An Unexpected Functional Link between Lysosomal Thiol Reductase and Mitochondrial Manganese Superoxide Dismutase

Abstract

Gamma interferon-inducible thiol reductase (GILT) is an enzyme involved in the initial steps of antigen processing and presentation. Recently we have shown that GILT is also expressed in mouse T cells, where it exerts an inhibitory role on T cell activation. In this study, we identified mitochondrial manganese superoxide dismutase (SOD2) as one of the key intermediaries affected by GILT expression in fibroblasts. Expression and activity of SOD2 is reduced in the absence of GILT because of reduced SOD2 protein stability. The forced increase in SOD2 expression in the absence of GILT restores fibroblast proliferation to wild-type levels. Thus, GILT appears to have a fundamental role in cellular proliferation mediated through its influence on SOD2 protein activity and expression.Enzymes of the thiol reductase family carry out reduction, oxidation, and isomerization of protein disulfide bonds in cytosol (for example, thioredoxin) (1, 2), mitochondria (3), endoplasmic reticulum (protein-disulfide isomerase) (2), and lysosomes (gamma interferon-inducible thiol reductase, GILT).2 The majority of these enzymes are functional at neutral or slightly alkaline conditions (4), they have similar three-dimensional structures, and all feature a conservative active site loop containing two cysteines in the sequence -CGPC-(5). GILT is a unique and unusual member of the thiol reductase family because its optimal enzymatic activity is at a low pH (4.5-5.5) (6 -8) and has an atypical active site (-CGAC-).GILT is synthesized as a 35-kDa soluble glycoprotein precursor and is transported to the endosomal compartment via the mannose-6-P receptor pathway (9). It is processed into the mature form (30 kDa) by proteolytic removal of N-and C-terminal peptides. The protein has an approximate molecular mass of 30 kDa and was therefore initially named IP-30 (6). In addition to endosomal/lysosomal localization, GILT is secreted in the tissue culture medium of the GILT-expressing cell lines (7,10), and is present in mouse sera.3 GILT is constitutively expressed in professional antigen-presenting cells (APCs), but it is also inducible by pro-inflammatory cytokines such as interferon ␥, tumor necrosis factor ␣, and interleukin 1␤ (9).Using GILT Ϫ/Ϫ mice as a model, we have shown that GILT catalyzes initial unfolding of antigenic protein (protein becomes more accessible for further processing by cathepsins) and therefore facilitates protein/peptide binding to MHC class II molecules (10). By changing the redox state of exogenous antigenic proteins with disulfide bonds, GILT initiates the adaptive immune response. However, we have shown that GILT is constitutively expressed in T cells and has a role in the regulation of T cell activation. This is so far the only known GILT function not related to MHC class II processing (11). GILT Ϫ/Ϫ T cells show increased proliferation and cytotoxic T cell activity in response to anti-CD3 stimulation. This observation suggests that GILT has a more fundamental role in cellular processes than just reduction of antigens...

Cutting Edge: Developmental Up-Regulation of IFN-γ-Inducible Lysosomal Thiol Reductase Expression Leads to Reduced T Cell Sensitivity and Less Severe Autoimmunity

“…Our previous study has shown that compared with wild type (WT), GILT Ϫ/Ϫ fibroblasts display lower steady-state levels of mitochondrial SOD2 but not cytoplasmic SOD1 (17). Consequently, the levels of reactive oxygen species were higher in GILT Ϫ/Ϫ fibroblasts.…”

“…Alternatively, GILT and SOD2 may interact directly in a common but minor subcellular compartment other than lysosomes and mitochondria. Even though the pathway that connects GILT and SOD2 remains to be elucidated, transfection experiments in fibroblasts demonstrate that SOD2 is a key intermediate between GILT and cellular proliferation, as transfection of SOD2 bypasses the effect of the absence or presence of GILT (17).…”

“…Flow cytometry, cell purification using magnetic beads, in vitro anti-CD3 stimulation cultures, superoxide anion detection, Western blotting for GILT, superoxide dismutase (SOD) 1 and SOD2, and quantitative realtime RT-PCR were performed as previously described (16,17). Primers for Egr-1 were 5Ј-gggagccgagcgaacaa-3Ј (forward) and 5Ј-tctcattattca gagcgatgtcaga-3Ј (reverse); TaqMan probe was 6-carboxyfluorescein-tgac cacagagtccttt-minor groove binder.…”

“…GILT is also expressed in T lymphocytes (16) and fibroblasts (17), leading to reduced cellular responses to TCR ligands or serum growth factors, respectively. In this study we show that the expression of GILT in T cells increases as a function of maturity, thereby reducing sensitivity of T cells and consequences of autoimmune responses.…”

Disulfide Reduction in the Endocytic Pathway: Immunological Functions of Gamma-Interferon-Inducible Lysosomal Thiol Reductase

“…GILT is constitutively expressed in most APCs, including monocytes=macrophages, B cells (primary and cell lines), and bone-marrow derived DCs (3,25,29,30,34,36). GILT is also constitutively expressed in thymocytes (37), mature T cells (5,37), and some fibroblasts (9,64). IFN-g plays an important role in inducing expression of MHC class II and other components of the class II-restricted processing pathway, including Ii and HLA-DM (10).…”