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 Cys102-Cys146), and one interchain disulfide bond (Cys31-Cys31). To examine the role of the seven cysteine residues in CSF-1(256) we r eplaced each half-cystine by a serine using site-directed mutagenesis, and stably expressed the mutated genes in mouse NIH 3T3 cells. We sho wed that each of the seven cysteines of CSF-1(256) is essential for it s biological activity. Our data further show that substitution of Cys4 8 or Cys139 totally blocked dimer formation and cell surface expressio n of CSF-1(256), and that substitution of Cys102 and Cys146 severely i mpaired CSF-1 dimer formation and cell surface expression. In contrast , substitution of Cys7 or Cys90 affected CSF-1 dimer formation to a le sser degree but did not significantly affect cell surface expression o f CSF-1. Furthermore, disruption of the interchain disulfide bond led to efficient cell surface expression of monomeric CSF-1. All of the ce ll surface expressed mutant CSF-1 proteins, either dimeric or monomeri c, still underwent efficient ectodomain cleavage. The electrophoretic mobilities of the cleaved dimeric ectodomains of these mutant CSF-1 pr oteins 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. T hese data suggest that replacement of either of a pair of intrachain h alf-cystine residues results in similar conformational changes, and ma y provide a novel method for mapping intrachain disulfide bonds in dim eric proteins. (C) 1996 Academic Press, Inc.