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Purification and bioactivity of the soluble form of the interleukin-6 receptor expressed in a baculovirus expression system
CYTOKINE,
556 I Abstructs
P4 October 2: Lymphokine/Cytokine A102
Vol. 6, No. 5 (September
1994: 539-582)
Receptors A105
Purification and Bioactivity of the Soluble Form of the Interleukin-6 Receptor Expressed in a Baculovirus Expression System. Vincent Thibault and Jack Gauldie, Department of Pathology, McMaster University, Hamilton, Ontario, Canada LBN 325 IL6 is a pleiotropic cytokine involved in the regulation of both immunity and inflammation. It mediates its effects through a multimeric receptor consisting of a binding protein (gp80) and a signal transducer (gp130). A soluble form of gp80 (slL-6R) or gp54 is found released from the surface of cells and appears to possess IL-6 agonist activity. The cDNA encoding a fusion protein consisting of a hexahistidine peptide linked to the rat sIL-6R (extracellular domain) through
a thrombin
cleavage
site has been cloned into a plasmid
for
the expression of the protein in insect cells using a baculovirus expression system. The presence of the signal sequence for a highly secreted protein upstream of the foreign gene induces the secretion of the rat sIL-6R in the culture media. The supernatant of infected cells is applied on a Ni-chelate affinity column and elu@d with an imidazole gradient. Bioactivity of the purified protein has been assessed both in v&o on B9 and H35 cells for proliferation and acute phase protein production respectively, and in viva to observe the effects of an increase in circulating sIL-6R. Antibodies have been raised against the recombinant material. A third approach is being developed with an adenovirus vector to deliver the recombinant soluble protein in viva. (Supported by MRC Canada and Rhone-Poulenc Rarer)
THE HIGH AFFINITY IL-6 RECEPTORS IS A HEXAMERIC COMPLEX CONSISTING OF TWO MOLECULES OF EACH IL-6, IL-6 RECEPTOR AND GP-130. L.D. Ward, G.J. Howl&t+, K. YasukawaC, A. Hammacher, R.L. Moritz and R.J. Simpson. Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research/The Walter and Eliza Hall Institute of Australia 3050: +Biochemistry Medical Research, Vie., Dept., Melbourne University, Australia 3050; #TOSOH Corporation, Ayase-shi Kanagawa 252, Japan. The high affinity human IL-6 receptor-complex consists of IL-6 and two transmembrane proteins-the IL-6 receptor (IL&R), which binds IL-6 with low affinity, and gp-130, which confers high affinity binding to the IL-6/IL-6R complex. To investigate the stoichiometry of binding in the low affinity and high affinity ternary receptorcomplexes, we have used the soluble domains of IL-6R and gp-130 (sIL-6R and sgp-130, respectively). The interaction of sIL-6R with 11-6 (low affinity complex) and sIL-6R/IL-6 with immobilised sgp-130 was characterised using a biosensor employing surface plasmon resonance detection. Usina a combination of SEC and analytical ultracentrifugation analysis, one molecule of IL-6 was shown to bind one molecule of sIL-6R in the low affinity receptor-complex, while the ternary comolex was shown to consist of two molecules of eachcomoonent.
Al 03
A106 GENERATION OF IL-6 RECEPTOR ANTAGONIST AND SUPERANTAGONIST BY MOLECULAR MODELLING AND SITE-DIRECTED MUTAGENESIS. C.Toniatti,A.Lahm,R.Savino,A.Cabibbo,L.Ciapponi,A.Demartis,G.Paoness a,A.L.Salvati,E. Sporeno,S. Altamura and G.Ciliberto. I.R.B.M. P. Anseletti, Rome, Italy. IL-6 exerts its-biological activity by Interacting with a complex receptor system constituted by two proteins: the a ligand-binding subunit, named IL&%, and gp130, referred to as the p signal-transducing chain. IL-6 first interacts with IL-6Ra: then. this low affinitv cvtokine-receotor comolex directly associate with the &racellular regi& Af gp130, thbs generating high-affinity binding sites responsible for signal transduction. We have used the X-ray structure of the human growth hormone (GH) receptor complex, in which two identical receptor subunits are brought together by a single GH molecule, as a general scaffold for modeling the human IL-6/IL6RcJql.1130 complex. The model predicts two distinct receptor binding sites locat& on oppdsite sides of the‘lL-6 molecule and the i&face between IL-6Ra and ao130. Residues in the three Proteins Dostulated to ConStitute the various &tact surfaces (IL-6/IL-6Ru, ‘IL-6/gpl$O. IL-6Rdgpl30) were subjected to site-direcetd mutagenesis and the resulting mutants were tested for their biological and biochemical properties. As predicted by the model, three different set of mutants were obtained: 1) IL-6 mutants in helix-D and AB-loop with increased affinity towards IL-6Ra 2) IL-6 mutants in helix A and C, selectively impaired in their ability to recruit gp130, which display a strongly dlminished biological activity and can act as IL-6 antagonist on human cells: 3) soluble IL-6Ra mutants that have a full IL-6 binding activity, reduced ability to interact with gp130 and display antagonistic instead of agonistic properties on human cells.
