Characterization of granzymes A and B isolated from granules of cloned human cytotoxic T lymphocytes.
A human CD8+ CTL clone with cytolytic potential was shown to express two serine proteases, a 50-kDa homodimer and a 27-kDa monomer, which were purified from cytoplasmic granules. N-terminal sequencing of the purified proteins revealed that the 50-kDa homodimer is the gene product of the human Hanukah factor cDNA clone and that it represents the human homologue to granzyme A. Similarly, the 27-kDa protein was shown to be the serine esterase encoded by the human lymphocyte protease cDNA clone and corresponds to granzyme B. There was no evidence for the presence of other granzymes, in particular for the human homologues to murine granzymes C, D, E, and F. The substrate best cleaved by granzyme A was Gly-Pro-Arg-amido-4-methyl-coumarin after the Arg residue and the pH optimum was 8 to 8.5. Upon triggering of the TCR-CD3 complex with an anti-CD3 mAb, granzyme A was released into the culture medium. Furthermore, a granule-associated hemolytic activity was detected after salt extraction and partial purification of granule proteins. This suggests that hemolytically active human perforin can be obtained from inactive granules.
Krähenbühl
O
O
Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland.
Rey
C
C
Jenne
D
D
Lanzavecchia
A
A
Groscurth
P
P
Carrel
S
S
Tschopp
J
J
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D002499:Centrifugation, Density Gradient; D002852:Chromatography, Ion Exchange; D002999:Clone Cells / Q000201:enzymology / Q000648:ultrastructure; D003594:Cytoplasmic Granules / Q000201:enzymology* / Q000502:physiology / Q000648:ultrastructure; D005089:Exocytosis; D053804:Granzymes; D006801:Humans; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000502:physiology; D013379:Substrate Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology* / Q000648:ultrastructure; D014158:Transcription, Genetic
D053804:Granzymes; D012697:Serine Endopeptidases; C509212:GZMA protein, human