Extended Cleavage Specificities of Two Mast Cell Chymase-Related Proteases and One Granzyme B-Like Protease from the Platypus, a Monotreme

et al. 2020

IJMS

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Serine proteases constitute the major protein content of the cytoplasmic granules of several hematopoietic cell lineages. These proteases are encoded from four different loci in mammals. One of these loci, the chymase locus, has in rats experienced a massive expansion in the number of functional genes. The human chymase locus encodes 4 proteases, whereas the corresponding locus in rats contains 28 such genes. One of these new genes has changed tissue specificity and has been found to be expressed primarily in vascular smooth muscle cells, and therefore been named rat vascular chymase (RVC). This β-chymase has been claimed to be a potent angiotensin-converting enzyme by cleaving angiotensin (Ang) I into Ang II and thereby having the potential to regulate blood pressure. To further characterize this enzyme, we have used substrate phage display and a panel of recombinant substrates to obtain a detailed quantitative view of its extended cleavage specificity. RVC was found to show a strong preference for Phe and Tyr in the P1 position, but also to accept Leu and Trp in this position. A strong preference for Ser or Arg in the P1’ position, just C-terminally of the cleavage site, and a preference for aliphatic amino acids in most other positions surrounding the cleavage site was also seen. Interesting also was a relatively strict preference for Gly in positions P3’ and P4’. RVC thereby shares similarity in its specificity to the mouse mucosal mast cell chymase mMCP-1, which efficiently converts Ang I to Ang II. This similarity adds support for the role of β-chymases as potent angiotensin converters in rodents, as their α-chymases, which have the capacity to efficiently convert Ang I into Ang II in other mammalian lineages, have become elastases. However, interestingly we found that RVC cleaved both after Arg2 and Phe8 in Ang I. Furthermore this cleavage was more than two hundred times less efficient than the consensus site obtained from the phage display analysis, indicating that RVC has a very low ability to cleave Ang I, raising serious doubts about its role in Ang I conversion.

Section: Figurementioning

confidence: 99%

Extended Cleavage Specificity of the Rat Vascular Chymase, a Potential Blood Pressure Regulating Enzyme Expressed by Rat Vascular Smooth Muscle Cells

Berglund

et al. 2020

IJMS

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“…The third platypus enzyme, granzyme BGH-like, is located on another contig (Figure 1). This enzyme was found to be a classical Asp-ase with a preference for negatively charged residues in the P1 position, similarly to human, rat and opossum granzyme B [52][53][54].…”

Section: Extended Cleavage Specificities Of Chymase-locus-expressed Serine Proteasesmentioning

confidence: 82%

“…We found that two of the platypus enzymes are chymases, named granzyme B and DDN-1-like, and exhibit very similar specificity to that of the human chymase. They both show preferences for Phe and Tyr over Trp and Leu in the P1 position and also show preferences for aliphatic amino acids both N-and C-terminally of the cleavage site [53]. The third platypus enzyme, granzyme BGH-like, is located on another contig (Figure 1).…”

Section: Extended Cleavage Specificities Of Chymase-locus-expressed Serine Proteasesmentioning

confidence: 99%

The Evolutionary History of the Chymase Locus -a Locus Encoding Several of the Major Hematopoietic Serine Proteases

