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.

Figure 1

Expression and titration of rTrxPurification of rTrx. (a) rTrx expression was induced in the M15 E. coli as described in Materials and Methods. Five microlitres of the bacterial lysate (1), the flow-through (2) and the successive fractions of washing and elution by the pH gradient phosphate buffer were analysed on 15% SDS–PAGE. The proteins were stained by Coomassie blue. (b) Activity of purified rTrx. Insulin was reduced by a commercial preparation of Trx (purified human thioredoxin, IMCO, Stockholm Sweden) (▪) or our purified rTrx (□) in a total volume of 250 μl containing 0·8 mm NADPH, 0·09 μm insulin, 80 mm HEPES pH 7·6, and variable amounts of Trx. Each reaction was started by adding 0·1 μm human TR at 37° and stopped after 40 min by adding 750 μl 0·4 mm DTNB-6M guanidinium–HCl. The abundance of SH groups was assessed by the absorbance at 412 nm. rTrx activity was similar as the control.

Figure 2

Kinetics of IgG proteolysis by subcellular fractions (a) Control of rTrx activity. [¹²⁵I]IgG (0·5 μg) was incubated with 0·4 μg rTrx in 50 μl 80 mm Na acetate buffer pH 5·5 at 37° for different times. [¹²⁵I]IgG (0·5 μg) was incubated in 50 μl endosome (b) or lysosome (c) 4.2 cell fractions in the presence of 0·1 μm TR and 0·8 mm NADPH at 37° for different times. The samples were analysed by 10–15% SDS–PAGE and exposed to PhosphorImager (NR, non-reduced IgG. R, reduced IgG). The lower mobility of free H and L chains in the assays compared to control results from different reducing treatment between the samples and the control IgG (see Materials and Methods).

Figure 3

Effects of reducing agents and rTrx on IgG proteolysis. [¹²⁵I]IgG (0·5 μg) was incubated with 50 μl lysosome fraction in the presence of 10 mm Cys, 10 mm GSH or 4 μm rTrx (100:1 rTrx:IgG ratio) for 18 hr. The samples were analysed in non-reducing conditions by 10–15% SDS–PAGE and exposed to PhosphorImager. NR, non-reduced IgG. R, reduced IgG.

Figure 4

Dose-dependence on rTrx of IgG proteolysis. (a) [¹²⁵I]IgG (0·5 μg) was incubated with 50 μl endosome fraction in the presence of 0–100:1 rTrx:IgG molar ratio in 80 mm Na acetate buffer pH 5·5 for 18 hr at 37°. The samples were analysed in non-reducing conditions by 10–15% SDS–PAGE and exposed to PhosphorImager. (b) The same experiment was carried out with the lysosome fraction. NR, non-reduced IgG. R, reduced IgG.

Figure 5

Effect of rTrx on cathepsin B activity in the lysosome fraction. Cathepsin B activity (arbitrary unit, a.u.) was measured in the lysosome fraction after addition of 0–5 μm of rTrx (•), as described in Materials and Methods. (▪) represents the data of the incubation experiment omitting addition of the lysosome fraction. The positive control containing 6 mm Cys and 1 mm DTT has a value of 630·4 a.u.

Figure 6

In vitro IgG proteolysis by purified cathepsin B. [¹²⁵I]IgG (0·5 μg) was incubated in the presence of 0·1 U cathepsin B in 80 mm Na acetate buffer pH 5·5 with addition of rTrx from 0 to 5 μm. A positive control was performed in the presence of 6 mm Cys and 1 mm DTT. The samples were analysed in non-reducing conditions by 10–15% SDS–PAGE and exposed to PhosphorImager. NR, non-reduced IgG. R, reduced IgG.