Proteins internalized into the endocytic pathway are usually degraded. Efficient proteolysis requires denaturation, induced by acidic conditions within lysosomes, and reduction of inter-and intrachain disulfide bonds. Cytosolic reduction is mediated enzymatically by thioredoxin, but the mechanism of lysosomal reduction is unknown. We describe here a lysosomal thiol reductase optimally active at low pH and capable of catalyzing disulfide bond reduction both in vivo and in vitro. The active site, determined by mutagenesis, consists of a pair of cysteine residues separated by two amino acids, similar to other enzymes of the thioredoxin family. The enzyme is a soluble glycoprotein that is synthesized as a precursor. After delivery into the endosomal͞lysosomal system by the mannose 6-phosphate receptor, N-and C-terminal prosequences are removed. The enzyme is expressed constitutively in antigen-presenting cells and induced by IFN-␥ in other cell types, suggesting a potentially important role in antigen processing. R eduction, oxidation, and isomerization of protein disulfide bonds in the cytosol and endoplasmic reticulum (ER) of eukaryotic cells are carried out by enzymes of the thioredoxin family (1). Protein disulfide isomerase and related molecules catalyze the formation and isomerization of protein disulfide bonds in the ER (2-4). Thioredoxin and glutaredoxin catalyze reduction of disulfide bonds in the cytosol and nucleus (1, 5). These enzymes use oxidized cofactors (e.g., oxidized glutathione) as electron sinks or reduced cofactors (e.g., glutathione) as electron donors for oxidation or reduction of protein disulfide bonds, respectively (5, 6). Members of the thioredoxin family often share little sequence similarity but do possess a common active site (WCGH͞PCK) and folding pattern (7-10). The cysteine residues in the active site are believed to act by transferring electrons between themselves and either the substrate protein or cofactors (9).Disulfide bond reduction also occurs in the endocytic pathway. Most proteins that enter the endocytic pathway are degraded in lysosomes to small peptides and free amino acids. Denaturation of proteins is a prerequisite for lysosomal proteolysis (reviewed in ref . 11) and is facilitated by the acidic pH of the lysosome (12). A reducing environment within the endocytic pathway also facilitates denaturation by cleaving disulfide bonds in substrate proteins (reviewed in ref. 13). Various groups have demonstrated reducing activity in lysosomes (14-17) and endosomes (18). Although the presence of excess cysteine favors the reduction of disulfide bonds (16), the process is not favored at low pH (19), and no enzyme(s) that catalyzes reduction in these compartments has been described. We have now defined such an enzyme and have named it Gamma interferon-inducible lysosomal thiol-reductase (GILT). GILT was originally described by Luster et al. (20) and called IP30. It is synthesized as a 224-aa precursor that is transported to endocytic compartments by mannose-6-phosphate receptors...

Processing of proteins for major histocompatibility complex (MHC) class II-restricted presentation to CD4-positive T lymphocytes occurs after they are internalized by antigen-presenting cells (APCs). Antigenic proteins frequently contain disulfide bonds, and their reduction in the endocytic pathway facilitates processing. In humans, a gamma interferon-inducible lysosomal thiol reductase (GILT) is constitutively present in late endocytic compartments of APCs. Here, we identified the mouse homolog of GILT and generated a GILT knockout mouse. GILT facilitated the processing and presentation to antigen-specific T cells of protein antigens containing disulfide bonds. The response to hen egg lysozyme, a model antigen with a compact structure containing four disulfide bonds, was examined in detail.

We recently identified a gamma-interferon-inducible lysosomal thiol reductase (GILT), constitutively expressed in antigen-presenting cells, that catalyzes disulfide bond reduction both in vitro and in vivo and is optimally active at acidic pH. GILT is synthesized as a 35-kDa precursor, and following delivery to major histocompatibility complex (MHC) class II-containing compartments (MIICs), is processed to the mature 30-kDa form via cleavage of N-and C-terminal propeptides. The generation of MHC class II epitopes requires both protein denaturation and reduction of intra-and interchain disulfide bonds prior to proteolysis. GILT may be important in disulfide bond reduction of proteins delivered to MIICs and consequently in antigen processing. In this report we show that, like its mature form, precursor GILT reduces disulfide bonds with an acidic pH optimum, suggesting that it may also be involved in disulfide bond reduction in the endocytic pathway. We also show that processing of precursor GILT can be mediated by multiple lysosomal proteases and provide evidence that the mechanism of action of GILT resembles that of other thiol oxidoreductases.