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. 2008 Jul;76(7):2862-71.
doi: 10.1128/IAI.00326-08. Epub 2008 Apr 14.

gp96 is a human colonocyte plasma membrane binding protein for Clostridium difficile toxin A

Xi Na  1 , Ho Kim, Mary P Moyer, Charalabos Pothoulakis, J Thomas LaMont
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"VSports注册入口" gp96 is a human colonocyte plasma membrane binding protein for Clostridium difficile toxin A

VSports app下载 - Xi Na et al. Infect Immun. 2008 Jul.

"VSports在线直播" Abstract

Clostridium difficile toxin A (TxA), a key mediator of antibiotic-associated colitis, requires binding to a cell surface receptor prior to internalization. Our aim was to identify novel plasma membrane TxA binding proteins on human colonocytes. TxA was coupled with biotin and cross-linked to the surface of HT29 human colonic epithelial cells. The main colonocyte binding protein for TxA was identified as glycoprotein 96 (gp96) by coimmunoprecipitation and mass spectrum analysis. gp96 is a member of the heat shock protein family, which is expressed on human colonocyte apical membranes as well as in the cytoplasm. TxA binding to gp96 was confirmed by fluorescence immunostaining and in vitro coimmunoprecipitation. Following TxA binding, the TxA-gp96 complex was translocated from the cell membrane to the cytoplasm. Pretreatment with gp96 antibody decreased TxA binding to colonocytes and inhibited TxA-induced cell rounding. Small interfering RNA directed against gp96 reduced gp96 expression and cytotoxicity in colonocytes. TxA-induced inflammatory signaling via p38 and apoptosis as measured by activation of BAK (Bcl-2 homologous antagonist/killer) and DNA fragmentation were decreased in gp96-deficient B cells. We conclude that human colonocyte gp96 serves as a plasma membrane binding protein that enhances cellular entry of TxA, participates in cellular signaling events in the inflammatory cascade, and facilitates cytotoxicity. VSports手机版.

