Purification of three cytotoxic lymphocyte granule serine proteases that induce apoptosis through distinct substrate and target cell interactions.
We recently reported the purification of a lymphocyte granule protein called "fragmentin," which was identified as a serine protease with the ability to induce oligonucleosomal DNA fragmentation and apoptosis (Shi, L., R. P. Kraut, R. Aebersold, and A. H. Greenberg. 1992. J. Exp. Med. 175:553). We have now purified two additional proteases with fragmentin activity from lymphocyte granules. The three proteases are of two types; one has the unusual ability to cleave a tripeptide thiobenzyl ester substrate after aspartic acid, similar to murine cytotoxic cell protease I/granzyme B, while two are tryptase-like, preferentially hydrolyzing after arginine, and bear some homology to human T cell granule tryptases, granzyme 3, and Hanukah factor/granzyme A. Using tripeptide chloromethyl ketones, the pattern of inhibition of DNA fragmentation corresponded to the inhibition of peptide hydrolysis. The Asp-ase fragmentin was blocked by aspartic acid-containing tripeptide chloromethyl ketones, while the tryptase fragmentins were inhibited by arginine-containing chloromethyl ketones. The two tryptase fragmentins were slow acting and were partly suppressed by blocking proteins synthesis with cycloheximide in the YAC-1 target cell. In contrast, the Asp-ase fragmentin was fast acting and produced DNA damage in the absence of protein synthesis. Using a panel of unrelated target cells of lymphoma, thymoma, and melanoma origin, distinct patterns of sensitivity to the three fragmentins were observed. Thus, these three granule proteases make up a family of fragmentins that activate DNA fragmentation and apoptosis by acting on unique substrates in different target cells.
Shi
L
L
Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Canada.
Kam
C M
CM
Powers
J C
JC
Aebersold
R
R
Greenberg
A H
AH
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000276:immunology / Q000502:physiology*; D003513:Cycloheximide / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000201:enzymology*; D004249:DNA Damage; D003851:Deoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology* / Q000276:immunology*; D014407:Tumor Cells, Cultured
D009842:Oligopeptides; D011480:Protease Inhibitors; D003513:Cycloheximide; D003851:Deoxyribonucleases; D012697:Serine Endopeptidases; C072501:fragmentin