Mast cells (MC) are resident tissue cells found primarily at the interphase between tissues and the environment. These evolutionary old cells store large amounts of proteases within cytoplasmic granules, and one of the most abundant of these proteases is tryptase. To look deeper into the question of their in vivo targets, we have analyzed the activity of the human MC tryptase on 69 different human cytokines and chemokines, and the activity of the mouse tryptase (mMCP-6) on 56 mouse cytokines and chemokines. These enzymes were found to be remarkably restrictive in their cleavage of these potential targets. Only five were efficiently cleaved by the human tryptase: TSLP, IL-21, MCP3, MIP-3b, and eotaxin. This strict specificity indicates a regulatory function of these proteases and not primarily as unspecific degrading enzymes. We recently showed that the human MC chymase also had a relatively strict specificity, indicating that both of these proteases have regulatory functions. One of the most interesting regulatory functions may involve controlling excessive TH2-mediated inflammation by cleaving several of the most important TH2-promoting inflammatory cytokines, including IL-18, IL-33, TSLP, IL-15, and IL-21, indicating a potent negative feedback loop on TH2 immunity.

Mast cell tryptases have crucial roles in allergic and inflammatory diseases. The mouse tryptase genes represent a cluster of loci on chromosome 16p3.3. While their functional studies have been extensively performed, transcriptional regulation of tryptase genes is poorly understood. In this study, we examined the molecular basis of the tryptase gene expression in bone marrow-derived mast cells (BMMCs) of C57BL/6 mice and in MEDMC-BRC6 mast cells. The expression of the Tpsb2 and Tpsg1 genes, which reside at the 3'-end of the tryptase locus, is significantly decreased by the reduction of the GATA transcription factors GATA1 or GATA2. Chromatin immunoprecipitation assays have shown that the GATA factors bind at multiple regions within the locus, including 1.0 and 72.8 kb upstream of the Tpsb2 gene, and that GATA1 and GATA2 facilitate each other's DNA binding activity to these regions. Deletion of the -72.8 kb region by genome editing significantly reduced the Tpsb2 and Tpsg1 mRNA levels in MEDMC-BRC6 cells. Furthermore, binding of CTCF and the cohesin subunit Rad21 was found upstream of the -72.8 kb region and was significantly reduced in the absence of GATA1. These results suggest that mouse tryptase gene expression is coordinately regulated by GATA1 and GATA2 in BMMCs.

Mast cells (MCs) are inflammatory cells primarily found in tissues in close contact with the external environment, such as the skin and the intestinal mucosa. They store large amounts of active components in cytoplasmic granules, ready for rapid release. The major protein content of these granules is proteases, which can account for up to 35 % of the total cellular protein. Depending on their primary cleavage specificity, they can generally be subdivided into chymases and tryptases. Here we present the extended cleavage specificities of two such proteases from the platypus. Both of them show an extended chymotrypsin-like specificity almost identical to other mammalian MC chymases. This suggests that MC chymotryptic enzymes have been conserved, both in structure and extended cleavage specificity, for more than 200 million years, indicating major functions in MC-dependent physiological processes. We have also studied a third closely related protease, originating from the same chymase locus whose cleavage specificity is closely related to the apoptosis-inducing protease from cytotoxic T cells, granzyme B. The presence of both a chymase and granzyme B in all studied mammals indicates that these two proteases bordering the locus are the founding members of this locus.

Serine proteases constitute the major protein content of mast cell (MC) secretory granules. These proteases can generally be subdivided into chymases and tryptases based on their primary cleavage specificity. Here, we presented the extended cleavage specificities of a rabbit β-chymase and a guinea pig α-chymase. Analyses by phage display screening and a panel of recombinant substrates showed a marked similarity in catalytic activity between the enzymes, both being strict Leu-ases (cleaving on the carboxyl side of Leu). Amino acid sequence alignment of a panel of mammalian chymotryptic MC proteases and 3D structural modeling identified an unusual residue in the rabbit enzyme at position 216 (Thr instead of more common Gly), which is most likely critical for the Leu-ase specificity. Almost all mammals studied, except rabbit and guinea pig, express classical chymotryptic enzymes with similarly extended specificities, indicating an important role of chymase in MC biology. The rabbit and guinea pig are the only two mammalian species currently known to lack a classical MC chymase. Key questions are now how this major difference affects their MC function, and if genes of other loci can rescue the loss of a chymotryptic activity in MCs of these two species.