Synthesis, cytotoxic properties and effects on early and late gene induction of a chimeric diphtheria toxin-leukemia-inhibitory factor protein.
Leukemia-inhibitory factor (LIF) is a neuropoietin able to regulate the differentiation and the survival of many cell types, which include some neuronal populations. The present study describes the genetic construction, expression, purification and properties of a diphtheria-toxin-related LIF gene fusion in which the native receptor-binding domain of diphtheria toxin was replaced with a gene encoding human LIF. The fusion protein expressed from the chimeric tox gene was designated DT-(1-389)-LIF-(2-184)-peptide. This fusion protein has a deduced molecular mass of 65980 Da and is formed by fusion of the first 389 amino acids of diphtheria toxin to amino acids 2-184 of mature human LIF, using a linker of 34 amino acids that includes six consecutive histidine residues. The latter span allows for single-step purification of the fusion protein by Ni(2+)-resin affinity chromatography. This linker provides a high degree of flexibility between the diphtheria toxin and LIF domains, thereby permitting aggregation-free refolding of the chimeric protein while bound to the affinity column. Both LIF and DT-(1-389)-LIF-(2-184)-peptide induced the phosphorylation of CLIP1 and CLIP2 in LIF-responsive neuroblastoma SH-N-BE cells. DT-(1-389)-LIF-(2-184)-peptide was selectively cytotoxic for cultured neuroblastoma cells bearing the LIF receptor, and for sympathetic neurons. The cytotoxic action of DT-(1-389)-LIF-(2-184)-peptide, like that of native diphtheria toxin, required receptor-mediated endocytosis, passage through an acidic compartment, and delivery of an ADP-ribosyltransferase to the cytosol of target cells. The latter point was confirmed by the fact that, while both LIF and DT-(1-389)-LIF-(2-184)-peptide increased c-fos mRNA expression in SH-N-BE cells, only LIF induced proenkephalin and c-fos promoter activities in cells transiently transfected with c-fos-chloramphenicol acetyltransferase and proenkephalin-chloramphenicol acetyltransferase fusion genes. Mutational analysis suggested that the C-terminal helix (helix D) of human LIF may, in part, constitute or contribute to the active site for LIF receptor binding and cell activation. The cytotoxic properties of DT-(1-389)-LIF-(2-184)-peptide may be useful in selectively depleting neuronal and immune cell populations that express the LIF beta receptor.
Department of Biological Chemistry, CRIBI Biotechnology Center, University of Padova, Italy.
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Skaper
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D004167:Diphtheria Toxin / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D004745:Enkephalins / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D016762:Genes, fos / Q000235:genetics; D006131:Growth Inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; D008222:Lymphokines / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D010766:Phosphorylation; D011498:Protein Precursors / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology
D017931:DNA Primers; D004167:Diphtheria Toxin; D004745:Enkephalins; D006131:Growth Inhibitors; D015850:Interleukin-6; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; D008222:Lymphokines; D010446:Peptide Fragments; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C029992:proenkephalin