The role of individual cysteine residues in the processing, structure, and function of human macrophage colony-stimulating factor.
The shortest form of human macrophage colony-stimulating factor (M-CSF alpha, CSF-1(256) is expressed on the cell surface as a homodimeric type I transmembrane glycoprotein. The seven cysteine residues in CSF-1(256) form three intrachain disulfide bonds (Cys7-Cys90, Cys48-Cys139, and Cys 102-Cys146), and one interchain disulfide bond (Cys31-Cys31). To examine the role of the seven cysteine residues in CSF-1(256), we replaced each half-cystine by a serine using site-directed mutagenesis, and stably expressed the mutated genes in mouse NIH 3T3 cells. We showed that each of the seven cysteines of CSF-1(256) is essential for its biological activity. Our data further show that substitution of Cys48 or Cys139 totally blocked dimer formation and cell surface expression of CSF-1(256), and that substitution of Cys102 and Cys146 severely impaired CSF-1 dimer formation and cell surface expression. In contrast, substitution of Cys7 or Cys90 affected CSF-1 dimer formation to a lesser degree but did not significantly affect cell surface expression of CSF-1. Furthermore, disruption of the interchain disulfide bond led to efficient cell surface expression of monomeric CSF-1. All of the cell surface expressed mutant CSF-1 proteins, either dimeric or monomeric, still underwent efficient ectodomain cleavage. The electrophoretic mobilities of the cleaved dimeric ectodomains of these mutant CSF-1 proteins on SDS-PAGE exhibited distinctly different patterns as compared with the wild type. Substitution of either Cys7 or Cys90 produced the same shift, while substitution of either Cys102 or Cys146 resulted in a shift distinct from that caused by substitution of Cys7 or Cys90. These data suggest that replacement of either of a pair of intrachain half-cystine residues results in similar conformational changes, and may provide a novel method for mapping intrachain disulfide bonds in dimeric proteins.
Deng
P
P
Department of Pathology, Childrens Hospital of Los Angeles, USA.
Wang
Y L
YL
Pattengale
P K
PK
Rettenmier
C W
CW
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003545:Cysteine; D019281:Dimerization; D006801:Humans; D015151:Immunoblotting; D016173:Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000737:chemistry* / Q000494:pharmacology; D008264:Macrophages; D051379:Mice; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D012694:Serine; D014162:Transfection
D000906:Antibodies; D011994:Recombinant Proteins; D012694:Serine; D016173:Macrophage Colony-Stimulating Factor; D003545:Cysteine