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A clathrin/dynamin- and mannose-6-phosphate receptor-independent pathway for granzyme B-induced cell death.

Trapani JA, Sutton VR, Thia KY, Li YQ, Froelich CJ, Jans DA, Sandrin MS, Browne KA - J. Cell Biol. (2003)

Bottom Line: Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays.Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR- L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it.Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Cancer Immunology Laboratory, Peter MacCallum Cancer Institute, Melbourne 8006, Australia. j.trapani@pmci.unimelb.edu.au

ABSTRACT
The 280-kD cation-independent mannose-6-phosphate receptor (MPR) has been shown to play a role in endocytic uptake of granzyme B, since target cells overexpressing MPR have an increased sensitivity to granzyme B-mediated apoptosis. On this basis, it has been proposed that cells lacking MPR are poor targets for cytotoxic lymphocytes that mediate allograft rejection or tumor immune surveillance. In the present study, we report that the uptake of granzyme B into target cells is independent of MPR. We used HeLa cells overexpressing a dominant-negative mutated (K44A) form of dynamin and mouse fibroblasts overexpressing or lacking MPR to show that the MPR/clathrin/dynamin pathway is not required for granzyme B uptake. Consistent with this observation, cells lacking the MPR/clathrin pathway remained sensitive to granzyme B. Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays. Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR- L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it. Contrary to previous findings, we also demonstrated that mouse tumor allografts that lack MPR expression were rejected as rapidly as tumors that overexpress MPR. Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.

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Granzyme B uptake into K44A mutant dynamin–overexpressing HeLa cells is not inhibited by a vast molar excess of M6P. Cytofluorographic analysis of HeLa cells overexpressing K44A mutant dynamin (−tet, bottom) or expressing only wild-type dynamin (+tet, top) after incubation with FITC–granzyme B (50 nM) at 37°C for 20 min or in the absence of FITC–granzyme B (filled traces). Cells were preincubated either with 5 mM M6P (red) or G6P (blue) or without competing monosaccharide (green) for 15 min at 37°C.
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fig2: Granzyme B uptake into K44A mutant dynamin–overexpressing HeLa cells is not inhibited by a vast molar excess of M6P. Cytofluorographic analysis of HeLa cells overexpressing K44A mutant dynamin (−tet, bottom) or expressing only wild-type dynamin (+tet, top) after incubation with FITC–granzyme B (50 nM) at 37°C for 20 min or in the absence of FITC–granzyme B (filled traces). Cells were preincubated either with 5 mM M6P (red) or G6P (blue) or without competing monosaccharide (green) for 15 min at 37°C.

Mentions: As described above, we postulated that the residual granzyme B uptake in K44A-dynamin–expressing cells was due to an alternative, clathrin/dynamin-independent uptake pathway. However, it was also plausible that the level of K44A-dynamin expression in the HeLa transfectants was insufficient to completely block granzyme B uptake through the clathrin-dependent MPR pathway. To distinguish between these possibilities, we incubated the K44A-dynamin–expressing cells with FITC–granzyme B in the presence of 5 mM mannose-6-phosphate, which represents a 100,000-fold molar excess of the monosaccharide over granzyme B (typically used at 25–75 nM). As a control, we added a similar concentration of glucose-6-phosphate (G6P), which is unable to bind to MPR (Motyka et al., 2000). As expected, uptake of FITC–granzyme B into HeLa cells expressing only wild-type dynamin was considerably reduced by M6P, whereas G6P had no effect (Fig. 2). By contrast, preincubation of cells with M6P did not diminish granzyme B uptake in K44A-dynamin–expressing HeLa cells, indicating that uptake of granzyme B through the MPR/clathrin pathway was already efficiently blocked in these cells. Overall, our results strongly suggested that granzyme B is able to enter the cell cytoplasm using a mechanism that is independent of the MPR/clathrin pathway.


