Extended cleavage specificities of two mast cell chymase-related proteases and one granzyme B-like protease from the platypus, a monotreme.

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.

Highly Selective Cleavage of TH2-Promoting Cytokines by the Human and the Mouse Mast Cell Tryptases, Indicating a Potent Negative Feedback Loop on TH2 Immunity.

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.

Chymase activities and survival in endotoxin-induced human chymase transgenic mice.

We examined the effects of overexpressed human chymase on survival and activity in lipopolysaccharide (LPS)-treated mice. Human chymase transgenic (Tg) and wild-type C57BL/6 (WT) mice were treated with LPS (0.03, 0.1 and 0.3 mg/day; intraperitoneal) for 2 weeks. Treatment with 0.03 mg LPS did not affect survival in either WT or Tg mice. WT mice were not affected by 0.1 mg/day of LPS, whereas 25% of Tg mice died. Survival of mice treated with 0.3 mg/day of LPS was 87.5% and 0% in WT and Tg, respectively. LPS-induced increases in chymase activity in the heart and skin were significantly greater in Tg than WT mice. These data suggest a possible contribution of human chymase activation to LPS-induced mortality.

Effects of Panax notoginseng flower extract on the TGF-ß/Smad signal transduction pathway in heart remodeling of human chymase transgenic mice.

Panax notoginseng is a common Chinese herb extensively used in Chinese medical practice for the treatment of cardiovascular diseases. The present study aimed to evaluate the effects of Panax notoginseng flower extract (PNFE) on the TGF-ß/Smad signal transduction pathway in heart remodeling of human chymase transgenic mice. After treatment with PNFE and soybean trypsin inhibitor (SBTI), the left ventricular mass indexes (LVMIs) of transgenic and normal C57 BL/6 mice were analyzed. The mRNA expression of chymase, TGF-ß1, Smad2, Smad3 and Smad7 in myocardium was assessed with RT-PCR, while the protein expression in myocardium was detected by western blotting. The results showed that PNFE and SBTI treatment led to a significant reduction in LVMIs in transgenic mice, indicating a beneficial effect on left ventricular remodeling. Mechanistically, PNFE and SBTI treatment attenuated the mRNA expression of chymase, TGF-ß1, Smad2 and Smad3, as well as the protein expression in the myocardium tissues of the transgenic mouse model. By contrast, PNFE and SBTI treatment markedly up-regulated the mRNA and protein expression of Smad7. It was concluded that PNFE was able to improve the ventricular hypertrophy state in human chymase transgenic mice through regulation of the expression of mRNA and protein of TGF-ß/Smad in ventricular tissues.