TWO DISTINCT SIGNALING REGIONS OF HUMAN GM-CSF RECEPTOR Sumiko Watanabel, Akihiko Mutol, Tohru Itol, Atsushi Miyajima2, Inst.Med.Sci., Takashi Yokotal, Ken-ichi Arail, 1Dept.MoLDev.BioL, Univ.Tokyo, *Dept.Mol.Biol., DNAX Res.Inst. hGM-CSFR consist of u and p subunits and GM-CSF induces activation of early response genes, protein tyrosine phosphorylation and proliferation in hematopoietic cells. Within the cytoplasmic region of the 0 subunit, we have identified two distinct regions, A which is required for induction of c-fos, c-jun, egr mRNAs and B for induction of clnD2, CDK, c-myc mRNAs and cell proliferation. Signaling from region A, the membrane proximal region including boxl, is sensitive to tyrosine kinase inhibitors herbimvcin and eenistein. In contrast. sianaline from region B, the membrane &al regi&, is insensitive to he;b&ycin&d genistein. More datailed analyses of p subunit mutants indicated that box1 is required for both signaling from A and B, and Tyr 577 is indespensable for signaling from B. These results indicated that different signals were generated by distinct regions of the p subunit and tyrosine kinases are differentially involved in multiple signal transduction cascade downstream of hGMR. The studies with chimeric receptor between CLand p subunits indicated homodimer of intracellular domain is sufficient to transduce signals of both A and B indicating intracellular domain of the a subunit connibuting to induce dimerization of the 0 subunit.
A104
A107 SCKEENlNGFOKPKOTElNSTHATRlNDTOTHElMKACELLULAKDOMAlNS OFTHE TWOTUMOK NECROSISFAnOK KECEPTOKSCINF-K) REVEALED EFFECTIVESELF ASSOCIATION OF THE 1’55 TNF-R “DEATH DOMAIN”. WHICH CAN TKlGGER SIGNALING. David Wallach Mark Boldin. Evgcni E. Varlolamccv. Yacek Bigda, Jacques. H. Camonis and Igor Mett. Department ol Membrane Rcscarch and Bionbvnlcs, Wclzlnann InsLilole or Science.
SOLUBLE IL-4 RECEPTOR (sIL-4R) MODULATES THE EFFECLS OF IL-4 ON HIV- 1 REPLICATION IN HUMAN MONOCYTES. N.D. Weber, K.A. Weih and K.A. Clouse. Division of Cytokine Biology, CBER, FDA, Bethesda, MD 20892 Interleukin 4 (IL-4) has been reported to either enhance or inhibit replication of human immunodeficiency virus trpe 1 (HIV-l) in human monocytes in vitro, depending on their state of differentiation. In this study, we tested a purified form of human sIL-4R for its ability to modulate these effects. Elutriated human monocytes were first cultured for 0 to 6 days in medium containing recombinant human IL4 (0 to 10 n&l) and/or sIL-4R (0 to 1 @ml), then infected with HIV-l and maintained in culture in the presence and absence of IL-4 and/or slL-4R. Low concentrations of sIL-4R (0.1 to 10 @ml) added post infection increased the IL-4 mediated inhibition of virus replication in differentiated monocytes (macrophages), as determined by reverse transcriptase assay. However, when sIL4R was present in loo-fold excess of IL-4, the inhibitory effects were reversed. The sIL4R alone had no effect on HIV-l replication. These results suggest that sIL-4R augments the biological effects of IL-4 when used at concentrations equivalent to IL-4, but at a high molar excess functions as an antagonist.
