title: Posttranslational modification of the glycosylation inhibiting factor (GIF) gene product generates bioactive GIF.
abstract: Glycosylation inhibiting factor (GIF) and macrophage migration inhibitory factor (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-purified 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 in vitro IgE antibody response and had high affinity for GIF receptors on the T helper hybridoma cells. In vitro treatment of wild-type recombinant human GIF/MIF with cystine resulted in preferential cysteinylation of Cys-60 in the molecules. The cysteinylated recombinant human GIF and the Ts hybridoma-derived cysteinylated GIF were comparable both in the affinity for the receptors and in the immunosuppressive activity. Polyclonal antibodies specific for a stretch of the amino acid sequence in alpha2-helix of GIF bound bioactive cysteinylated GIF but failed to bind wild-type GIF/MIF. These results strongly suggest that cysteinylation of Cys-60 and consequent conformational changes in the GIF/MIF molecules are responsible for the generation of GIF bioactivity.
authors.affiliation: Pharmaceutical Research Laboratory, Kirin Brewery Company, Ltd., 3. Miyahara-cho, Takasaki 370-1295, Gunma, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Watarai
authors.forename: R
authors.initials: R
authors.lastname: Nozawa
authors.forename: A
authors.initials: A
authors.lastname: Tokunaga
authors.forename: N
authors.initials: N
authors.lastname: Yuyama
authors.forename: M
authors.initials: M
authors.lastname: Tomas
authors.forename: A
authors.initials: A
authors.lastname: Hinohara
authors.forename: K
authors.initials: K
authors.lastname: Ishizaka
authors.forename: Y
authors.initials: Y
authors.lastname: Ishii
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D006825:Hybridomas; D007073:Immunoglobulin E / Q000276:immunology; D008222:Lymphokines / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008822:Mice, Transgenic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D029584:Prostatic Secretory Proteins; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013154:Spleen / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*; D050378:T-Lymphocytes, Regulatory / Q000276:immunology
chemical_list: D008222:Lymphokines; D029584:Prostatic Secretory Proteins; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011994:Recombinant Proteins; C043076:beta-microseminoprotein; C013059:immunoglobulin-binding factors; D007073:Immunoglobulin E; D003545:Cysteine