Characterization of three serine esterases isolated from human IL-2 activated killer cells.
Human peripheral blood mononuclear cells, activated for 14 to 20 days with 1000 U/ml rIL-2, develop strong cytotoxicity for NK sensitive and resistant targets. This process is accompanied by the acquisition of cytoplasmic granules in approximately 60% of the cells and by the expression of esterase activity cleaving the synthetic substrate BLT. The esterase activity, localized in the cytoplasmic granules, was purified and characterized. Three proteins with 3H-DFP binding activity were isolated and had the following properties. Following the proposed nomenclature by Masson et al., the esterases were named human granzymes 1, 2, and 3. Human granzyme 1 on SDS-PAGE has an unreduced relative m.w. of 43,000 and can form disulfide-linked oligomers of relative higher m.w. All forms of granzyme 1 bind 3H-DFP. Upon reduction, granzyme 1 migrates with Mr 30,000 on SDS-PAGE. Additional proteolytic fragments of Mr 24,000 and Mr 28,000 are observed in some reduced preparations. Granzyme 1 cleaves the substrate BLT and appears homologous with murine granzyme A. Human granzyme 2 has an unreduced relative m.w. of 30,000; after reduction, it migrates at Mr 32,000. Even though granzyme 2 binds 3H-DFT, it does not cleave BLT. Human granzyme 2 has properties similar to those of murine granzymes B-H. Human granzyme 3 has unreduced and reduced relative m.w. of 25,000 and 28,000, respectively. It is active in cleaving the substrate BLT. A murine analog for human granzyme 3 has not been described previously. N-terminal sequencing of the purified human granzymes revealed that human granzyme 1 is the gene product of human Hanuka factor cDNA clone and that it represents the human homolog to murine granzyme A. Similarly, human granzyme 2 revealed absolute identity with cDNA-derived N-terminal sequence of a putative human lymphocyte protease cDNA clone.
Hameed
A
A
Department of Microbiology and Immunology, University of Miami School of Medicine, FL 33101.
Lowrey
D M
DM
Lichtenheld
M
M
Podack
E R
ER
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D053804:Granzymes; D006801:Humans; D007376:Interleukin-2; D007694:Killer Cells, Natural / Q000201:enzymology* / Q000276:immunology; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D012697:Serine Endopeptidases / Q000302:isolation & purification*; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology
D007376:Interleukin-2; D011994:Recombinant Proteins; D053804:Granzymes; D012697:Serine Endopeptidases; C509212:GZMA protein, human