Immunosuppressive activities of recombinant glycosylation-inhibiting factor mutants.
We have shown previously that glycosylation-inhibiting factor (GIF) in culture supernatants of suppressor T cell (Ts) hybridomas had bioactivity, while the same cells contained a substantial quantity of inactive GIF in cytosol. Mass-spectrometric analysis of GIF in the culture supernatant and cytosol of a Ts hybridoma provided direct evidence that GIF protein was posttranslationally modified in the Ts cells, and that the GIF bioactivity is associated with the posttranslationally modified species. Assuming that conformational changes induced by the posttranslational modifications are responsible for generation of bioactivity, we constructed cysteine mutants of human rGIF (rhGIF) in which cysteine at position 57, 60, or 81 was replaced with Ala, and the mutants were expressed in Escherichia coli. Replacement of Cys57 or Cys60 with Ala resulted in generation of bioactivity, while replacement of Cys81 with Ala failed to do so. It was also found that replacement of Cys57 with Ala and carboxymethylation of a sulfhydryl group in Cys60 synergistically increased the GIF bioactivity of the GIF derivatives. A mutated GIF protein, in which Cys57 and Asn106 in the rhGIF were replaced with Ala and Ser, respectively, had immunosuppressive effects on the IgE 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.
Tomura
T
T
Pharmaceutical Research Laboratory, Kirin Brewery Co. Ltd., Takasaki, Gunma, Japan.
Watarai
H
H
Honma
N
N
Sato
M
M
Iwamatsu
A
A
Kato
Y
Y
Kuroki
R
R
Nakano
T
T
Mikayama
T
T
Ishizaka
K
K
D000818:Animals; D002460:Cell Line; D005260:Female; D006031:Glycosylation; D006801:Humans; D008222:Lymphokines / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D029584:Prostatic Secretory Proteins; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology*; D013491:Suppressor Factors, Immunologic / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D050378:T-Lymphocytes, Regulatory / Q000737:chemistry
D008222:Lymphokines; D029584:Prostatic Secretory Proteins; D011994:Recombinant Proteins; D013491:Suppressor Factors, Immunologic; C043076:beta-microseminoprotein; C013059:immunoglobulin-binding factors