Cathepsin H Is an Additional Convertase of Pro-granzyme B

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.

Neutrophilic Cathepsin C Is Maturated by a Multistep Proteolytic Process and Secreted by Activated Cells during Inflammatory Lung Diseases

“…Our data support the conclusions of Méthot et al (25,26) who reported that very high fractional inhibition of the CatC activity by chronic administration of a CatC inhibitor is required to prevent the activation of NSPs in vivo. CatH has been shown to serve as a backup for CatC-mediated granzyme B activation in cytotoxic lymphocytes (38). In human neutrophil precursor cell lines we did not observe any additional convertase activity when CatC is inhibited in keeping with the results of Pham et al (19).…”

Cystatins, cysteine peptidase inhibitors, as regulators of immune cell cytotoxicity

“…(93). As noted above, the main cathepsin responsible for granzyme activation is cathepsin C but, at least for granzyme B, the alternative pro-granzyme convertase is cathepsin H. Furthermore, even cathepsin C and H null mice exhibit some residual granzyme B activity, suggesting another mechanism of activation (40).…”

“…These results suggested that there are other peptidases involved in granzyme activation and, for granzyme B, cathepsin H was found as an additional peptidase. However, cytotoxic lymphocytes from cathepsin C and H null mice still retain residual granzyme B activity, implying that further peptidases are involved in granzyme B activation (40). In addition to activation of granule-localized serine peptidases, cathepsins are also involved in perforin processing in cytotoxic cells.…”

“…Inside cytotoxic granules this dipeptide is cleaved off and the granzymes are activated. Cleavage of the dipeptide is carried out primarily by cathepsin C however, at least for granzyme B, it can be catalysed also by cathepsin H (40,94). The expression of granzymes is in generally limited to lymphoid cells and the only cells known to synthesize and store granzymes constitutively are NK cells, natural killer T cells (NKT), and gd T cells, whereas in other cells granzymes are expressed only after stimulation, e.g.…”

“…In addition to perforin and granzymes, the cytotoxic granules contain several other molecules, such as cysteine peptidases cathepsins C, H and L and lysosomal membrane proteins Lamp-1, Lamp-2 and Lamp-3 (40,86).…”