Immunosuppressive activities of recombinant glycosylation-inhibiting factor mutants

We have shown previously that glycosylation-inhibiting factor (GIF) in cult ure supernatants of suppressor T cell (Ts) hybridomas had bioactivity, whil e the same cells contained a substantial quantity of inactive GIF in cytoso l, Mass-spectrometric analysis of GIF in the culture supernatant and cytoso l of a Ts hybridoma provided direct evidence that GIF protein was posttrans lationally modified in the Ts cells, and that the GIF bioactivity is associ ated with the posttranslationally modified species. Assuming that conformat ional changes induced by the posttranslational modifications are responsibl e for generation of bioactivity, we constructed cysteine mutants of human r GIF (rhGIF) in which cysteine at position 57, 60, or 81 was replaced with A la, and the mutants were expressed in Escherichia coli. Replacement of Cys( 57) or Cys(60) with Ala resulted in generation of bioactivity, while replac ement of Cys(81) with Ala failed to do so. It was also found that replaceme nt of Cys(57) with Ala and carboxymethylation of a sulfhydryl group in Cys( 60) synergistically increased the GIF bioactivity of the GIF derivatives. A mutated GIF protein, in which Cys(57) and Asn(106) in the rhGIF were repla ced with Ala and Ser, respectively, had immunosuppressive effects on the Ig E and IgG1 Ab responses of BDF1 mice to DNP-OVA, while wild-type rhGIF did not. Evidence was obtained that the mutated GIF suppressed Ag priming of Th cells for the Ab responses and proliferative response.