556 I Abstructs
P4 October 2: Lymphokine/Cytokine A102
Vol. 6, No. 5 (September
1994: 539-582)
Receptors A105
Purification and Bioactivity of the Soluble Form of the Interleukin-6 Receptor Expressed in a Baculovirus Expression System. Vincent Thibault and Jack Gauldie, Department of Pathology, McMaster University, Hamilton, Ontario, Canada LBN 325 IL6 is a pleiotropic cytokine involved in the regulation of both immunity and inflammation. It mediates its effects through a multimeric receptor consisting of a binding protein (gp80) and a signal transducer (gp130). A soluble form of gp80 (slL-6R) or gp54 is found released from the surface of cells and appears to possess IL-6 agonist activity. The cDNA encoding a fusion protein consisting of a hexahistidine peptide linked to the rat sIL-6R (extracellular domain) through
a thrombin
cleavage
site has been cloned into a plasmid
for
the expression of the protein in insect cells using a baculovirus expression system. The presence of the signal sequence for a highly secreted protein upstream of the foreign gene induces the secretion of the rat sIL-6R in the culture media. The supernatant of infected cells is applied on a Ni-chelate affinity column and elu@d with an imidazole gradient. Bioactivity of the purified protein has been assessed both in v&o on B9 and H35 cells for proliferation and acute phase protein production respectively, and in viva to observe the effects of an increase in circulating sIL-6R. Antibodies have been raised against the recombinant material. A third approach is being developed with an adenovirus vector to deliver the recombinant soluble protein in viva. (Supported by MRC Canada and Rhone-Poulenc Rarer)
THE HIGH AFFINITY IL-6 RECEPTORS IS A HEXAMERIC COMPLEX CONSISTING OF TWO MOLECULES OF EACH IL-6, IL-6 RECEPTOR AND GP-130. L.D. Ward, G.J. Howl&t+, K. YasukawaC, A. Hammacher, R.L. Moritz and R.J. Simpson. Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research/The Walter and Eliza Hall Institute of Australia 3050: +Biochemistry Medical Research, Vie., Dept., Melbourne University, Australia 3050; #TOSOH Corporation, Ayase-shi Kanagawa 252, Japan. The high affinity human IL-6 receptor-complex consists of IL-6 and two transmembrane proteins-the IL-6 receptor (IL&R), which binds IL-6 with low affinity, and gp-130, which confers high affinity binding to the IL-6/IL-6R complex. To investigate the stoichiometry of binding in the low affinity and high affinity ternary receptorcomplexes, we have used the soluble domains of IL-6R and gp-130 (sIL-6R and sgp-130, respectively). The interaction of sIL-6R with 11-6 (low affinity complex) and sIL-6R/IL-6 with immobilised sgp-130 was characterised using a biosensor employing surface plasmon resonance detection. Usina a combination of SEC and analytical ultracentrifugation analysis, one molecule of IL-6 was shown to bind one molecule of sIL-6R in the low affinity receptor-complex, while the ternary comolex was shown to consist of two molecules of eachcomoonent.
Al 03
A106 GENERATION OF IL-6 RECEPTOR ANTAGONIST AND SUPERANTAGONIST BY MOLECULAR MODELLING AND SITE-DIRECTED MUTAGENESIS. C.Toniatti,A.Lahm,R.Savino,A.Cabibbo,L.Ciapponi,A.Demartis,G.Paoness a,A.L.Salvati,E. Sporeno,S. Altamura and G.Ciliberto. I.R.B.M. P. Anseletti, Rome, Italy. IL-6 exerts its-biological activity by Interacting with a complex receptor system constituted by two proteins: the a ligand-binding subunit, named IL&%, and gp130, referred to as the p signal-transducing chain. IL-6 first interacts with IL-6Ra: then. this low affinitv cvtokine-receotor comolex directly associate with the &racellular regi& Af gp130, thbs generating high-affinity binding sites responsible for signal transduction. We have used the X-ray structure of the human growth hormone (GH) receptor complex, in which two identical receptor subunits are brought together by a single GH molecule, as a general scaffold for modeling the human IL-6/IL6RcJql.1130 complex. The model predicts two distinct receptor binding sites locat& on oppdsite sides of the‘lL-6 molecule and the i&face between IL-6Ra and ao130. Residues in the three Proteins Dostulated to ConStitute the various &tact surfaces (IL-6/IL-6Ru, ‘IL-6/gpl$O. IL-6Rdgpl30) were subjected to site-direcetd mutagenesis and the resulting mutants were tested for their biological and biochemical properties. As predicted by the model, three different set of mutants were obtained: 1) IL-6 mutants in helix-D and AB-loop with increased affinity towards IL-6Ra 2) IL-6 mutants in helix A and C, selectively impaired in their ability to recruit gp130, which display a strongly dlminished biological activity and can act as IL-6 antagonist on human cells: 3) soluble IL-6Ra mutants that have a full IL-6 binding activity, reduced ability to interact with gp130 and display antagonistic instead of agonistic properties on human cells.
