Assignment of the inter- and intramolecular disulfide linkages in recombinant human macrophage colony stimulating factor using fast atom bombardment mass spectrometry.
The disulfide bridges in recombinant human macrophage colony stimulating factor (rhM-CSF), a 49-kDa homodimeric protein, were assigned. The 18 cysteines in the dimer form three intermolecular and two sets of three intramolecular disulfide bonds. The intermolecular disulfide bridges hold the dimer together and form symmetric bonds in which Cys31 and Cys157/Cys159 from one monomer unit are linked to the corresponding cysteines of the second monomer. The intramolecular disulfide bonds are located between Cys7-Cys90, Cys48-Cys139, and Cys102-Cys146, respectively. The resistance of native M-CSF to proteolytic cleavage was overcome by an initial chemical cleavage reaction using BrCN. The close proximity of four cysteines (Cys139, Cys146, Cys157, and Cys159) results in a tight core complex that makes the protein undigestable for most proteases. Digestion using endoprotease Asp-N resulted in cleavage at Asp156 near the C-terminal end of this region, thereby opening the complex structure.
Glocker
M O
MO
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331.
Arbogast
B
B
Schreurs
J
J
Deinzer
M L
ML
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016339:Spectrometry, Mass, Fast Atom Bombardment
D004220:Disulfides; D011994:Recombinant Proteins; D016173:Macrophage Colony-Stimulating Factor