Granzyme A binding to target cell proteins. Granzyme A binds to and cleaves nucleolin in vitro.
The physiologic substrates of cytotoxic T lymphocyte granule-associated serine esterases (referred to hereafter as proteases or "granzymes"), and the role of these enzymes in cell-mediated activity remain unclear. We have developed an assay for possible ligands of the trypsin-like dimeric serine protease granzyme A based on Western immunoblotting techniques. This protein-binding assay demonstrates the selective binding of granzyme A to several proteins present in the target cell P815. The binding specificity is preserved when enzyme binding is performed in the presence of excess competing proteins, including such cationic species as lysozyme and RNase. Enzyme binding is inhibited, however, by heat or detergent inactivation of granzyme A. Subcellular fractionation of target cells shows that the nuclear fraction contains most granzyme A binding reactivity, which is recovered in the nuclear salt wash fraction. A protein with Mr = 100,000 and two closely migrating proteins with Mr = 35,000 and 38,000 are the predominant reactive moieties, and the N-terminal sequence of the 100-kDa protein confirmed that this protein was murine nucleolin. Incubation of granzyme A with nucleolin generates a discrete proteolytic cleavage product of Mr = 88,000. Since nucleolin is known to shuttle between nucleus and cytoplasm, the interaction of granzyme A and nucleolin may be important in the process of apoptosis which accompanies cytotoxic T lymphocyte-mediated lysis of target cells.
Pasternack
M S
MS
Infectious Disease Units (Children's and Medical Services), Massachusetts General Hospital, Boston 02129.
Bleier
K J
KJ
McInerney
T N
TN
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D053804:Granzymes; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D016601:RNA-Binding Proteins; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors; D013379:Substrate Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism*; D014357:Trypsin; D000097796:Nucleolin
D053804:Granzymes; D009687:Nuclear Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors; D014357:Trypsin; D000097796:Nucleolin