A clathrin/dynamin- and mannose-6-phosphate receptor-independent pathway for granzyme B-induced cell death.

Trapani JA, Sutton VR, Thia KY, Li YQ, Froelich CJ, Jans DA, Sandrin MS, Browne KA - J. Cell Biol. (2003)

Granzyme B uptake into K44A mutant dynamin–overexpressing HeLa cells is not inhibited by a vast molar excess of M6P. Cytofluorographic analysis of HeLa cells overexpressing K44A mutant dynamin (−tet, bottom) or expressing only wild-type dynamin (+tet, top) after incubation with FITC–granzyme B (50 nM) at 37°C for 20 min or in the absence of FITC–granzyme B (filled traces). Cells were preincubated either with 5 mM M6P (red) or G6P (blue) or without competing monosaccharide (green) for 15 min at 37°C.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172645&req=5

fig2: Granzyme B uptake into K44A mutant dynamin–overexpressing HeLa cells is not inhibited by a vast molar excess of M6P. Cytofluorographic analysis of HeLa cells overexpressing K44A mutant dynamin (−tet, bottom) or expressing only wild-type dynamin (+tet, top) after incubation with FITC–granzyme B (50 nM) at 37°C for 20 min or in the absence of FITC–granzyme B (filled traces). Cells were preincubated either with 5 mM M6P (red) or G6P (blue) or without competing monosaccharide (green) for 15 min at 37°C.
Mentions: As described above, we postulated that the residual granzyme B uptake in K44A-dynamin–expressing cells was due to an alternative, clathrin/dynamin-independent uptake pathway. However, it was also plausible that the level of K44A-dynamin expression in the HeLa transfectants was insufficient to completely block granzyme B uptake through the clathrin-dependent MPR pathway. To distinguish between these possibilities, we incubated the K44A-dynamin–expressing cells with FITC–granzyme B in the presence of 5 mM mannose-6-phosphate, which represents a 100,000-fold molar excess of the monosaccharide over granzyme B (typically used at 25–75 nM). As a control, we added a similar concentration of glucose-6-phosphate (G6P), which is unable to bind to MPR (Motyka et al., 2000). As expected, uptake of FITC–granzyme B into HeLa cells expressing only wild-type dynamin was considerably reduced by M6P, whereas G6P had no effect (Fig. 2). By contrast, preincubation of cells with M6P did not diminish granzyme B uptake in K44A-dynamin–expressing HeLa cells, indicating that uptake of granzyme B through the MPR/clathrin pathway was already efficiently blocked in these cells. Overall, our results strongly suggested that granzyme B is able to enter the cell cytoplasm using a mechanism that is independent of the MPR/clathrin pathway.

Bottom Line: Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays.Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR- L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it.Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Cancer Immunology Laboratory, Peter MacCallum Cancer Institute, Melbourne 8006, Australia. j.trapani@pmci.unimelb.edu.au

ABSTRACT
The 280-kD cation-independent mannose-6-phosphate receptor (MPR) has been shown to play a role in endocytic uptake of granzyme B, since target cells overexpressing MPR have an increased sensitivity to granzyme B-mediated apoptosis. On this basis, it has been proposed that cells lacking MPR are poor targets for cytotoxic lymphocytes that mediate allograft rejection or tumor immune surveillance. In the present study, we report that the uptake of granzyme B into target cells is independent of MPR. We used HeLa cells overexpressing a dominant-negative mutated (K44A) form of dynamin and mouse fibroblasts overexpressing or lacking MPR to show that the MPR/clathrin/dynamin pathway is not required for granzyme B uptake. Consistent with this observation, cells lacking the MPR/clathrin pathway remained sensitive to granzyme B. Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays. Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR- L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it. Contrary to previous findings, we also demonstrated that mouse tumor allografts that lack MPR expression were rejected as rapidly as tumors that overexpress MPR. Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.

Show MeSH
Related in: MedlinePlus