cell-specific molecule (called ESM-1) cloned from a human umbilical vein endothelial cell (HUVEC) cDNA library. Constitutive ESM-1 gene expression (as demonstrated by Northern blot and reverse transcription-polymerase chain reaction analysis) was found in HUVECs but not in the other human cell lines tested. The cDNA sequence contains an open reading frame of 552 nucleotides and a 1398-nucleotide 3′-untranslated region including several domains involved in mRNA instability and five putative polyadenylation consensus sequences. The deduced 184-amino acid sequence defines a cysteine-rich protein with a functional NH2-terminal hydrophobic signal sequence. Searches in several data bases confirmed the unique identity of this sequence. A rabbit immune serum raised against the 14-kDa COOH-terminal peptide of ESM-1 immunoprecipitated a 20-kDa protein only in ESM-1-transfected COS cells. Immunoblotting and immunoprecipitation of HUVEC lysates revealed a specific 20-kDa band corresponding to ESM-1. In addition, constitutive ESM-1 gene expression was shown to be tissue-restricted to the human lung. Southern blot analysis suggests that a single gene encodes ESM-1. A time-dependent up-regulation of ESM-1 mRNA was seen after addition of tumor necrosis factor α (TNFα) or interleukin (IL)-1β but not with IL-4 or interferon γ (IFNγ) alone. In addition, when IFNγ was combined with TNFα, IFNγ inhibited the TNFα-induced increase of ESM-1 mRNA level. These data suggest that ESM-1 may have potent implications in the areas of vascular cell biology and human lung physiology.

The Novel Nucleoside Analog R1479 (4′-Azidocytidine) Is a Potent Inhibitor of NS5B-dependent RNA Synthesis and Hepatitis C Virus Replication in Cell Culture

Hepatitis C virus (HCV) polymerase activity is essential for HCV replication. Targeted screening of nucleoside analogs identified R1479 (4′-azidocytidine) as a specific inhibitor of HCV replication in the HCV subgenomic replicon system (IC50 = 1.28 μm) with similar potency compared with 2′-C-methylcytidine (IC50 = 1.13 μm). R1479 showed no effect on cell viability or proliferation of HCV replicon or Huh-7 cells at concentrations up to 2 mm. HCV replicon RNA could be fully cleared from replicon cells after prolonged incubation with R1479. The corresponding 5′-triphosphate derivative (R1479-TP) is a potent inhibitor of native HCV replicase isolated from replicon cells and of recombinant HCV polymerase (NS5B)-mediated RNA synthesis activity. R1479-TP inhibited RNA synthesis as a CTP-competitive inhibitor with a Ki of 40 nm. On an HCV RNA-derived template substrate (complementary internal ribosome entry site), R1479-TP showed similar potency of NS5B inhibition compared with 3′-dCTP. R1479-TP was incorporated into nascent RNA by HCV polymerase and reduced further elongation with similar efficiency compared with 3′-dCTP under the reaction conditions. The S282T point mutation in the coding sequence of NS5B confers resistance to inhibition by 2′-C-MeATP and other 2′-methyl-nucleotides. In contrast, the S282T mutation did not confer cross-resistance to R1479.

Antigenic drift between the haemagglutinin of the Hong Kong influenza strains A/Aichi/2/68 and A/Victoria/3/75

A DNA copy of the gene coding for the influenza A/Aichi/2/68 haemagglutinin protein was cloned in the plasmid pBR322 and the complete nucleotide sequence determined. Comparison of this primary structure and the deduced amino acid sequence with the haemagglutinin gene and protein of strains belonging to the same (H3) subtype and to different subtypes, of both human (H2) and avian (Hav1) origin, documents further at the molecular level the two independent modes of antigenic variation of the virus—drift and shift.

