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. 2009 Oct 26;206(11):2429-40.
doi: 10.1084/jem.20090782. Epub 2009 Oct 6.

Subtilase cytotoxin cleaves newly synthesized BiP and blocks antibody secretion in B lymphocytes

Chih-Chi Andrew Hu  1 , Stephanie K Dougan, Sebastian Virreira Winter, Adrienne W Paton, James C Paton, Hidde L Ploegh
Affiliations

Subtilase cytotoxin cleaves newly synthesized BiP and blocks antibody secretion in B lymphocytes

Chih-Chi Andrew Hu et al. J Exp Med. .

Abstract

Shiga-toxigenic Escherichia coli (STEC) use subtilase cytotoxin (SubAB) to interfere with adaptive immunity. Its inhibition of immunoglobulin secretion is both rapid and profound. SubAB favors cleavage of the newly synthesized immunoglobulin heavy chain-binding protein (BiP) to yield a C-terminal fragment that contains BiP's substrate-binding domain. In the absence of its regulatory nucleotide-binding domain, the SubAB-cleaved C-terminal BiP fragment remains tightly bound to newly synthesized immunoglobulin light chains, resulting in retention of light chains in the endoplasmic reticulum (ER). Immunoglobulins are thus detained in the ER, making impossible the secretion of antibodies by SubAB-treated B cells. The inhibitory effect of SubAB is highly specific for antibody secretion, because other secretory proteins such as IL-6 are released normally from SubAB-treated B cells. Although SubAB also causes BiP cleavage in HepG2 hepatoma cells, (glyco)protein secretion continues unabated in SubAB-exposed HepG2 cells. This specific block in antibody secretion is a novel means of immune evasion for STEC. The differential cleavage of newly synthesized versus "aged" BiP by SubAB in the ER provides insight into the architecture of the ER compartments involved.

