Protein disulphide-isomerase reduces ricin to its A and B chains in the endoplasmic reticulum

Abstract

Cells expressing ricin B chain within the secretory pathway are significantly more resistant to intoxication by ricin holotoxin but not to other cytotoxins that exploit similar endocytic routes to the cytosol. Furthermore, cells expressing the related B chain of abrin are protected against both incoming abrin and ricin. These phenotypes can be correlated with the abilities of the respective B chains to form disulphide-linked A-B holotoxins, since abrin B chain forms heterodimers with either abrin or ricin A chains, whereas ricin B chain forms heterodimers with ricin A chain only. In the ricin B-expressing cells, this newly made lectin disappears with biphasic kinetics comprising a retention phase followed by slow turnover and disposal after disengagement from calnexin cycle components. Interference with ricin cytotoxicity occurs during the early retention phase when ricin B chain is associated with PDI (protein disulphide-isomerase). The data show that retrotranslocation of incoming toxin is impeded by PDI-catalysed formation of heterodimers between endogenous B and A chains derived from reduced holotoxin, thus proving that reduction of ricin occurs in the endoplasmic reticulum. In contrast with other toxins, ricin does not appear to require either proteolytic cleavage or unfolding for PDI-catalysed reduction.

Figure 1. RTB is expressed in HeLa cells without toxic effects

Detergent-extracted proteins (10 μg) from vector-controlled HeLa cells expressing lacZ (lane 2), or from HeLa expressing ricin B (RTB) clone H2 (lane 4) or clone H12 (lane 6) together with corresponding Tn-treated samples (lanes 3, 5 and 7) were immunoblotted in parallel with RTB purified from R. communis seeds (lane 1). The black arrowhead represents the mass of expressed RTB and the open arrowhead represents the mass of mature, non-glycosylated RTB. Positions of migration of molecular-mass standards are displayed on the left.

Figure 2. Expressed RTB protects cells against ricin

(A) Non-transfected HeLa cells (○) and transfected HeLa cells expressing lacZ (▴), hygromycin (▵), RTB-H2 (•) or RTB-H12 (grey circles) were treated with ricin, and their ability to synthesize proteins subsequently was determined. (B) Non-transfected HeLa cells (□) and RTB-H2 cells (▪) were treated with recombinant RTA and their ability to synthesize proteins subsequently was determined.

Figure 3. RTB and ATB form particular cross-heterodimers with toxin A chains in vitro

Silver-stained reducing SDS/PAGE showing reassociations of combinations of toxin A chains (open arrowheads) and B chains (black arrowheads). After incubations (see the Materials and methods section), the mixtures were loaded on to immobilized α-lactose columns and bound B chains/disulphide-linked holotoxins were eluted with 100 mM lactose. In each case, F represents flowthrough; E1–E3 represent samples of the eluted fractions. ATA was from a bacterially expressed abrin F isoform; ATB was from an abrin A isoform purified from Abrus precatorius seeds; RTA was bacterially expressed [46]; RTB was purified from R. communis seeds. The asterisks denote a proteolytic breakdown product of ATA.

Figure 4. Most of the RTB was retained in the early secretory pathway from where it disappeared with time

(A) Pulse-labelled RTB (black arrowhead) from cell extracts and conditioned medium was immunoprecipitated after chase (t) for up to 12 h, and electrophoresed under reducing conditions. (B) Immunoprecipitates, as generated in (A), were incubated with or without endo H as described in the Materials and methods section. Such a treatment generated a faster migrating form (open arrowhead) that co-migrated with the Tn-treated samples. (C) RTB, recovered by immunoprecipitation from conditioned growth medium (black arrowhead), was treated with endo H to generate two faster migrating species (open and grey arrowheads). (D) Cells treated with BFA were lysed and RTB immunoprecipitated from cell and media fractions. Molecular-mass standards are shown on the left (A–C) or right (D).

Figure 5. Expressed RTB was partially stabilized when proteasomes were inhibited

The relative amount of label incorporated into RTB was quantified after electrophoresis using a PhosphorImager. Results are displayed graphically, plotting the proportion of RTB retained in cell extracts against the time of chase, for untreated cells (○) and cells treated with clasto-lactacystin β-lactone (□).

Figure 6. Disposal of RTB involved interaction with the CNX cycle in a manner that did not interfere with incoming ricin

(A) Quantification of RTB in cell extracts after electrophoresis using a PhosphorImager. Results are displayed graphically, plotting the proportion of RTB retained in cell extracts against the time of chase, for cells treated with CST (○) or DMM (□), compared with untreated cells from Figure 5 (- - - -). (B) Effect of CST and DMM on cytotoxicity of ricin. HeLa cells (grey triangles) and HeLa/RTB-H2 cells (grey circles) were challenged for 4 h with increasing concentrations of ricin and their ability to incorporate [³⁵S]methionine into proteins was determined and expressed as the proportion of remaining protein synthesis. In parallel, cells were treated with CST (▵, HeLa; ○, HeLa/RTB-H2) or DMM (grey squares, HeLa; □, HeLa/RB-H2) during the intoxication process.

Figure 7. Disulphide bond reduction perturbs the protection provided by RTB against a ricin challenge

HeLa cells (A) and HeLa/RTB-H2 (B) cells were treated with fresh medium (○) or with fresh medium containing 0.25 mM (grey triangles), 0.5 mM (grey squares) or 1 mM (•) DTT and challenged with increasing concentrations of ricin, and their ability to synthesize proteins subsequently was measured. (C) The concentrations of ricin required to reduce protein synthesis by 50% (IC₅₀) and the effect (fold) of different DTT concentrations.

Figure 8. PDI can dissociate and reassociate ricin subunits in vitro

(A) Ricin (70 nM) was incubated with GSH (1 mM), GSSG (1 mM) or neither, in the presence of BSA (3.5 μM) or purified mammalian PDI (3.5 μM) at 30 °C for 30 min. Samples were analysed by non-reducing SDS/PAGE and immunoblotting with antibodies to RTA and RTB. (B) RTA (70 nM) was incubated with either RTB (70 nM) or ATB (70 nM) with or without GSH (1 mM) in the presence of BSA or purified mammalian PDI (3.5 μM) at 30 °C for 30 min. Samples were analysed by non-reducing SDS/PAGE and immunoblotting with antibodies to RTB and ATB.

Figure 9. PDI interacts with RTB

Pulse-labelled cell extracts from untreated HeLa/RTB-H2 cells or cells treated with NEM or after cross-linking with BMH were immunoprecipitated (IP) using sheep antiserum (Sh) specific for RTB or rabbit antisera (Rb) raised against either RTB or PDI. TCEP, Tris[2-carboxyethyl]phosphine; black arrowhead, RTB; open arrowhead, PDI; grey arrowhead, an irrelevant crossreacting protein (58 kDa) that is immunoselected by the rabbit anti-RTB serum but not by the sheep anti-RTB serum; *, RTB co-immunoprecipitated with anti-PDI antibodies. Positions of molecular-mass standards are shown on the right.