Entry and trafficking of granzyme B in target cells during granzyme B-perforin-mediated apoptosis.
In the widely accepted model of granule-mediated killing by cytotoxic lymphocytes, granzyme B entry into the target cell is facilitated by the pore forming molecule, perforin. Using indirect immunofluorescence and also direct visualization of fluorescein isothiocyanate (FITC)-conjugated granzyme B, we demonstrate internalization in the absence of perforin. Induction of the lytic pathway, however, required a second signal that was provided by perforin or adenovirus (Ad2). The combination of agents also resulted in a dramatic relocalization of the granzyme. Microinjection of granzyme B directly into the cytoplasm of target cells resulted in apoptosis without the necessity of a second stimulus. This suggested that the key event is the presence of granzyme B in the cytoplasm, and that when the enzyme is internalized by a target cell, it trafficks to an intracellular compartment and accumulates until release is stimulated by the addition of perforin. We found that the proteinase passed through rab5-positive vesicles and then accumulated within a novel compartment. On the basis of these results, we propose a new model for granzyme-perforin-induced target cell lysis in which granzyme B is subjected to trafficking events in the target cell that control and contribute to cell death.
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
M J
MJ
Pinkoski
M
M
Hobman
J A
JA
Heibein
K
K
Tomaselli
F
F
Li
P
P
Seth
C J
CJ
Froelich
R C
RC
Bleackley
D000256:Adenoviridae / Q000502:physiology; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001692:Biological Transport; D019556:COS Cells / Q000187:drug effects / Q000378:metabolism; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology; D053804:Granzymes; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells / Q000166:cytology / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000494:pharmacology / Q000502:physiology*; D008845:Microinjections; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000494:pharmacology / Q000502:physiology*; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D020696:rab5 GTP-Binding Proteins
D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins; D054353:Perforin; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases; D019204:GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins