Importance of thioredoxin in the proteolysis of an immunoglobulin G as antigen by lysosomal Cys‐proteases

Abstract

For disulphide-bonded antigens, reduction has been postulated to be a prerequisite for proteolytic antigen processing, with subsequent production of major histocompatibility complex (MHC) class II binding fragments. The murine monoclonal immunoglobulin G (IgG) CE25/B7 was used as a multimeric antigen in a human model. Native IgG is highly resistant to proteolysis and has been previously found to be partially reduced at early steps of cell processing to become a suitable substrate for endopeptidases. The role of the oxidoreductase thioredoxin (Trx) was assessed in the reduction of the IgG by cleavage of H-L and H-H disulphide bonds. Recombinant human Trx (rTrx) has been assayed in a proteolytic in vitro system on IgG using endosomal and lysosomal subcellular fractions from B lymphoblastoid cells. rTrx is required in a dose-dependent manner for development of efficient proteolysis, catalysed by thiol-dependent Cys-proteases, such as cathepsin B. We demonstrated that cathepsin B activity was stimulated by the addition of rTrx. Thus, we propose that Trx-dependent IgG proteolysis occurred, on the one hand by means of the unfolding of the IgG after disulphide reduction, becoming a substrate of lysosomal proteases, and on the other hand by Cys-proteases such as cathepsin B that are fully active upon the regeneration of their activity by hydrogen donors.

Differential Requirements for Endosomal Reduction in the Presentation of Two H2-Ed-Restricted Epitopes from Influenza Hemagglutinin

“…It will be of interest to determine the source of reductive activity that is necessary for the generation of S1 and almost certainly other epitopes as well. Enzymes such as protein disulfide isomerase, that has been detected in endosomes (48), and thioredoxin oxidoreductase, that has been shown to enhance in vitro proteolysis of an Ag by cysteine-proteases (49), are some candidates, but their involvement in endosomal reduction in intact cells has not been demonstrated and they do not appear to be active at low pH.…”

Cathepsin B Responsiveness to Glutathione and Lipoic Acid Redox

“…Three well known protein reductive reactions have been previously distinguished in defined cysteine protease assays (2,9,11,20,25,27,36,37,38) (A) PrS ox + 2 GSH « PrS red + GSSG (19,38). Due to the large change in free energy upon intramolecular oxidation of vicinal disulfhydryls, the back reactions are small after reduction of electron acceptors such as GSSG (18).…”

“…A remarkable diversity of redoxresponsive proteolytic effector mechanisms is now well established (20,25,33,40).…”

γ-Interferon-inducible Lysosomal Thiol Reductase (GILT) Maintains Phagosomal Proteolysis in Alternatively Activated Macrophages

“…Whether or not reductive machinery present in the endo/lysosomal system can maintain cysteine cathepsin activity in a mechanistically similar fashion is currently unknown. In reconstituted systems cathepsin B activity can be maintained with glutathione as well as thioredoxin (16,17) and papain by NADPH through the thioredoxin reductase/thioredoxin system (18). However, glutathione and thioredoxin have not been shown to be active in the lysosome.…”