Expression of Human Recombinant Granzyme A Zymogen and Its Activation by the Cysteine Proteinase Cathepsin C

Abstract

Human granzyme A is one of the serine proteinases present in the granules of cytotoxic T lymphocytes and natural killer cells. Granzymes are synthesized as inactive proenzymes with an amino-terminal prodipeptide, which is processed during transport of granzymes to the cytotoxic granules, where they are stored as active proteinases. In this study, we explored the possibility of producing recombinant granzymes. Recombinant human granzyme A zymogen was expressed in several eukaryotic cell lines (HepG2, Jurkat, and COS-1) after infection with a recombinant vaccinia virus containing full-length granzyme A cDNA. Immunoblot analysis of cell lysates showed that all infected cells produced a disulfide-linked homodimer of identical molecular weight as natural granzyme A. Infected HepG2 cells produced the largest amount of this protease (approximately 160 times more than lymphokine activated killer (LAK) cells). The recombinant protein only had high mannose type oligosaccharides as did the natural protein. Although infected HepG2 and COS cells contained high granzyme A antigen levels, lysates from these cells did not show any granzyme A proteolytic activity. However, the inactive proenzyme could be converted into active granzyme A by incubation with the thiol proteinase cathepsin C (dipeptidyl peptidase I). This study is the first to demonstrate expression of an active recombinant human cytotoxic lymphocyte proteinase and conversion of inactive progranzyme A into an active enzyme by cathepsin C. We suggest that a similar approach can be used for the production of other granzymes and related proteinases.

Selective Inhibition of Dipeptidyl Peptidase I, Not Caspases, Prevents the Partial Processing of Procaspase-3 in CD3-activated Human CD8+ T Lymphocytes

“…We first used GF.dmk, a synthetic peptide that readily enters the cells and that specifically and irreversibly inhibits the activity of DPPI, a lysosomal thiol protease with dipeptidyl aminopeptidase activity that is required for post-transational processing and activation of many myeloid and lymphoid granule-associated serine proteases (13,(23)(24)(25)(26). Enriched in cytolytic granules of CTL (27) and coordinately expressed with GrA during CD8 ϩ T cell development and differentiation (28), DPPI is the sole protease that performs in vivo the proteolytic activation of the proenzyme forms of GrA and GrB (29).…”

“…DPPI is a thiol protease with aminodipeptidase activity required for the activation of many granule-associated serine proteases. In T lymphocytes, it is predominantly located in the cytolytic granules of CTL (27,28), where it performs the proteolytic maturation/activation of proGrA and proGrB (13,23,24,29). We therefore assumed that a granule-associated serine protease dependent on DPPI activity was in charge of procaspase-3 processing.…”

The conversion of recombinant human mast cell prochymase to enzymatically active chymase by dipeptidyl peptidase I is inhibited by heparin and histamine

“…Evidence that amount of prochymase secreted into the culture supernatant by High Five cells was 6.3-times and 13-times greater than that DPP I might be the enzyme responsible for the processing of granulocyte proteases in vivo comes from its ability to catalyse produced by Sf9 and Sf21 cells, respectively. For production of recombinant prochymase, 500 ml cells at a density of 1.8Ϫ this reaction in vitro [20,21,25], its greater abundance in cells which secrete them [22,26], and the inhibition of granulocyte 2.2ϫ10 6 ml Ϫ1 were inoculated with the transformed baculovirus at a multiplicity of infection of 5, and the supernatant harvested protease activation by the addition of the specific DPP I inhibitor Gly-Phe-diazomethyl ketone to cells in culture [18,26,27].…”

Molecular Cloning, Chromosomal Localization, and Expression of Murine Dipeptidyl Peptidase I

“…In addition, DPPI activity was found to colocalize with serine protease activity (such as granzyme A tryptase activity) in the specialized granule fraction of CTL (14). Several studies have also demonstrated the ability of DPPI to directly cleave the prodipeptide of various serine proteases, thereby activating them (16,17). To examine whether the expression of DPPI is regulated during in vitro lymphocyte activation, we examined DPPI mRNA and protein levels, and DPPI activity (using a defined synthetic substrate) in resting splenocytes, or splenocytes activated with various protocols (Figs.…”

“…Several reports have also suggested that DPPI activity, although present in a wide variety of tissues, is significantly higher in cytotoxic lymphocytes and myeloid cells (13)(14)(15); in these cells, DPPI localizes to the secretory granule compartment and may be the major enzyme responsible for processing serine proteases from the proenzyme form to the catalytically active form (14,15). Finally, two different reports have shown that DPPI can directly cleave the activation prodipeptides of granzymes A and B (16,17), serine proteases that are found in the secretory granules of activated cytotoxic lymphocytes (CTL) and natural killer cells (18).…”