Posttranslational modification of the glycosylation inhibiting factor (GIF) gene product generates bioactive GIF

Glycosylation inhibiting factor (GIF) and macrophage migration inhibitory f actor (MIF) share an identical structure gene. Here we unravel two steps of posttranslational modifications in GIF/MIF molecules in human suppressor T (Ts) cell hybridomas. Peptide mapping and MS analysis of the affinity-puri fied GIF from the Ts cells revealed that one modification is cysteinylation at Cys-60, and the other is phosphorylation at Ser-91, Cysteinylated GIF, but not the wild-type GIF/MIF, possessed immunosuppressive effects on the i n vitro IgE antibody response and had high affinity for GIF receptors on th e T helper hybridoma cells. In vitro treatment of wild-type recombinant hum an GIF/MIF with cystine resulted in preferential cysteinylation of Cys-60 i n the molecules. The cysteinylated recombinant human GIF and the Ts hybrido ma-derived cysteinylated CIF were comparable both in the affinity for the r eceptors and in the immunosuppressive activity. Polyclonal antibodies speci fic for a stretch of the amino acid sequence in alpha2-helix of GIF bound b ioactive cysteinylated GIF but failed to bind wildtype GIF/MIF, These resul ts strongly suggest that cysteinylation of Cys-60 and consequent conformati onal changes in the GIF/MIF molecules are responsible for the generation of GIF bioactivity.