Structure-function studies on human macrophage colony-stimulating factor (M-CSF).
M-CSF (CSF-1) can be produced in a variety of structural forms that may affect function in vivo. Truncated, nonglycosylated forms of recombinant M-CSF (rM-CSF) from E. coli have been refolded in vitro in high yield and shown to be functionally equivalent in vitro to glycosylated rM-CSF secreted from mammalian cells. An N-terminal domain of 149 amino acids is produced by all of the known M-CSF mRNA splice variants and is the region responsible for bioactivity observed in vitro. Heterodimeric rM-CSFs from different splice variants containing this domain were produced in pure form by refolding in vitro, and are fully active, but have yet to be observed in vivo. The circulating half-life of truncated M-CSF forms injected intravenously into rats increased with the MW of the M-CSF used. Large increases in half-life in vivo were observed following chemical addition of a single molecule of 10 kD polyethylene glycol to rM-CSF in vitro. The crystal structure of rM-CSF revealed that M-CSF is a member of a family of molecules related by having a distinctive four-helical-bundle structural core. Site-directed mutagenesis showed that residues in or near helix A and helix C are involved in receptor binding, as reflected by decreased bioactivity and receptor binding of certain mutants. A soluble form of the M-CSF receptor, c-fms, was produced in a baculovirus/Sf9 expression system and purified to homogeneity. The MW of rM-CSF saturated with this soluble receptor was determined by molecular sieve chromatography and light scattering. Each dimeric M-CSF molecule appears to bind two soluble receptor molecules in vitro, supporting the observation that M-CSF signaling is linked to receptor dimerization.
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Chiron Corporation, Emeryville, California 94608-2916, USA.
D000818:Animals; D018360:Crystallography, X-Ray; D006207:Half-Life; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000493:pharmacokinetics / Q000502:physiology; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000493:pharmacokinetics; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D016173:Macrophage Colony-Stimulating Factor; D016186:Receptor, Macrophage Colony-Stimulating Factor