Functional Requirements for the Lysosomal Thiol Reductase GILT in MHC Class II-Restricted Antigen Processing

Abstract

Ag processing and presentation via MHC class II is essential for activation of CD4+ T lymphocytes. γ-IFN-inducible lysosomal thiol reductase (GILT) is present in the MHC class II loading compartment and has been shown to facilitate class II Ag processing and recall responses to Ags containing disulfide bonds such as hen egg lysozyme (HEL). Reduction of proteins within the MHC class II loading compartment is hypothesized to expose residues for class II binding and protease trimming. In vitro analysis has shown that the active site of GILT involves Cys46 and Cys49, present in a CXXC motif that shares similarity with the thioredoxin family. To define the functional requirements for GILT in MHC class II Ag processing, a GILT-deficient murine B cell lymphoma line was generated and stably transduced with wild-type and cysteine mutants of GILT. Intracellular flow cytometric, immunoblotting, and immunofluorescence analyses demonstrated that wild-type and mutant GILT were expressed and maintained lysosomal localization. Transduction with wild-type GILT reconstituted MHC class II processing of a GILT-dependent HEL epitope. Mutation of either Cys46 or Cys49 abrogated MHC class II processing of a GILT-dependent HEL epitope. In addition, biochemical analysis of these mutants suggested that the active site facilitates processing of precursor GILT to the mature form. Precursor forms of GILT-bearing mutations in Cys200 or Cys211, previously found to display thiol reductase activity in vitro, could not mediate Ag processing. These studies demonstrate that the thiol reductase activity of GILT is its essential function in MHC class II-restricted Ag processing.

Dendritic Cells Inhibit the Progression ofListeria monocytogenesIntracellular Infection by Retaining Bacteria in Major Histocompatibility Complex Class II-Rich Phagosomes and by Limiting Cytosolic Growth

“…The LLO point mutation (G486D) in the DP-L4044 strain used in the current study resides in the cholesterol-binding domain of the toxin (45), suggesting the interesting possibility that DC phagosomes present a different composition or configuration of membrane cholesterol, reducing toxin binding. Alternatively, the access of L. monocytogenes to the active form of GILT, a host-derived thiol reductase involved in antigen processing (17) that recently has been shown to be required for the activation of LLO (40), may be restricted in early DC phagosomes. Finally, exposure to lowlevel, NOX2-generated reactive oxygen species immediately upon the arrival of L. monocytogenes into DC phagosomes could inactivate LLO, similarly to a scenario proposed for activated macrophages (28).…”

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

“…Mutational analyses have demonstrated that Cys-222 is responsible for disulfide-mediated dimerization of secreted precursor GILT (45). Mutations of Cys-91, -98, -200, or -211 impair processing to the mature form (23,45). Remarkably, although Cys-211 is present in the C-terminal pro-peptide and presumably nonessential to the function of the mature form, C211S GILT and a mutant in its proposed disulfide partner, C200S GILT, are impaired in processing to the mature form, although the mutant precursors remain active especially at neutral pH (45).…”

Inactivation of Thioredoxin Reductases Reveals a Complex Interplay between Thioredoxin and Glutathione Pathways in Arabidopsis Development

“…Such proteins should present special characteristics: the redox center of the Arabidopsis NTRs is situated in a groove and is only accessible to proteins with a disulfide bridge on the surface of a small molecule or on an appendage of a large molecule (Dai et al, 1996). To date, such structures have been described only for the thioredoxin superfamily and, more recently, for the g-interferon-inducible thiol reductases (Hastings et al, 2006), with six homologs in the Arabidopsis genome, one of them implicated in high O-acetyl-LSer accumulation (Ohkama-Ohtsu et al, 2004).…”