Expression of a fusion protein of human ciliary neurotrophic factor and soluble CNTF-receptor and identification of its activity.
Ciliary neurotrophic factor (CNTF) has pleiotropic actions on many neuronal populations as well as on glia. Signal transduction by CNTF requires that it bind first to CNTF-R, permitting the recruitment of gp130 and LIF-R, forming a tripartite receptor complex. Cells that only express gp130 and LIF-R, but not CNTF-R are refractory to stimulation by CNTF. On many target cells CNTF only acts in the presence of its specific agonistic soluble receptors. We engineered a soluble fusion protein by linking the COOH-terminus of sCNTF-R to the NH2-terminus of CNTF. Recombinant CNTF/sCNTF-R fusion protein (Hyper-CNTF) was successfully expressed in COS-7 cells. The apparent molecular mass of the Hyper-CNTF protein was estimated from western blots to be 75 kDa. Proliferation assays of transfected BAF/3 cells in response to CNTF and Hyper-CNTF were used to verify the activity of the cytokines. The proliferative results confirmed that CNTF required homodimerization of the gp130, CNTF-R and LIF-R receptor subunit whereas Hyper-CNTF required heterodimerization of the gp130 and LIF-R receptor subunit. We concluded that the fusion protein Hyper-CNTF had superagonistic activity on target cells expressing gp130 and LIF-R, but lacking membrane-bound CNTF-R.
Sun
Yi
Y
College of Life Sciences, Zhejiang University, Hangzhou 310027, China. sun_yi@mail.hz.zj.cn
Pia
März
M
Uwe
Otten
O
Ge
Ji-Guang
JG
Stefan
Rose-John
RJ
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D019556:COS Cells / Q000166:cytology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002522:Chlorocebus aethiops; D020934:Ciliary Neurotrophic Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D050822:Cytokine Receptor gp130; D016207:Cytokines / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008970:Molecular Weight; D015202:Protein Engineering / Q000379:methods; D020801:Receptor, Ciliary Neurotrophic Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018121:Receptors, Cytokine / Q000378:metabolism*; D053606:Receptors, OSM-LIF; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D012995:Solubility
D015703:Antigens, CD; D020934:Ciliary Neurotrophic Factor; D016207:Cytokines; C494199:IL6ST protein, human; C507840:LIFR protein, human; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D008562:Membrane Glycoproteins; D020801:Receptor, Ciliary Neurotrophic Factor; D018121:Receptors, Cytokine; D053606:Receptors, OSM-LIF; D011993:Recombinant Fusion Proteins; D050822:Cytokine Receptor gp130