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A study of intermediates involved in the folding pathway for recombinant human macrophage colony-stimulating factor
J A Wilkins1, J Cone, Z I Randhawa
1Department of Protein Chemistry, Otsuka America Pharmaceutical Co., Rockville, Maryland 20850.
Abstract:
The folding pathway for a 150-amino acid recombinant form of the dimeric cytokine human macrophage colony-stimulating factor (M-CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a non-native dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer. The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0. The blocked intermediates are stable under conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges. Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M-CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell-based assay. The results presented here strongly suggest that M-CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates.
Insights
Human macrophage colony-stimulating factor (M-CSF) folding occurs through distinct pathways. Intermediates reveal rapid dimer formation followed by slow progression to the native dimer via monomeric or dimeric routes.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Folding
Background:
- Human macrophage colony-stimulating factor (M-CSF) is a dimeric cytokine crucial for immune cell development.
- Understanding the protein folding pathway is essential for elucidating its biological function and potential therapeutic applications.
Purpose of the Study:
- To investigate the folding pathway of a recombinant human M-CSF homodimer.
- To characterize the structural intermediates involved in the M-CSF folding process.
Main Methods:
- Utilized iodoacetamide to block folding intermediates.
- Employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for intermediate stability analysis.
- Applied peptide mapping and electrospray ionization mass spectrometry for structural characterization.
Main Results:
- Identified rapid formation of non-native dimeric intermediates followed by slow progression to the native dimer.
- Demonstrated that folding can proceed through both monomeric and dimeric intermediates.
- A folded monomeric M-CSF species with three disulfide bridges retained partial biological activity.
Conclusions:
- Human M-CSF folding can occur via at least two distinct pathways: one involving monomeric intermediates and another exclusively involving dimeric intermediates.
- The existence of biologically active monomeric intermediates suggests complex folding dynamics.