5] Disulfide reduction in major histocompatibility complex class II-restricted antigen processing by interferon-γ-inducible lysosomal thiol reductase

Abstract

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.