Inhibition of dipeptidyl peptidase I in the human mast cell line HMC-1: blocked activation of tryptase, but not of the predominant chymotryptic activity

Abstract

The mast cell proteases tryptase and chymase are synthesised as inactive precursors, but are stored and secreted as active enzymes. The cysteinyl protease dipeptidyl peptidase I (DPPI, cathepsin C) can activate the corresponding proenzymes in cell-free systems, but it is unknown whether it fulfils this role within the intact cell. We, therefore, tested the effect the DPPI-selective inhibitor Gly-Phe diazomethyl ketone (Gly-Phe-CHN₂) on the tryptic and chymotryptic activity of the human mast cell-like cell line, HMC-1, and monitored any changes in the amount of immunodetectable enzymes by flow cytometry. Culture in Gly-Phe-CHN₂ produced a significant decrease in tryptase activity in cell lysates within 24 hr and further decreases during continued culturing to 216 hr with periodic replenishment of Gly-Phe-CHN₂-containing media. Flow cytometry showed no significant change in the levels of immunoreactive tryptase. In contrast, chymotryptic activity in treated cells did not differ significantly from untreated cells at any time point. Treatment of 216 hr cell lysates with DPPI revealed significant amounts of activatable protryptase in Gly-Phe-CHN₂-treated cells, but not in controls, whereas activatable prochymotryptic activity was found in both treated and control cells. Chymase was detected immunologically, though small differences in substrate specificity and molecular mass were observed. These results strongly suggest that DPPI plays a role in the activation of tryptase, but not of the predominant chymotryptic activity of HMC-1 cells. As inhibitors of tryptase have proven efficacious in models of allergic disease, these results also indicate that inhibitors of DPPI might provide an additional point of therapeutic control.

Introduction

Mast cells are phenotypically and functionally versatile tissue-dwelling cells capable of secreting a wide variety of mediators [1]. Their role in provoking the immediate hypersensitivity response of allergic reactions is well established and there is increasing evidence for their involvement in chronic inflammation and tissue remodelling, including fibrosis, development of atherosclerotic plaques and angiogenesis in tumours. They are characteristically activated through allergen-dependent cross-linking of high-affinity receptors for IgE (FcεRI), but can also be activated by superoxide, complement proteins, opioids, neuropeptides and lipoproteins. Of particular importance amongst the products secreted by activated mast cells are a number of proteases including tryptase, chymase, carboxypeptidase and cathepsin G, which can rapidly process a variety of biologically active peptides and proteins or their precursors [2].

Tryptase (EC 3.4.21.59), a serine protease with trypsin-like specificity, and chymase (EC 3.4.21.39), a serine protease with chymotrypsin-like specificity, are particularly abundant in the mast cell granule, with up to 30 and 5 pg per cell, respectively, compared with 2 pg per cell histamine [2]. Cathepsin G (EC 3.4.21.20), which, like chymase, is also a serine protease with chymotrypsin-like specificity, is less abundant, being present at about one-tenth the levels of chymase [3]. These three proteases have been shown to act on specific extracellular proteins and peptides, as well as to alter the behaviour of various cell types [2], [4], [5], [6]. Tryptase inhibitors can block allergen-induced airway inflammatory responses in allergic sheep [7], [8], and one such inhibitor, APC-366, was able to reduce late phase bronchoconstriction in a Phase II clinical trial with asthmatic patients [9].

An alternative to therapeutic regulation of an enzyme by its inhibitors is to block the processing of an inactive precursor form of that enzyme. Tryptase, chymase and cathepsin G are synthesised as inactive precursors, but stored and released in the fully active mature forms [2]. Chymase [10], [11] and cathepsin G [12], [13] are synthesised as proenzymes with a two-amino acid extension at the N terminus, similar to other granulocyte proteases including granzyme A, granzyme B, and neutrophil elastase [12], [13], [14]. The proforms of these proteases are catalytically inactive, but removal of the N-terminal dipeptide with the cysteinyl protease DPPI (also known as cathepsin C or dipeptidylaminopeptidase I) (EC 3.4.14.1) has been shown to restore full catalytic activity. DPPI is a ubiquitous granule/lysosomal protein that sequentially removes dipeptides from the amino terminus of proteins and is particularly abundant in cells that are rich in granulocyte proteases [15]. It is unique amongst members of the papain family of cysteine proteases in that it exists as a tetramer with each monomeric unit composed of three chains: the pro-chain, the heavy chain and the light chain. The association with the pro-chain confers the dipeptidyl aminopeptidase specificity on the catalytic chains [16], [17].

Tryptase is not a single enzyme, but a family of closely related proteases. On the basis of amino acid sequence homology, these have been identified as α-tryptase and three very closely related β-tryptases, βI, βII, and βIII [2], [18]. The prosequence for all tryptases is substantially longer than that of chymase (14 amino acids). In the presence of heparin, recombinant βII-tryptase was found to process itself autocatalytically to remove the first 12 amino acids [18]. The resulting pro′enzyme was inactive but removal of the remaining dipeptide with dipeptidyl peptidase I yielded an active product. Recombinant α-tryptase was unable to carry out this autocatalytic step, and so doubts have been expressed as to whether it is activated in vivo[18].

The ability of an enzyme to catalyse a reaction in a cell-free system does not necessarily indicate that it has this role in the intact cell. Indeed, in a previous study we have shown that heparin and histamine, compounds present in the mast cell granule along with chymase, strongly inhibit the activation of recombinant prochymase by dipeptidyl peptidase I from a commercially available bovine source [11]. The availability of recombinant human DPPI [19] has permitted a re-examination in the present study as to whether any species-specific factors might be involved. Furthermore, we have been able to follow the expression and activation of both tryptase and chymase in an ex vivo cell culture system utilising the human mast cell line HMC-1. This cell line, derived from a patient with mast cell leukaemia, expresses several mast cell-related markers, including tryptase [20], [21], [22] and chymase [22]. RT-PCR studies have identified the tryptase expressed as a β-tryptase [22], consistent with the recent finding that HMC-1 cells lack the gene for α-tryptase [23]. We have tested the effects on these cells of the DPPI-selective inhibitor Gly-Phe-CHN₂[12], [13], [14], [24], and here present evidence for the first time that DPPI is responsible for processing mast cell tryptase within the mast cell. In contrast, the detectable chymotryptic activity displayed a different response to Gly-Phe-CHN₂, which raises the possibility that it might be activated by an alternative pathway.