Complete structure of the hemagglutinin gene from the human influenza A/Victoria/3/75 (H3N2) strain as determined from cloned DNA

The complete sequence of a hemagglutinin (HA) gene of a recent human influenza A strain, A/Victoria/3/75, is 1768 nucleotides long and contains the information for 567 amino acids. It codes for a signal peptide of 16 amino acids, the HA1 chain of the mature hemagglutinin of 329 amino acids, a connecting region between HA1 and HA2 consisting of a single arginine residue and the HA2 portion of 221 amiino acids. The sequence is compared with the hemagglutinin of two members of other subtypes, the human H2 strain A/Jap/305/57 and the avian Hav1 strain A/FPV/Rostock/34, and with one of the same H3 subtype, A/Memphis/3/72. To align the HA1 chain of different major subtypes several deletions/insertions of single amino acids must be invoked, but two more extensive differences are found at both ends, one leading to an extension of the amino terminal sequence of HA1 and the other (four residues) occurring in the region processed away between HA1 and HA2. Comparison of the HA1 of two H3 strains suggests that drift probably depends on single base mutations, some of which change antigenic determinants. The HA2 region, which apparently is not involved in the immune response, is highly conserved even between different subtypes, and single base substitutions account for all the observed diversity. A hydrophobic segment of 24 residues is present in the same position close to the carboxyl terminus of HA2 in both Victoria and FPV, and presumably functions in implantation into the lipid bilayer. The many conserved features not only in HA2 but also in HA1 suggest a rather rigid architecture for the whole hemagglutinin molecule.

LIGAND-INDEPENDENT DIMERIZATION OF THE EXTRACELLULAR DOMAIN OF THE LEPTIN RECEPTOR AND DETERMINATION OF THE STOICHIOMETRY OF LEPTIN BINDING

The leptin receptor is a class I transmembrane protein with either a short or a long cytoplasmic domain. Using chemical cross-linking we have analyzed the binding of leptin to its receptor. Cross-linking of radiolabeled leptin to different isoforms of the leptin receptor expressed on COS-1 cells reveals leptin receptor monomer, homodimer, and oligomer complexes. Cotransfection of the long and short form of the leptin receptor did not provide any evidence for the formation of heterodimer complexes. Soluble forms consisting of either the entire extracellular domain or the two cytokine receptor homologous domains of the leptin receptor were purified to homogeneity from recombinant baculovirus-infected insect cells by leptin affinity chromatography. Gel filtration chromatography showed that these proteins exist in a dimeric form. Analysis of the complex formed between soluble leptin receptor and leptin by native polyacrylamide gel electrophoresis, and data obtained from the amino acid composition of the complex provide direct evidence that the extracellular domain of the leptin receptor binds leptin in a 1:1 ratio.

Complete structure of A/duck/Ukraine/63 influenza hemagglutinin gene: Animal virus as progenitor of human H3 Hong Kong 1968 influenza hemagglutinin

Abstract We have explored the possibility that an animal viral reservoir contained a direct ancestor gene for the H3 hemagglutinin type present in influenza A viruses in humans since 1968. For this purpose, the duck/Ukraine/1/63 hemagglutinin gene was cloned and sequenced. From the comparison of its complete primary structure with that of several human H3 hemagglutinins as well as those of an H2 and an H7 hemagglutinin, we conclude that the duck/Ukraine/63 hemagglutinin sequence fully corroborates its previous identification by immunological and other methods as belonging to the H3 subtype. Moreover, the duck/Ukraine/63 amino acid sequence is more closely related structurally and presumably antigenically to the human Aichi/68 hemagglutinin, which formed the beginning of the H3N2 pandemic in humans, than to that of Victoria/75, which has undergone an additional 7 year drift period in humans. This observation could best be explained by a common ancestor hemagglutinin gene for duck/Ukraine/63 and human Aichi/68. On the basis of silent, accumulated base changes, we estimate that the strain carrying this postulated common progenitor hemagglutinin gene was circulating in the period 1949–1953 in the animal reservoir. This relatively recent divergence, as well as the closer kinship between the duck/Ukraine/63 and the human Aichi/68 hemagglutinin, as compared with the later Victoria/75, strongly suggests that the influenza A virus of the H3N2 subtype circulating in the human population since 1968 has derived its hemagglutinin gene from a strain in the animal reservoir. Undoubtedly, this occurred by reassortment between previously present human H2N2 virus and this animal strain. These results provide support at the molecular level for the general idea that the wide variety of influenza viruses known to be present in animals can serve as a gene reservoir for human influenza A viruses.