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

Abstract

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.

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

“…All of these MMC-specific, or blood vessel wall (RVC)-specific β-chymases have chymotrypsin-like activity, showing a preference for aromatic amino acids in the P1 position, and similarly to the primate α-chymases they also show a preference for aliphatic amino acids at several positions, both N-and C-terminally of the cleavage site. However, they lack the preference for negatively charged amino acids in the P2 position that is characteristic of the primate α-chymases (Figures 6 and 7) [36,43,45,48] and furthermore, they have preferences for Ser or Arg in the P1 position, just C-terminally of the cleavage site, which is not seen in primate α-chymases (Figures 4, 6 and 7) [36,43,45].…”

“…Members of this category are the mouse mMCP-1 and the rat rMCP-2, rMCP-3 and rMCP-4 (Figure 6) [36,43,45]. In the rat there is also a β-chymase that is not expressed in hematopoietic cells but in vascular smooth muscle cells, which is therefore named the rat vascular chymase (RVC) [48]. All of these MMC-specific, or blood vessel wall (RVC)-specific β-chymases have chymotrypsin-like activity, showing a preference for aromatic amino acids in the P1 position, and similarly to the primate α-chymases they also show a preference for aliphatic amino acids at several positions, both N-and C-terminally of the cleavage site.…”

“…and similarly to the primate α-chymases they also show a preference for aliph at several positions, both N-and C-terminally of the cleavage site. Howeve preference for negatively charged amino acids in the P2´ position that is char primate α-chymases (Figures 6 and 7) [36,43,45,48] and furthermore, they have Ser or Arg in the P1´ position, just C-terminally of the cleavage site, which primate α-chymases (Figures 4, 6 and 7) [36,43,45]. Interestingly when analyzing the β-chymase in rabbits and the α-chymase in guine pigs (Cma1-like and Cma1), we can see that they have both become strict Leu-ases an are no longer classical chymases (Figure 7) [50,49].…”

Mast Cell Chymase and Kidney Disease

“…The situation is more complicated in rats, as rat MC chymase rMCP-1 showed Ang II-degrading activity [ 53 ]. However, other enzymes not produced by MCs, such as rat vascular chymase (RVC) or rat mesenteric arterial bed elastase-2, may provide a relay in this species [ 54 , 55 , 56 ]. The possibility to have ACE-independent Ang II generation launched a series of studies evoking a possible role of chymase-dependent Ang II formation in various human tissues, such as the vasculature [ 51 ], heart [ 12 ] and kidneys after a high salt intake [ 57 ] and in patients with diabetic nephropathy [ 58 ].…”

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

“…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.…”