RECOMBINANT HUMAN GRANZYME-A BINDS TO 2 PUTATIVE HLA-ASSOCIATED PROTEINS AND CLEAVES ONE OF THEM

The release of cytotoxic granule contents by cytotoxic T lymphocytes t riggers apoptotic target cell death, Cytotoxic granules contain a pore -forming protein, perforin, and a group of serine proteases called gra nzymes. We expressed human granzyme A in bacteria as a proenzyme capab le of in vitro activation by enterokinase, The recombinant activated e nzyme has catalytic activity against substrates with Arg, preferably, or Lys at the P1 position, comparable to trypsin, An enzymatically ina ctive recombinant granzyme A, with the active site Ser mutated to Ala, was produced and used with affinity chromatography to identify potent ial substrates, Two granzyme A-binding cytoplasmic proteins of molecul ar mass 33 and 44 kDa were isolated and identified by tryptic fragment sequencing as PHAP I and II, ubiquitous putative HLA-associated prote ins, previously coisolated by binding to an HLA class Il peptide. PHAP II forms an SDS-stable complex with recombinant mutant granzyme A and coprecipitates with it from cytoplasmic extracts. PHAP II, either pur ified or in cell lysates, is cleaved by the recombinant enzyme at nano molar concentrations to a 25-kDa fragment. PHAP II begins to be degrad ed within minutes of initiation of cytotoxic T lymphocyte attack PHAP I and LI are candidate participants in the granzyme A pathway of cell- mediated cytotoxicity.