Wernersson

et al. 2021

IJMS

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Several hematopoietic cells of the immune system store large amounts of proteases in cytoplasmic granules. The absolute majority of these proteases belong to the large family of chymotrypsin-related serine proteases. The chymase locus is one of four loci encoding these granule-associated serine proteases in mammals. The chymase locus encodes only four genes in primates, (1) the gene for a mast-cell-specific chymotryptic enzyme, the chymase; (2) a T-cell-expressed asp-ase, granzyme B; (3) a neutrophil-expressed chymotryptic enzyme, cathepsin G; and (4) a T-cell-expressed chymotryptic enzyme named granzyme H. Interestingly, this locus has experienced a number of quite dramatic expansions during mammalian evolution. This is illustrated by the very large number of functional protease genes found in the chymase locus of mice (15 genes) and rats (18 genes). A separate expansion has also occurred in ruminants, where we find a new class of protease genes, the duodenases, which are expressed in the intestinal region. In contrast, the opossum has only two functional genes in this locus, the mast cell (MC) chymase and granzyme B. This low number of genes may be the result of an inversion, which may have hindered unequal crossing over, a mechanism which may have been a major factor in the expansion within the rodent lineage. The chymase locus can be traced back to early tetrapods as genes that cluster with the mammalian genes in phylogenetic trees can be found in frogs, alligators and turtles, but appear to have been lost in birds. We here present the collected data concerning the evolution of this rapidly evolving locus, and how these changes in gene numbers and specificities may have affected the immune functions in the various tetrapod species.

“…We, therefore, wanted to look deeper into its specificity. We started with phage display as this technique can give a detailed picture of the extended specificity of a protease [ 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 41 , 42 , 43 , 44 , 45 , 46 , 47 ]. We tried more than 10 times with the phage display technique and also with a large panel of both chromogenic and recombinant substrates to identify the cleavage specificity of mMCP-8, however, without success why we doubted if our protease was active.…”

Section: Resultsmentioning

confidence: 99%

“…Cleavage of the anticoagulant proteins, hirudin and anophelin, by a panel of mammalian hematopoietic serine proteases.The efficiency in cleavage of three anticoagulant proteins were assayed in a 15 µL sample volume with approximately 1 µg of the anticoagulant protein and varying amount of protease depending on the activity as determined previously. All proteases except mMCP-8 have been described in previous publications (Human chymase[30], Dog chymase[31], rMCP-1[32], Rat vascular chymase (RVC)[33], rMCP-2[34], Hamster chymase[35], Rabbit and Guinea pig Leu-ases[29], Opossum chymase[36], Platypus chymases[37], Human cathepsin G[38], Human proteinase 3 and N-elastase[39], Human mast cell tryptase and mMCP-6[40]. Panel (A) shows the amino acid sequence of hirudin where the cysteines have been marked in red and all negatively charged residues in green.…”

mentioning

confidence: 99%

Mast Cells and Basophils in the Defense against Ectoparasites: Efficient Degradation of Parasite Anticoagulants by the Connective Tissue Mast Cell Chymases

Olsson

et al. 2021

IJMS

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Ticks, lice, flees, mosquitos, leeches and vampire bats need to prevent the host’s blood coagulation during their feeding process. This is primarily achieved by injecting potent anticoagulant proteins. Basophils frequently accumulate at the site of tick feeding. However, this occurs only after the second encounter with the parasite involving an adaptive immune response and IgE. To study the potential role of basophils and mast cells in the defense against ticks and other ectoparasites, we produced anticoagulant proteins from three blood-feeding animals; tick, mosquito, and leech. We tested these anticoagulant proteins for their sensitivity to inactivation by a panel of hematopoietic serine proteases. The majority of the connective tissue mast cell proteases tested, originating from humans, dogs, rats, hamsters, and opossums, efficiently cleaved these anticoagulant proteins. Interestingly, the mucosal mast cell proteases that contain closely similar cleavage specificity, had little effect on these anticoagulant proteins. Ticks have been shown to produce serpins, serine protease inhibitors, upon a blood meal that efficiently inhibit the human mast cell chymase and cathepsin G, indicating that ticks have developed a strategy to inactivate these proteases. We show here that one of these tick serpins (IRS-2) shows broad activity against the majority of the mast cell chymotryptic enzymes and the neutrophil proteases from human to opossum. However, it had no effect on the mast cell tryptases or the basophil specific protease mMCP-8. The production of anticoagulants, proteases and anti-proteases by the parasite and the host presents a fascinating example of an arms race between the blood-feeding animals and the mammalian immune system with an apparent and potent role of the connective tissue mast cell chymases in the host defense.