Binding of Granzyme B in the Nucleus of Target Cells
RECOGNITION OF AN 80-KILODALTON PROTEIN (*)
- From the (1)Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada and the
- (2)Cell and Molecular Biology Graduate Program and Department of Microbiology, University of Nevada, Reno, Nevada 89557
- ¶ Recipient of an Alberta Heritage Foundation for Medical Research Scientist Award. To whom correspondence should be addressed. Tel.: 403-492-3968; Fax: 403-492-0882; Chris_Bleackley{at}darwin.biochem.ualberta.ca.
Abstract
Granzyme B (cytotoxic cell proteinase 1) is a serine proteinase that has been implicated in cytotoxic T lymphocyte-induced apoptosis. In order to understand how granzyme B is involved in mechanisms of target cell destruction, characterization and identification of substrates are required. We have developed an in situ binding assay using permeabilized cells and recombinant granzyme B that allows us to visualize potential substrates after immunostaining with anti-granzyme B antiserum.
Confocal laser scanning microscopy and immunoelectron microscopic analyses demonstrate that granzyme B recognizes a nuclear substrate. The labeling pattern observed corresponds with regions of positive staining with uranyl acetate which binds to heterochromatin in the nucleus. Positive labeling of target cells with granzyme B is dependent on the presence of a catalytically active proteinase, since an inactive proenzyme form of granzyme B fails to give rise to any binding in the target cells. Far-Western blotting and immunoprecipitation of subcellular fractions of target cells have shown that the putative substrate of catalytically active granzyme B is an 80-kDa nuclear protein. Minor cytosolic bands of 50 and 94 kDa are also observed. A cytoplasmic band of 69 kDa is detected by both active and zymogen forms of granzyme B.
Footnotes
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↵§ Recipient of an Alberta Heritage Foundation for Medical Research Studentship.
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↵* Funding for these studies was provided in part by the Medical Research Council of Canada and the National Cancer Institute of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- CTL
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cytotoxic T lymphocytes
- α-CD3
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antibody to CD3
- ConA
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concanavalin A
- α-CCP
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antiserum to granzyme B
- FITC
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fluorescein isothiocyanate
- CLSM
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confocal laser scanning microscopy
- Ig
-
immunoglobulin.
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- Received September 20, 1995.
- Revision received January 9, 1996.
