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J. Biol. Chem., Vol. 269, Issue 28, 18359-18365, 07, 1994
JA Trapani, MJ Smyth, VA Apostolidis, M Dawson and KA Browne
One mechanism by which cytotoxic T lymphocytes and natural killer cells
inflict target cell death depends upon secreting the contents of their
specialized cytoplasmic granules, containing a pore-forming protein,
perforin, and a family of homologous serine proteases ("granzymes") with
various enzyme activities. We used a granzyme B-specific mouse anti-human
monoclonal antibody 2C5 and Western blotting to demonstrate that nuclear
extracts of human interleukin-2-stimulated peripheral blood mononuclear
cells, the human NK leukemia cell line YT, and the rat NK leukemia cell
line RNK-16 contain abundant granzyme B. In interleukin-2-activated
peripheral blood mononuclear cells, more than 50% of the total cellular
granzyme B was present in the nuclear lysate. Nuclear granzyme B had an
apparent molecular mass of approximately 32 kDa in human cells and
approximately 30 kDa in RNK-16 and was eluted from immobilized heparin at
the same NaCl concentration as granzyme B from cytoplasmic granules.
Granzyme B that was affinity-purified with 2C5 from the nuclei of YT or
human LAK cells was capable of efficiently cleaving synthetic peptide
thiobenzyl ester substrates with the same specificity (peptide cleavage
after aspartic acid) as granule-localized granzyme B. By contrast perforin,
which colocalizes with granzymes in cytotoxic granules, was not detectable
in nuclear lysates. Granzyme B was also demonstrated to be present in the
nucleus and cytoplasmic granules of YT by immunohistochemical staining with
monospecific anti- granzyme B antisera. Other protease activities (tryptase
and peptide cleavage after methionine) were also readily detectable in
nuclear and cytoplasmic lysates of YT, RNK-16, and LAK cells, as determined
by the cleavage of the synthetic substrates N
alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) and
Boc-Ala-Ala-Met-S-benzyl, except that BLT- esterase activity was absent
from the nucleus of YT. The localization of serine proteases in the nucleus
was restricted to lymphocytes with cytotoxic capacity, as non-cytotoxic
cell lines expressed high levels of peptide cleavage after methionine and
tryptase activities in their cytoplasm, but possessed no nuclear serine
protease activity. Furthermore, non-cytotoxic monkey kidney COS-7 cells
transfected with an SV40-driven expression plasmid incorporating
full-length human granzyme B cDNA contained abundant cytoplasmic granzyme
B, but demonstrated minimal nuclear granzyme B accumulation. We conclude
that serine proteases of NK cells are not restricted to cytolytic granules
and, further, that their capacity to access the nucleus may have
implications for the role of these enzymes in eliciting target cell death.
Granule serine proteases are normal nuclear constituents of natural killer cells
Cellular Cytotoxicity Laboratory, Austin Research Institute, Heidelberg, Victoria, Australia.
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