Posttranslational modification of the glycosylation inhibiting factor (GIF) gene product generates bioactive GIF.
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.
Watarai
H
H
Pharmaceutical Research Laboratory, Kirin Brewery Company, Ltd., 3. Miyahara-cho, Takasaki 370-1295, Gunma, Japan.
Nozawa
R
R
Tokunaga
A
A
Yuyama
N
N
Tomas
M
M
Hinohara
A
A
Ishizaka
K
K
Ishii
Y
Y
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
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