Mast Cell and Basophil Granule Proteases - In Vivo Targets and Function

Abstract

Proteases are stored in very large amounts within abundant cytoplasmic granules of mast cells (MCs), and in lower amounts in basophils. These proteases are stored in their active form in complex with negatively charged proteoglycans, such as heparin and chondroitin sulfate, ready for rapid release upon MC and basophil activation. The absolute majority of these proteases belong to the large family of chymotrypsin related serine proteases. Three such enzymes are found in human MCs, a chymotryptic enzyme, the chymase, a tryptic enzyme, the tryptase and cathepsin G. Cathepsin G has in primates both chymase and tryptase activity. MCs also express a MC specific exopeptidase, carboxypeptidase A3 (CPA3). The targets and thereby the functions of these enzymes have for many years been the major question of the field. However, the fact that some of these enzymes have a relatively broad specificity has made it difficult to obtain reliable information about the biologically most important targets for these enzymes. Under optimal conditions they may cleave a relatively large number of potential targets. Three of these enzymes, the chymase, the tryptase and CPA3, have been shown to inactivate several venoms from snakes, scorpions, bees and Gila monster. The chymase has also been shown to cleave several connective tissue components and thereby to be an important player in connective tissue homeostasis. This enzyme can also generate angiotensin II (Ang II) by cleavage of Ang I and have thereby a role in blood pressure regulation. It also display anticoagulant activity by cleaving fibrinogen and thrombin. A regulatory function on excessive T_H2 immunity has also been observed for both the chymase and the tryptase by cleavage of a highly selective set of cytokines and chemokines. The chymase also appear to have a protective role against ectoparasites such as ticks, mosquitos and leeches by the cleavage of their anticoagulant proteins. We here review the data that has accumulated concerning the potential in vivo functions of these enzymes and we discuss how this information sheds new light on the role of MCs and basophils in health and disease.

Keywords: CPA3; chymase; mast cell; serine protease; tryptase.

Figure 1

Nomenclature of the amino acids surrounding the cleaved peptide bond and the amino acids forming the active site pocket. Panel A shows the amino acids N-terminal from the cleaved bond are termed P1 (where cleavage occurs, depicted by scissors), P2, P3 etc. Amino acids C-terminal of the cleaved bond are termed P1’ (adjacent to P1), P2’, P3’ etc. The corresponding interacting sub-sites in the enzyme are denoted with S. Panel B shows the S1 pocket, which is important in determining the primary specificity of the chymotrypsin family. The important residues are shown, which determine chymotrypsin-, trypsin- or elastase-like specificity. Three residues corresponding to positions 189, 216 and 226 in bovine pancreatic chymotrypsinogen has been found to be the amino acids forming the major part of this pocket and thereby giving the primary specificity of the enzyme (24).

Figure 2

The chymase locus of three mammalian species. An in-scale figure of the chymase locus in three mammalian species; human, mouse and rat. The genes are color coded. Granzymes are shown in dark blue, α-chymases in light blue and β-chymases as blue with a darker tint. mMCP-8-related genes are in cyan, duodenases in red and cathepsin G in green. The general scale bar of 50 kB is valid for most of the genes. However, the rat locus is much larger in size why we have reduced the size to half. For rat we have therefore introduced a 100 kB size marker.

Figure 3

Angiotensin cleavage. Panel A shows the N-terminal end of angiotensinogen including the 10 amino acid and the cleavage sites in angiotensinogen for renin and for the angiotensin converting enzyme (ACE). Panel B shows the Ang I peptide with the cleavage site for the majority of MC chymase at Phe 8 generating Ang II and for a few additional MC chymases from some species where their chymases also degrade Ang II by cleavage at Tyr 4.

Figure 4

Cleavage of adhesion molecules and thrombin by the human chymase. An SDS-PAGE analysis of a cleavage analysis of a panel of different cell adhesion molecules and of the coagulation factor thrombin by the human MC chymase. C marks the target protein without addition of the chymase, as control, and HC marks the cleavage of the target molecule by the human chymase. As a control for the activity of the human chymase is also a recombinant 2xTrx substrate included, where an optimal cleavage site for this enzyme is inserted between two copies of the E. coli redox protein thioredoxin (43). In panel (A) forty ng of HC was used in each of the cleavage reactions. In panel (B) which is a second cleavage reaction included of thrombin, there we used five times higher amount, or 200 ng, of the human chymase.

Figure 5

A T_H2 immune response. A number of cytokines have been shown to be potent inducers of T_H2 mediated immunity. The most well characterized are thymic stromal lymphopoietin (TSLP), IL-33, IL-18, IL-25 and IL-4. IL-18 has been shown to be potent inducers of T_H2 mediated immunity when present alone and not in combination with IL-12. Interestingly when present together with IL-12, IL-18 acts instead as an enhancer of T_H1 mediated immunity (85). IL-4 and IL-13 are the only two cytokines known to induce isotype switching in B cells to IgE (86). IL-5 is important for eosinophil infiltration, activation and proliferation, and IL-31 acts as an inducer of itch in skin of atopic dermatitis patients. IL-9 is, in mice, an inducer of mucosal mast cell differentiation (87). Both IL-15 and IL-21 has been found to have T_H2 promoting activity as described in the text. Cleavage of the T_H2 initiating early cytokines would most likely result in a dampening effect on T_H2 mediated immunity.

Figure 6

Targets for the human MC-Chymase. A number of potential targets for the human MC chymase is summarized.

Figure 7

Targets for the human MC-Tryptase. A number of potential targets for the human MC tryptase is summarized.

Figure 8

Targets for the human MC-CPA3. A number of potential targets for the human MC carboxypeptidase A3 (CPA3) is summarized.

Figure 9

Targets for the mouse basophil proteases mMCP-8 and mMCP-11. A number of potential targets for the mouse basophil proteases mMCP-8 and mMCP-11 is summarized. It is only for mMCP-8 that we so far have any targets identified in mouse PDGF-B, MIP-3a and tubulin. For both mMCP-8 and mMCP-11 we know that they induce inflammation and immune cell infiltration. Human basophils express the MC-tryptase and CPA3. Mouse basophils also express CPA3.