Disulfide reduction in major histocompatibility complex class II-restricted antigen processing by interferon-gamma-inducible lysosomal thiol reductase.
Constitutively expressed in antigen-presenting cells (APCs), interferon-gamma-inducible lysosomal thiol reductase (GILT) catalyzes disulfide bond reduction under acidic conditions. GILT contains a CXXC motif similar to the WCGH/PCK motif of proteins in the thioredoxin family. This class of enzymes catalyzes, at a neutral pH, dithiol oxidation, disulfide bond reduction, and disulfide bond isomerization. A well-established assay spectrophotometrically measures interchain disulfide bond reduction of insulin via the precipitation of aggregating free B chains. However, the insolubility of insulin at low pH limits the use of this assay. To assess the thiol reductase activity of GILT, we employed an assay that uses denatured [125I]F(ab')2 as a substrate, which is detailed in this article. In addition, we discuss approaches used to demonstrate the mechanism of action of GILT and to identify substrates.
Phan
Uyen T
UT
Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Maric
Maja
M
Cresswell
Peter
P
D000818:Animals; D017951:Antigen Presentation; D019556:COS Cells; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D000949:Histocompatibility Antigens Class II / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000378:metabolism; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000494:pharmacology; D008954:Models, Biological; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D050862:Oxidoreductases Acting on Sulfur Group Donors; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D004220:Disulfides; D000949:Histocompatibility Antigens Class II; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D007371:Interferon-gamma; D010088:Oxidoreductases; C056859:IFI30 protein, human; D050862:Oxidoreductases Acting on Sulfur Group Donors