Cathepsin H Is an Additional Convertase of Pro-granzyme B

Michael E D'Angelo,Phillip I Bird,Christoph Peters,Thomas Reinheckel,Joseph A Trapani,Vivien R Sutton

doi:10.1074/jbc.m109.094573

Michael E D'Angelo, Phillip I Bird + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m109.094573

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Abstract

The serine protease granzyme B (GrB) is the most potent proapoptotic cytotoxin of the granule exocytosis pathway of cytotoxic lymphocytes. GrB is synthesized as a zymogen (proGrB) and activated in cytotoxic granules by the lysosomal cysteine protease cathepsin C (CatC) which removes the N-terminal dipeptide Gly-Glu. It has been shown recently that mice lacking CatC nonetheless express significant residual GrB activity, indicating the presence of additional proGrB convertases. Here, we describe an assay to assess activation of proGrB and show that the amino-peptidase cathepsin H (CatH) has proGrB convertase activity in vitro, whereas dipeptidylpeptidase II does not. We generated mice lacking both CatC and CatH expression (CatCH(-/-)) and found that their lymphocytes have reduced convertase activity compared with those from CatC-deficient mice. Despite this, cytotoxic lymphocytes from CatCH(-/-) mice retain cytotoxic activity and some residual GrB activity. We conclude that CatH can act as an additional proGrB convertase and that other protease/s (apart from dipeptidylpeptidase II) must also possess convertase activity. This indicates a great deal of functional redundancy in GrB maturation, which would prevent pathogen-mediated immune suppression by via convertase inhibition.

Highlights

Cytotoxic lymphocytes (CLs)6 include natural killer and CD8-positive T cells and are responsible for eliminating virusinfected and cancerous cells
Perforin is critical for granzyme-mediated cell death, as it facilitates entry of proteases into the target cell cytosol, where they can access and cleave substrates to bring about target cell death [4]
We found that cathepsin H (CatH), but not dipeptidylpeptidase II (DPPII), has granzyme B (GrB) convertase activity; lymphocytes deficient in both cathepsin C (CatC) and CatH are still capable of generating active GrB, indicating that additional GrB convertases exist in intact CLs

Summary

EXPERIMENTAL PROCEDURES

Enzymes and Reagents—All reagents were obtained from either Merck or Sigma unless otherwise specified. Production of proGrB—Recombinant human ProGrB was produced in Pichia pastoris and purified from culture supernatant as described previously for GrB [14], except that the construct was designed to include the natural two-residue prodomain (Gly-Glu) downstream of the enterokinase (EK) cleavage site. Removal of the His tag by EK was verified by submitting 2 ␮g of proGrB for amino acid sequencing via Edman degradation (Monash Proteomics Facility) as described previously [15]. 10 ␮l of lysate was incubated overnight with 10 ␮l of buffer containing 20 pmol of proGrB, 50 mM MES, 150 mM NaCl, and 5 mM dithiothreitol, pH 5.5 This reaction was assayed for GrB activity as above.

RESULTS

Activation of proGrB with Mouse

DISCUSSION