Conversion of inactive glycosylation inhibiting factor to bioactive derivatives by modification of a SH group.
Escherichia coli-derived recombinant human glycosylation inhibiting factor (rhGIF) contains three cysteine residues (Cys-57, -60, and -81). All SH groups in the cysteine residues are free, and the GIF molecule had no biologic activity. Carboxymethylation of the SH group of Cys-60 in the molecule resulted in the generation of bioactivity, although the activity of the carboxymethylated GIF was 10- to 20-fold less than that of suppressor T cell (Ts)-derived GIF. However, treatment of the inactive rhGIF with ethylmercurithiosalicylate or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) resulted in the generation of derivatives whose bioactivity was comparable to that of the Ts-derived bioactive GIF. The activity of these derivatives was lost by treatment with DTT. Isolation and chemical analysis of the DTNB-treated GIF derivative revealed that binding the 5-thio-2-nitrobenzoic acid group with Cys-60 was responsible for the generation of the highly bioactive derivative. Inactive cytosolic GIF from mammalian cells could also be converted to bioactive derivative by treatment with the SH reagent, while Ts-derived bioactive GIF was inactivated by DTT. These results, together with an x-ray crystal structure of GIF molecules, strongly suggest that the generation of bioactivity of GIF in Ts cells is due to posttranslational modifications that result in conformational changes in the molecule.
Nakano
T
T
La Jolla Institute for Allergy and Immunology, CA 92037, USA.
Watarai
H
H
Liu
Y C
YC
Oyama
Y
Y
Mikayama
T
T
Ishizaka
K
K
D000595:Amino Acid Sequence; D001681:Biological Assay; D004220:Disulfides; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D008222:Lymphokines / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000494:pharmacology*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D029584:Prostatic Secretory Proteins; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D004220:Disulfides; D008222:Lymphokines; D029584:Prostatic Secretory Proteins; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; C043076:beta-microseminoprotein; C013059:immunoglobulin-binding factors