Binding of granzyme B in the nucleus of target cells. Recognition of an 80-kilodalton protein.
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.
Pinkoski
M J
MJ
Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
Winkler
U
U
Hudig
D
D
Bleackley
R C
RC
D000818:Animals; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D053804:Granzymes; D007939:Leukemia L1210; D008855:Microscopy, Electron, Scanning; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology*
D004792:Enzyme Precursors; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D053804:Granzymes; D012697:Serine Endopeptidases