Gamma-Interferon-inducibleLysosomal Thiol Reductase (GILT): MATURATION, ACTIVITY, AND MECHANISM OF ACTION

Uyen T Phan,Balasubramanian Arunachalam,Peter Cresswell

doi:10.1074/jbc.m003459200

Uyen T Phan, Balasubramanian Arunachalam + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m003459200

Copy DOI

Abstract

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 inter-chain 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.

Highlights

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
In this report we show that, like its mature form, precursor GILT reduces disulfide bonds with an acidic pH optimum, suggesting that it may be involved in disulfide bond reduction in the endocytic pathway
We 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

Summary

EXPERIMENTAL PROCEDURES

Cells and Antibodies—The human B lymphoblastoid line (B-LCL), Pala [13], has been described previously. The rabbit antisera R.GILT [5], R.IP30N [12], R.IP30C [12], and the mouse monoclonal antibodies MAP.IP30 [12], DA6.147 [14], and IG12 [15], have been previously described. Cathepsins D and L and N-(benzyloxycarbonyl)-L-phenyl-alanyl-L-tyrosinal (Z-FY-CHO) were purchased from Calbiochem. Cathepsin S was generously provided by Boehringer Ingelheim Pharmaceuticals (Ridgefield, CT), and morpholinurea-leucine-homophenylalanine-vinylsulfone phenyl (LHVS) was generously provided by Dr Hidde Ploegh, Harvard Medical School. GILT Purification—Human GILT was affinity-purified from B-LCLs using the monoclonal antibody MAP.IP30 coupled to Bio-Gel A15m beads as described previously [5]. Polymerase Chain Reaction-based Mutagenesis—The generation of the cysteine to serine mutant GILT derivatives (C46S, C49S, C46S/ C49S) and the cloning of wild-type and mutant GILT cDNAs into the expression vector pcDNA 3.1 puro(Ϫ) was described previously [5]. Transient Transfections in COS-7 Cells—COS-7 cells were seeded overnight in T75 flasks, grown to 80% confluence, and transfected with 20 ␮g of GILT or mutant GILT cDNAs using CellFECTIN

GILT Maturation and Mechanism

RESULTS

DISCUSSION