Synthesis and cytotoxic profile of a diphtheria toxin-neurotrophin-4 chimera.
A diphtheria toxin-neurotrophin-4/5 (NT-4/5) chimera (DAB389-NT4), in which the native receptor binding domain of diphtheria toxin was replaced with a synthetic gene encoding rat NT-4/5, was expressed, refolded, and purified. This fusion toxin has a deduced molecular mass of 60,163 and is formed by joining the first 389 amino acids of diptheria toxin to amino acids 1-130 of mature rat NT-4/5, using an NH2-terminal bridge of 33 additional amino acids including six consecutive histidines. Neural cell types expressing only p75LNGFR or p75LNGFR and full-length or truncated TrkB were used to evaluate the cytotoxic efficacy of DAB389-NT4. The fusion toxin produced a concentration-dependent killing of all cell populations, with LC50 values that largely reflected the known NT-4/5 binding affinities for these receptor proteins. Mean LC50 values ranged from 2,960 pM in p75LNGFR-expressing neuro-2a neuroblastoma cells to 1,075 and 70 pM, respectively, in hippocampal astrocytes (p75LNGFR+/truncated TrkB+) and cerebellar granule cells (p75LNGFR+/TrkB+). The LC50 for DAB389-NT4 in receptor-negative 3T3 fibroblasts was 20 nM. NT-4/5 and brain-derived neurotrophic factor but not ciliary neurotrophic factor added in excess neutralized DAB389-NT4 cytotoxicity. NT-4/5, however, did not reduce the cytotoxicity of intact diphtheria toxin.
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Department of Biological Chemistry, CRIBI, University of Padova, Italy.
Skaper
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D016475:3T3 Cells / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular; D003603:Cytotoxins / Q000494:pharmacology; D004167:Diphtheria Toxin / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression / Q000502:physiology; D051379:Mice; D009414:Nerve Growth Factors / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D009447:Neuroblastoma; D009474:Neurons / Q000737:chemistry / Q000187:drug effects; D018696:Neuroprotective Agents / Q000737:chemistry* / Q000494:pharmacology; D009620:Nodose Ganglion / Q000166:cytology; D017434:Protein Structure, Tertiary; D051381:Rats; D020801:Receptor, Ciliary Neurotrophic Factor; D011956:Receptors, Cell Surface / Q000378:metabolism; D017475:Receptors, Nerve Growth Factor / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology
D003603:Cytotoxins; D004167:Diphtheria Toxin; D009414:Nerve Growth Factors; D018696:Neuroprotective Agents; D020801:Receptor, Ciliary Neurotrophic Factor; D011956:Receptors, Cell Surface; D017475:Receptors, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; C073922:neurotrophin 4