High-affinity binding of bioactive glycosylation-inhibiting factor to antigen-primed T cells and natural killer cells

Sugie, Katsuji; Nakano, Tatsumi; Tomura, Takafumi; Takakura, Kenji; Mikayama, Toshifumi; Ishizaka, Kimishige

doi:10.1073/pnas.94.10.5278

Cited by 18 publications

(19 citation statements)

References 31 publications

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“…Purified C57A͞N106S was radiolabeled with 125 I by the method described (20). SDS͞PAGE and Immunoblotting.…”

Section: Methodsmentioning

confidence: 99%

“…Previous experiments have shown that bioactive GIF from Ts cells and bioactive derivatives of rhGIF bind to the receptors on Th hybridomas, activated T and B cells with high affinity, whereas inactive, cytosolic GIF and E. coli-derived wild-type rhGIF failed to do so (20,21). High affinity binding capacity of GIF molecules for the target cells was generated by replacement of Cys-57 with Ala, and of Asn-106 with Ser or by binding of 5-thio(2-nitrobenzoic acid) group to Cys-60 in the C57A molecules.…”

Section: Binding Capacity Of Cysteinylated Gif Derivatives To Target mentioning

confidence: 99%

“…The affinity of GIF and its derivatives for the high affinity receptor on the 12H5 cells were compared by their ability to inhibit the binding of 1 nM 125 I-labeled C57A͞N106S to the cells (20). Serial dilutions of a GIF sample to be tested were mixed with 125 I-labeled C57A͞N106S, and each mixture was added to a cell suspension in duplicate.…”

Section: Inhibition Of High Affinity Binding Of Gif Derivatives To Gimentioning

confidence: 99%

“…Serial dilutions of a GIF sample to be tested were mixed with 125 I-labeled C57A͞N106S, and each mixture was added to a cell suspension in duplicate. After incubation for 20 min at 37°C, cell-bound radioactivity was determined by the procedures described (20). Nonspecifically bound radioactivity was determined by the addition of 100-fold excess of unlabeled C57A͞ N106S to the system.…”

Section: Inhibition Of High Affinity Binding Of Gif Derivatives To Gimentioning

confidence: 99%

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

Watarai

Nozawa

Tokunaga

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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Glycosylation inhibiting factor (GIF) and macrophage migration inhibitory factor (MIF) share an identical structure gene. Here we unravel two steps of posttranslational modifications in GIF͞MIF 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 GIF͞MIF, 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 GIF͞MIF 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 ␣2-helix of GIF bound bioactive cysteinylated GIF but failed to bind wildtype GIF͞MIF. These results strongly suggest that cysteinylation of Cys-60 and consequent conformational changes in the GIF͞MIF molecules are responsible for the generation of GIF bioactivity.

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“…Purified C57A͞N106S was radiolabeled with 125 I by the method described (20). SDS͞PAGE and Immunoblotting.…”

Section: Methodsmentioning

confidence: 99%

Section: Binding Capacity Of Cysteinylated Gif Derivatives To Target mentioning

confidence: 99%

Section: Inhibition Of High Affinity Binding Of Gif Derivatives To Gimentioning

confidence: 99%

Section: Inhibition Of High Affinity Binding Of Gif Derivatives To Gimentioning

confidence: 99%

See 2 more Smart Citations

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

Watarai

Nozawa

Tokunaga

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…The gene product of GIF/MIF becomes cysteinylated at C60 in human suppressor T hybridoma cells (6), although the MIF nomenclature in the literature generally refers to the unmodified form of the cytokine. The modification is required for GIF/MIF (hereafter, GIF) to bind to target cells because in contrast to the modified GIF the unmodified form fails to show saturable binding to any cell types (6)(7)(8). The modified GIF seems to be uniquely immunoregulatory because injection of C60-modified recombinant GIF at the time of immunization with DNP-Ova suppressed the formation of anti-DNP IgE and IgG1, whereas unmodified GIF showed no such effect (9).…”

mentioning

confidence: 99%