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Figures

Figure 1.
Figure 1.
SubAB cleaves BiP and induces the UPR in B cells. (A) BiP is a specific target for SubAB in B cells. B cells purified from spleens of MD4 mice were cultured in LPS (20 µg/ml) to induce differentiation. 3-day LPS-stimulated B cells were treated with 0.1 µg/ml mutant or native SubAB for 3 h before lysis. Lysates were immunoblotted using antibodies against the BiP N terminus or C terminus; PDI; AAA-ATPase (p97); calnexin; calreticulin; ERdj3 (an HSP40 family protein); and actin. (B) IRE-1/XBP-1 and PERK pathways are activated in B cells in response to SubAB treatment. Similar lysates were immunoblotted using antibodies against BiP at its N terminus, IRE-1, XBP-1, phospho-eIF2α, eIF2α, p97, and actin. (C) 1-d LPS-stimulated B cells were treated with 0.1 µg/ml mutant or native SubAB or dithiothreitol (DTT) for 3 h before lysis. Lysates were immunoblotted using antibodies against IRE-1, XBP-1, p97, and actin. Results shown in each panel are representative of three independent experiments. For each experiment, B cells were pooled from at least two mouse spleens.
Figure 2.
Figure 2.
SubAB preferentially cleaves newly synthesized BiP in B cells. (A) BiP cleavage by SubAB reaches its maximum within 30 min. B cells purified from spleens of μS−/− mice were cultured in LPS (20 µg/ml) to induce differentiation. 3-day LPS-stimulated B cells were treated with 0.1 µg/ml mutant or native SubAB for the times indicated before lysis. Lysates were immunoblotted for full-length BiP, N-terminal BiP, C-terminal BiP, p97, and actin. (B) 3-day LPS-stimulated μS−/− B cells were labeled with [35S]methionine and [35S]cysteine for 4 h, chased for the indicated times in the presence of mutant or native SubAB, and lysed. Full-length BiP was immunoprecipitated from the lysates using an anti-KDEL antibody. Radioactive polypeptides were quantified using a phosphorimager. (C) Newly synthesized BiP is cleaved completely by SubAB. 3-day LPS-stimulated μS−/− B cells were radiolabeled for 10, 30, 60, or 240 min, chased for the indicated times in the presence of SubAB, and lysed. Full-length BiP was immunoprecipitated from the lysates using the anti-KDEL antibody. Radioactive polypeptides were quantified using a phosphorimager. (D) Aged BiP is not cleaved efficiently by SubAB. 3-day LPS-stimulated B cells were pretreated with CHX (100 µM) for 0, 1, 2, or 3 h and subsequently exposed to 0.1 µg/ml mutant or native SubAB for 2 h before lysis. Lysates were immunoblotted for full-length BiP, N-terminal BiP, p97, and actin. Intensities of protein bands in the full-length BiP immunoblot were determined using ImageJ (National Institutes of Health) and data were plotted. Results shown in each panel are representative of three independent experiments. For each experiment, B cells were pooled from at least two mouse spleens.
Figure 3.
Figure 3.
SubAB blocks secretion of IgM and free κ light chains. (A) 3-day LPS-stimulated MD4 B cells were pretreated with mutant or native SubAB for 30 min, labeled with [35S]methionine and [35S]cysteine for 10 min, and chased for the indicated times. Secreted IgM was immunoprecipitated from the culture media using an anti-μ or -κ antibody. (B) 3-day LPS-stimulated MD4 B cells, treated and pulse-chased as described in A, were lysed. IgM was immunoprecipitated from the lysates using anti-μ antibody. A longer exposed autoradiogram is shown for SubAB-treated samples. (C) 3-day LPS-stimulated μS−/− B cells were pretreated with mutant or native SubAB for 30 min, radiolabeled for 10 min, and chased. The secreted free κ chains were immunoprecipitated from the culture media using anti-κ antibody. Approximately seven times as much starting material was used for immunoprecipitations for the SubAB-treated samples to emphasize the differences in levels of secretion. (D) HepG2 cells were treated with mutant or native SubAB during the 30-min radiolabeling period, and chased. Lysates were used for immunoprecipitation with an anti-KDEL antibody. (E) Culture media from toxin-treated, radiolabeled HepG2 cells were immunoprecipitated for α1-antitrypsin (AAT) and albumin. Results shown in each panel are representative of three independent experiments. For each experiment shown in panels A–C, B cells were pooled from at least two mouse spleens.
Figure 4.
Figure 4.
SubAB blocks secretion of IgM, but not intracellular transport of class I MHC molecules. (A) 3-day LPS-stimulated MD4 B cells were labeled with [35S]methionine and [35S]cysteine for 10 min in the presence of mutant or native SubAB, chased for the indicated times, and lysed. Total lysates were analyzed by SDS-PAGE. (B) Lysates (A) were used for immunoprecipitations with antibodies against μ or κ. (C) Band intensities of μ (immunoprecipitated with α-μ) and κ (immunoprecipitated with α-κ) were quantified using a phosphorimager, and the ratio was determined by comparing to the total μ or κ signal at time point zero. (D) 3-day LPS-stimulated B cells were radiolabeled for 10 min in the presence of mutant or native SubAB, and chased for the indicated times. Culture media from each chase point were used for immunoprecipitations with antibodies against μ or κ. (E) Band intensities of μ (immunoprecipitated with α-μ) and κ (immunoprecipitated with α-κ) were quantified using a phosphorimager, and the obtained raw numbers were plotted. (F) Lysates (A) were also used for immunoprecipitations of the class I MHC heavy chains. CHO, high-mannose glycans; CHO*, complex-type glycans. Results shown in A, B, D, and F are representative of three independent experiments. For each experiment shown in A, B, D, and F, B cells were pooled from at least two mouse spleens.
Figure 5.
Figure 5.
SubAB blocks the ER-exit of membrane IgM, but not class I MHC molecules. 3-day LPS-stimulated μS−/− B cells were pretreated with mutant or native SubAB for 30 min, labeled with [35S]methionine and [35S]cysteine for 10 min, chased, and lysed. Membrane IgM was immunoprecipitated from lysates using anti-μ and -κ antibodies (A). Class I MHC molecules were immunoprecipitated using an anti–class I heavy chain antibody (B). Asterisk represents molecules bearing complex-type glycans. Longer exposed gels are shown for SubAB-treated samples to emphasize the absence of complex-type glycan modification on IgM. Results shown in each panel are representative of three independent experiments. For each experiment, B cells were pooled from at least two mouse spleens.
Figure 6.
Figure 6.
The C-terminal BiP cleavage fragment retains κ light chains in the ER. (A) 3-day LPS-stimulated μS−/− B cells were labeled with [35S]methionine] and [35S]cysteine for 4 h, washed with PBS, and treated with mutant or native SubAB for the indicated times. Immunoprecipitations were performed using an anti-KDEL antibody. In addition to BiP, the anti-KDEL antibody retrieves additional polypeptides, but none of these are affected by SubAB. (B) In a similar but independent experiment, proteins immunoprecipitated with the anti-KDEL antibody were eluted and reimmunoprecipitated with an anti-κ antibody. (C) 3-day LPS-stimulated μS−/− B cells were labeled with [35S]methionine and [35S]cysteine for 10 min and chased in the presence of mutant or native SubAB for the indicated times. Lysates were immunoprecipitated with the anti-KDEL antibody. Asterisk marks an unidentified polypeptide. Results shown in each panel are representative of three independent experiments. For each experiment, B cells were pooled from at least two mouse spleens.
Figure 7.
Figure 7.
SubAB blocks the secretion of antibodies, but not of IL-6. 3-day LPS-stimulated wild-type and μS−/− B cells were washed, counted, aliquoted into 96-well plates with fresh media containing mutant or native SubAB, and incubated for 4 h (black bars) or 24 h (gray bars). The levels of secreted IgM (A), IgG1 (B), IgG2a (C), IgG2b (D), IgA (E), Igκ (F), or Igλ (G) in culture media at each time point were determined by ELISA. Separate aliquots of cells were treated for 24 h with SubAB plus an antibody that blocks IL-6 receptor (IL-6R) to prevent internalization of secreted IL-6 via the IL-6R. The levels of IL-6 in culture media were measured by ELISA (H). Results (means ± SD) shown in each panel are representative of two independent experiments. For each experiment, B cells were pooled from two spleens of each genotype.
Figure 8.
Figure 8.
A model for the mode of action of SubAB. SubAB cleaves BiP into two segments, one containing the NBD, and the other containing the SBD. The SBD consists of short stretches of hydrophobic amino acids that allow its binding to unfolded/misfolded proteins. SubAB cleaves BiP, resulting in sequestration of immunoglobulin light chains through interactions with its cleaved product, the SBD of BiP.
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