Recombinant human granzyme A binds to two putative HLA-associated proteins and cleaves one of them.
The release of cytotoxic granule contents by cytotoxic T lymphocytes triggers apoptotic target cell death. Cytotoxic granules contain a pore-forming protein, perforin, and a group of serine proteases called granzymes. We expressed human granzyme A in bacteria as a proenzyme capable of in vitro activation by enterokinase. The recombinant activated enzyme has catalytic activity against substrates with Arg, preferably, or Lys at the P1 position, comparable to trypsin. An enzymatically inactive recombinant granzyme A, with the active site Ser mutated to Ala, was produced and used with affinity chromatography to identify potential substrates. Two granzyme A-binding cytoplasmic proteins of molecular mass 33 and 44 kDa were isolated and identified by tryptic fragment sequencing as PHAP I and II, ubiquitous putative HLA-associated proteins, previously coisolated by binding to an HLA class II peptide. PHAP II forms an SDS-stable complex with recombinant mutant granzyme A and coprecipitates with it from cytoplasmic extracts. PHAP II, either purified or in cell lysates, is cleaved by the recombinant enzyme at nanomolar concentrations to a 25-kDa fragment. PHAP II begins to be degraded within minutes of initiation of cytotoxic T lymphocyte attack. PHAP I and II are candidate participants in the granzyme A pathway of cell-mediated cytotoxicity.
Beresford
P J
PJ
Center for Blood Research, Harvard Medical School, Boston, MA 02115, USA.
Kam
C M
CM
Powers
J C
JC
Lieberman
J
J
D000818:Animals; D001665:Binding Sites; D003602:Cytotoxicity, Immunologic; D053804:Granzymes; D000949:Histocompatibility Antigens Class II / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D011506:Proteins / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D012697:Serine Endopeptidases / Q000276:immunology / Q000378:metabolism*; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000276:immunology
D000949:Histocompatibility Antigens Class II; D011506:Proteins; D011994:Recombinant Proteins; D053804:Granzymes; D012697:Serine Endopeptidases; C509212:GZMA protein, human