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Figures

FIG. 1.
FIG. 1.
Immunoprecipitation with TxA-biotin. (A) Native and biotinylated TxA (2 μg each) were resolved on SDS-PAGE gels for either Coomassie blue staining or Western blotting (WB) with streptavidin to detect biotinylated TxA. (B) HT29 cells were exposed to either TxA (1 nM) or TxA-biotin (1 nM) for 60 min (60′) at 37°C, and cell rounding was assessed microscopically. (C) HT29 cells were incubated with TxA-biotin (1 nM) at 4°C for 1 h in the presence of BS3. TxA-biotin-associated proteins were immunoprecipitated (IP) against streptavidin. Immunoprecipitates were resolved on SDS-PAGE gels and subjected to Western blotting (WB) with streptavidin-AP. TxA-biotin was loaded as a control.
FIG. 2.
FIG. 2.
gp96 interacts with TxA. (A) Peptide sequences (red) matching gp96 that were identified in the protein interacting with TxA on HT29 cells. (B) NCM460 cells were incubated with TxA (1 nM) for 1 h at 4°C. Cells were then lysed and immunoprecipitated (IP) with gp96 antibody or control IgG. Immunoprecipitates were resolved on SDS-PAGE gels and subjected to Western blotting (WB) with gp96 and TxA. Whole-cell lysates were used as a positive control. (C) Native TxA or heat-inactivated TxA (10 μg) was incubated with or without gp96 (10 μg) at either 4 or 37°C. The mixture was immunoprecipitated with gp96 antibody after a 60-min incubation. Immunoprecipitates were then resolved on SDS-PAGE gels and immunoblotted with antibodies to gp96 or TxA.
FIG. 3.
FIG. 3.
Cellular localization of gp96. (A) gp96 colocalized with TxA. HT29 cells were exposed to TxA (1 nM) for 5 (5′) or 15 (15′) min at 37°C. Endogenous gp96 or TxA was detected by double immunostaining with gp96 rat monoclonal antibody and TxA goat polyclonal antibody, followed by Texas red-conjugated secondary antibody for gp96 and FITC green-conjugated secondary antibody for TxA. The localizations of gp96 and TxA were analyzed by confocal immunofluorescence laser microscopy. (B) Human colon tissue was immunostained with gp96, which revealed expression (red) mainly in the epithelial cells. Magnification, ×10 (left) and ×20 (right).
FIG. 4.
FIG. 4.
gp96 antibody inhibits cell surface TxA binding. (A) HT 29 cells were incubated with either control IgG (40 μg/ml) or gp96 (40 μg/ml) monoclonal antibody for 10 min at 4°C prior to TxA (0.3 μg/ml, or 1 nM) exposure. Membrane-bound TxA was detected by immunostaining with TxA goat polyclonal antibodies, followed by FITC green-conjugated secondary antibody. (B) HT29 cells were incubated with either a 100-fold excess of unlabeled TxA, control IgG (40 μg/ml), gp96 (40 μg/ml), or medium, along with TxA-biotin at the concentration indicated at 4°C for 1 h. After a washing step, cells were incubated with streptavidin-AP for 1 h. The amount of TxA-biotin bound to the cell surface was estimated as AP optical density (OD). Results are expressed as mean OD values ± standard error of the mean of three separate determinations. Ab, antibody.
FIG. 5.
FIG. 5.
gp96 antibody inhibits TxA-induced cell rounding. (A) HT29 cells were incubated with cell medium, TxA (1 nM), gp96 antibody (40 μg/ml) plus TxA (1 nM), or control IgG (40 μg/ml) plus TxA (1 nM). Cell rounding was measured after a 60-min (60′) incubation at 37°C and expressed as mean percent cell rounding ± standard error of the mean of three experiments per group. *, P < 0.05 for the percentage of cell rounding in cells exposed to TxA antibody plus gp96 versus cells exposed to TxA plus control IgG. (B) HT29 cells were incubated with antibodies to gp96, Hsp75, Hsp60, Hsp90α, or Hsp90β or with control IgG prior to TxA exposure. Cell rounding and statistical analyses were performed as described for panel A. Ab, antibody.
FIG. 6.
FIG. 6.
Silencer gp96 (gp96si) blocks TxA-induced cell rounding. (A) NCM460 cells were transfected with control (3 μg/ml) or silencer gp96 (3 μg/ml) for 24 or 48 h. Cells were then exposed to TxA (0.3 μg/ml, or 1 nM) for 60 min (60′). gp96 expression was analyzed by Western blotting (WB) using gp96 and actin antibodies, followed by signal quantifications. (B) Cells transfected with control sequence or silencer gp96 were examined for cell rounding induced by TxA. *, P < 0.05 for the relative gp96 expression and percentage of cell rounding in cells transfected with silencer gp96 versus cells transfected with the control sequence with TxA stimulation.
FIG. 7.
FIG. 7.
gp96 and TxA-mediated cell signaling. (A) Control (wild type) and E4.126 (gp96-deficient B lymphocytes) cells were exposed to either LPS (10 μg/ml) (a) or TxA (10 nM) (b). Cell lysates were resolved on SDS-PAGE gels and subjected to Western blotting (WB) against phosphorylated p38 or gp96. Antibodies against total p38 demonstrated equal protein loading. (B) Control (wild type) and E4.126 (gp96-deficient B lymphocytes) cells were incubated with TxA (10 nM) for different time periods (a). Cell lysates were resolved on SDS-PAGE gels and immunoblotted against BAK. Antibodies against actin were used to compare protein loading. Control and E4.126 cells were exposed to TxA (10 or 50 nM) for different time periods (b). TxA-induced DNA degradation (laddering) was examined in 1% agarose gels. Staurosporine (Stauro; 1 μM) was used as a positive control. An equal amount of DNA (2 μg) was loaded on each lane. Diminished apoptosis in E4.126 cells exposed to TxA is evident by the large amount of residual genomic DNA present at the origin (arrow) compared to the complete degradation of genomic DNA after toxin exposure in control B cells. Control and E4.126 cells were exposed to a 10 nM concentration of inactivated TxA or TxA for 24 h (c). TxA-induced DNA degradation (laddering) was examined in 1% agarose gels as described for part b.
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VSports - References

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