Disulfide reduction in the endocytic pathway: immunological functions of gamma-interferon-inducible lysosomal thiol reductase

Abstract

Gamma-interferon-inducible lysosomal thiol reductase (GILT) is constitutively expressed in most antigen presenting cells and is interferon γ inducible in other cell types via signal transducer and activator of transcription 1. Normally, N- and C-terminal propeptides are cleaved in the early endosome, and the mature protein resides in late endosomes and lysosomes. Correspondingly, GILT has maximal reductase activity at an acidic pH. Monocyte differentiation via Toll-like receptor 4 triggers secretion of a disulfide-linked dimer of the enzymatically active precursor, which may contribute to inflammation. GILT facilitates major histocompatibility complex (MHC) class II-restricted processing through reduction of protein disulfide bonds in the endocytic pathway and is hypothesized to expose buried epitopes for MHC class II binding. GILT can also facilitate the transfer of disulfide-containing antigens into the cytosol, enhancing their cross-presentation by MHC class I. A variety of antigens are strongly influenced by GILT-mediated reduction, including hen egg lysozyme, melanocyte differentiation antigens, and viral envelope glycoproteins. In addition, GILT is conserved among lower eukaryotes and likely has additional functions. For example, GILT expression increases the stability of superoxide dismutase 2 and decreases reactive oxygen species, which correlates with decreased cellular proliferation. It is also a critical host factor for infection with Listeria monocytogenes.

FIG. 1.

GILT in the MHC class II processing pathway. In the ER, MHC class II α (red) and β (light blue) chains are synthesized, form heterodimers, and associate with Ii (dark blue). The cytoplasmic tail of Ii directs the class II-Ii complex into the endocytic pathway. Cathepsins (represented by scissors) are responsible for the proteolysis of protein antigens and the sequential cleavage of Ii leaving CLIP bound in the class II peptide binding groove. Mature GILT (yellow) is localized to the late endosomes and lysosomes where it catalyzes reduction of protein disulfide bonds. HLA-DM (green) catalyzes the exchange of CLIP for locally generated peptides and functions as a peptide editor. MHC class II-peptide complexes are directed to the cell surface where they can stimulate CD4⁺ T cells. CLIP, class II-associated invariant chain peptide; ER, endoplasmic reticulum; GILT, gamma-interferon-inducible lysosomal thiol reductase; Ii, invariant chain. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 2.

Alignment of GILT homologs. GILT protein homologs from multiple representative organisms were aligned using NCBI Cobalt. The signal sequences predicted by SignalP 3.0 are shown in italics. N-linked glycosylation sites predicted using NetNGlyc 1.0 are shaded. The N- and C-terminal pro-peptides determined in human GILT are underlined. The 10 cysteine residues that are conserved in the majority of species; the reductase active site and the GILT motif are shown in bold. Residue numbers correspond to human GILT. Cys-46 and Cys-49 are part of the CXXC reductase active site. Cys-91 through Cys-106 defines the GILT motif CQHGX₂ECX₂NX₄C.

FIG. 3.

Proposed mechanism of GILT-mediated reduction. Similar to thioredoxin, the thiol group of the N-terminal cysteine (Cys-46 in human GILT) initiates a nucleophilic attack on the substrate disulfide bond. There is formation of a mixed disulfide GILT-substrate intermediate, which can be isolated using a trapping mutant in which the C-terminal cysteine has been mutated. Subsequent intramolecular attack by the Cys-49 thiol results in the release of the reduced substrate. Lysosomal cysteine is a physiological reducing agent that is capable of reducing GILT, so that it can catalyze the next reaction. Adapted from (44).

FIG. 4.

GILT trafficking and secretion. Lysosomal enyzmes are synthesized in the ER and transported through the Golgi complex to the trans-Golgi network. From the trans-Golgi network, proteins such as GILT can follow the constitutive secretory pathway (left) to the plasma membrane with possible endocytosis to reach the lysosome, or they can directly traffic to the lysosome via the M6PR (right) [reviewed in (48, 55)]. The specific N-glycans that are mannose 6-phosphorylated to mediate the lysosomal trafficking of GILT have not been characterized. In the ER and early Golgi compartments, GNPT catalyzes the transfer of GlcNAc-1-phosphate from UDP-GlcNAc to certain C6 hydroxyl groups of mannose sugars on the α-1,6 branch. A subsequent phosphorylation may occur on the α-1,3 branch. In the TGN, UCE hydrolyzes the phosphodiester bond releasing GlcNAc and exposing the M6P, which is recognized by M6PRs. M6PRs mediate transport of M6P-tagged lysosomal enzymes to endosomes. M6PRs dissociate from their ligand in the mildly acidic environment of the early endosomes and return to the TGN to mediate additional rounds of transport. Following Toll-like receptor-mediated signals via nuclear factor kappa B, reduced transcription of GNPT and UCE reduces M6P tagging and results in a shift toward the constitutive secretory pathway. GNPT, N-acetylglucosamine-1-phosphotransferase; M6PR, mannose-6-phosphate receptor; UCE, uncovering enzyme.

FIG. 5.

Hen egg lysozyme structure and position of I-A^b restricted epitopes. Ribbon diagram (left) and schematic (right) of X-ray crystal structure of hen egg lysozyme demonstrating the location of disulfide bonds and I-A^b-restricted epitopes: residues 20–35 in yellow, 30–53 in orange, 46–61 in green, and 74–88 in purple. Cysteine residues, all of which are involved in disulfide bonds, are shown with balls in red, unless they are part of an epitope as described above. Reprinted with permission from Maric et al. (36). (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 6.

The role of GILT in the antigen cross-presentation pathway. The cytosolic pathway of cross-presentation in dendritic cells involves the transfer of exogenous proteins or protein fragments (purple) from the phagosome into the cytosol [reviewed in (2)]. Large proteins in the phagosome must be unfolded and/or partially cleaved before retrotranslocation into the cytosol, possibly via Sec61 or Derlin-1 recruited from the ER. For disulfide bond containing antigens, reduction by GILT facilitates the unfolding, proteolysis, and retrotranslocation steps. Once in the cytosol, these proteins are degraded by the proteosome, and the resulting peptides are translocated into the ER via the TAP transporter (orange). Peptides of the appropriate sequence and length bind MHC class I molecules in the peptide loading complex composed of class I heavy chain (light blue), β₂m (pink), tapasin (red), ERp57 (green), and calreticulin (brown). Peptide binding triggers dissociation of the peptide loading complex, and the class I:peptide complexes are directed to the plasma membrane. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 7.

gB from herpes simplex virus 1. gB is a trimeric glycoprotein that contains a peptide (residues 498–505) that binds to the murine MHC class I molecule H2-K^b and is recognized by CD8⁺ T cells. The location of the peptide is indicated by a red box in (A), representing the protein in linear form, and in the structural depiction of a single gB subunit in (B). Generation of this peptide in association with H2-K^b by the pathway shown in Figure 6 is dependent on the presence of GILT in the phagosome. (C) and (D) show the structure of the gB trimer as a ribbon diagram and a space filling model, respectively, and indicate that the H2-K^b-binding peptide is buried in the three-dimensional structure of the gB molecule. Reprinted with permission from Heldwein et al. (24). gB, glycoprotein B. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).