TWO DISTINCT SIGNALING REGIONS OF HUMAN GM-CSF RECEPTOR Sumiko Watanabel, Akihiko Mutol, Tohru Itol, Atsushi Miyajima2, Inst.Med.Sci., Takashi Yokotal, Ken-ichi Arail, 1Dept.MoLDev.BioL, Univ.Tokyo, *Dept.Mol.Biol., DNAX Res.Inst. hGM-CSFR consist of u and p subunits and GM-CSF induces activation of early response genes, protein tyrosine phosphorylation and proliferation in hematopoietic cells. Within the cytoplasmic region of the 0 subunit, we have identified two distinct regions, A which is required for induction of c-fos, c-jun, egr mRNAs and B for induction of clnD2, CDK, c-myc mRNAs and cell proliferation. Signaling from region A, the membrane proximal region including boxl, is sensitive to tyrosine kinase inhibitors herbimvcin and eenistein. In contrast. sianaline from region B, the membrane &al regi&, is insensitive to he;b&ycin&d genistein. More datailed analyses of p subunit mutants indicated that box1 is required for both signaling from A and B, and Tyr 577 is indespensable for signaling from B. These results indicated that different signals were generated by distinct regions of the p subunit and tyrosine kinases are differentially involved in multiple signal transduction cascade downstream of hGMR. The studies with chimeric receptor between CLand p subunits indicated homodimer of intracellular domain is sufficient to transduce signals of both A and B indicating intracellular domain of the a subunit connibuting to induce dimerization of the 0 subunit.
A104
A107 SCKEENlNGFOKPKOTElNSTHATRlNDTOTHElMKACELLULAKDOMAlNS OFTHE TWOTUMOK NECROSISFAnOK KECEPTOKSCINF-K) REVEALED EFFECTIVESELF ASSOCIATION OF THE 1’55 TNF-R “DEATH DOMAIN”. WHICH CAN TKlGGER SIGNALING. David Wallach Mark Boldin. Evgcni E. Varlolamccv. Yacek Bigda, Jacques. H. Camonis and Igor Mett. Department ol Membrane Rcscarch and Bionbvnlcs, Wclzlnann InsLilole or Science.
SOLUBLE IL-4 RECEPTOR (sIL-4R) MODULATES THE EFFECLS OF IL-4 ON HIV- 1 REPLICATION IN HUMAN MONOCYTES. N.D. Weber, K.A. Weih and K.A. Clouse. Division of Cytokine Biology, CBER, FDA, Bethesda, MD 20892 Interleukin 4 (IL-4) has been reported to either enhance or inhibit replication of human immunodeficiency virus trpe 1 (HIV-l) in human monocytes in vitro, depending on their state of differentiation. In this study, we tested a purified form of human sIL-4R for its ability to modulate these effects. Elutriated human monocytes were first cultured for 0 to 6 days in medium containing recombinant human IL4 (0 to 10 n&l) and/or sIL-4R (0 to 1 @ml), then infected with HIV-l and maintained in culture in the presence and absence of IL-4 and/or slL-4R. Low concentrations of sIL-4R (0.1 to 10 @ml) added post infection increased the IL-4 mediated inhibition of virus replication in differentiated monocytes (macrophages), as determined by reverse transcriptase assay. However, when sIL4R was present in loo-fold excess of IL-4, the inhibitory effects were reversed. The sIL4R alone had no effect on HIV-l replication. These results suggest that sIL-4R augments the biological effects of IL-4 when used at concentrations equivalent to IL-4, but at a high molar excess functions as an antagonist.