Dipeptidyl Peptidase I Is Essential for Activation of Mast Cell Chymases, but Not Tryptases, in Mice

Abstract

Dipeptidyl peptidase I (DPPI) is the sole activator in vivo of several granule-associated serine proteases of cytotoxic lymphocytes. In vitro, DPPI also activates mast cell chymases and tryptases. To determine whether DPPI is essential for their activation in vivo, we used enzyme histochemical and immunohistochemical approaches and solution-based activity assays to study these enzymes in tissues and bone marrow-derived mast cells (BMMCs) from DPPI ؉/؉ and DPPI ؊/؊ mice. We find that DPPI ؊/؊ mast cells contain normal amounts of immunoreactive chymases but no chymase activity, indicating that DPPI is essential for chymase activation and suggesting that DPPI ؊/؊ mice are functional chymase knockouts. The absence of DPPI and chymase activity does not affect the growth, granularity, or staining characteristics of BMMCs and, despite prior predictions, does not alter IgE-mediated exocytosis of histamine. In contrast, the level of active tryptase (mMCP-6) in DPPI ؊/؊ BMMCs is 25% that of DPPI ؉/؊ BMMCs. These findings indicate that DPPI is not essential for mMCP-6 activation but does influence the total amount of active mMCP-6 in mast cells and therefore may be an important, but not exclusive mechanism for tryptase activation.Granule-associated serine proteases of cytotoxic lymphocytes, neutrophils, and mast cells (i.e. granzymes, elastase, cathepsin G, and chymases) are structurally related (1). They have a two-amino acid propeptide (also referred to as the "activation dipeptide") and an isoleucine at the NH 2 terminus of the activated enzyme (2). The activation dipeptide maintains the protease in an inactive state. After removal of the dipeptide, the new NH 2 -terminal isoleucine moves from its position on the surface of the protease to a region in the interior of the enzyme, where it interacts with an aspartate residue near the catalytic center (3). These movements are thought to change the enzyme substrate binding cleft and catalytic apparatus to a catalytically competent conformation.Initial studies investigating the activation of the granuleassociated serine proteases focused on the cysteine protease dipeptidyl peptidase I (DPPI), 1 also known as cathepsin C (2, 4 -8). DPPI was a logical candidate activator of these proteases because it is found in the same cells and is a promiscuous exopeptidase that hydrolyzes most NH 2 -terminal dipeptides (9). Studies using the DPPI inhibitor Gly-Phe-diazomethyl ketone reported that DPPI inhibition reduces granzyme A, neutrophil elastase, and cathepsin G activity in the cells (2). However, because the inhibitor is not entirely specific for DPPI and does not eliminate the activity of the proteases in question, the possibility remained that another protease was responsible for their activation. This possibility was tested in a DPPI knockout mouse (10). Cytotoxic lymphocytes of these animals produce normal amounts of granzymes A and B, but both enzymes are inactive and present in their pro-forms. These findings suggest that DPPI is the sole activator of granzymes A and B in vivo...

Protease-Activated Receptor 2, Dipeptidyl Peptidase I, and Proteases Mediate Clostridium difficile Toxin A Enteritis

“…9 Mice lacking DPPI exhibit a 75% reduction in the normal tryptase levels in mast cells and show increased survival from sepsis, which is directly due to the lack of DPPI in mast cells. 8,10 Proteases that activate PAR 2 are released and activated during inflammation and injury, and PAR 2 controls responses to these insults, with effects on gastrointestinal secretion, motility, inflammation, and pain. 1,3,5,[11][12][13] We investigated the role of PAR 2 and proteases that activate PAR 2 in enteritis induced by toxin A (TxA) from Clostridium difficile.…”

Diverse regulatory roles for lysosomal proteases in the immune response

“…These enzymes are initially made as inactive zymogens and depend on the amino dipeptidase cathepsin C (also known as DPPI), which removes two N-terminal residues to trigger activation [56]. Mice lacking cathepsin C fail to activate neutrophil and mast cell granule proteases [57,58]. Defects in cytotoxic T-cell function have also been reported in these mice [59].…”

Critical role of dipeptidyl peptidase I in neutrophil recruitment during the development of experimental abdominal aortic aneurysms

“…These serine proteases share a high degree of homology at the cDNA and amino acid levels, and all are synthesized as preproenzymes requiring activation by DPPI granules. Indeed, mice with a loss-of-function mutation in DPPI are systematically deficient in neutrophil elastase, cathepsin G, and proteinase 3 activities, as well as cytotoxic T cell granzymes and mast cell chymase (21)(22)(23). DPPI Ϫ/Ϫ mice thereby provide a powerful tool to investigate the role of granule-associated serine proteases in murine models of inflammatory diseases.…”