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.
Highlights
Granzyme B is a serine proteinase that has been implicated in cytotoxic T lymphocyte-induced apoptosis
We present evidence to demonstrate that catalytically active granzyme B will recognize a nuclear protein that co-localizes with heterochromatin in target cells
This protein is not recognized by inactive granzyme B zymogen, which differs from the mature proteinase by an additional dipeptide (Gly-Glu) at the N terminus
Summary
(Received for publication, September 20, 1995, and in revised form, January 9, 1996). Apoptosis induced in target cells by cytotoxic T lymphocytes is believed to occur after CTL1-target conjugation which is facilitated by engagement of the T-cell receptor and its accessory molecules [1]. This cell-cell contact induces the exocytosis, from the CTL, of granules which contain the potential cytolytic effector molecules These include the pore-forming protein perforin (cytolysin) and a family of serine proteases collectively known as granzymes. Granzyme B has been isolated as the activity known as fragmentin [7] This activity is necessary for degradation of DNA in target cells by CTL. We present evidence to demonstrate that catalytically active granzyme B will recognize a nuclear protein that co-localizes with heterochromatin in target cells
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