Expression profile of novel members of the rat mast cell protease (rMCP)-2 and (rMCP)-8 families, and functional analyses of mouse mast cell protease (mMCP)-8.
Four hematopoietic serine proteases are common to the mast cell chymase locus of all analyzed mammals: alpha-chymase, cathepsin G, granzyme B, and granzyme C/H. Apart from these common genes, the mouse and rat loci hold additional granzyme-, beta-chymase-, and Mcpt8-like genes. To better understand the functional consequences of these additional enzymes and to be able to compare human and rodent immune functions, we have analyzed the expression of novel beta-chymase- and Mcpt8-like genes in the rat. Four novel genes, i.e., Mcpt2-rs2a, Mcpt2-rs2c, Mcpt8-rs1, and Mcpt8-rs4 were transcribed in tissues holding mucosal mast cells (MMC), where also the classical MMC protease Mcpt2 was expressed. We also found transcripts of rat vascular chymase (rVch) in some of these tissues. RVch is a beta-chymase that converts angiotensin I, like the human chymase. Rat MMC may therefore have similar angiotensin-converting properties as chymase-positive human mast cells, although these are mostly regarded the counterpart of rat connective tissue mast cells. The human mast cells that are considered the counterpart of rat MMC express, however, only tryptase, whereas rat MMC express various proteases, but no tryptase. We further studied the proteolytic activity of mMCP-8 as a first representative for the Mcpt8-subfamily. Based on sequence comparison and molecular modeling, mMCP-8 may prefer aspartic acid in substrate P1 position. However, we could not detect hydrolysis of chromogenic substrates or phage-displayed random nonapeptides despite numerous trials. On the other hand, we have obtained evidence that the function of the Mcpt8-like proteases depends on proteolytic activity. Namely, the expression of the only Mcpt8-family member with a mutation in the catalytic triad, Mcpt8-rs3, was strongly reduced. Thus, the substrate specificity of mMCP-8 may be too narrow to be detected with the employed methods, or the enzyme may require a substrate conformation that is not provided by the analyzed peptides.
Gallwitz
Maike
M
Department of Cell and Molecular Biology, The Biomedical Center, Uppsala University, Box 596, SE-751 24, Uppsala, Sweden.
Enoksson
Mattias
M
Hellman
Lars
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D053818:Chymases / Q000145:classification / Q000235:genetics*; D015870:Gene Expression; D006868:Hydrolysis; D051379:Mice; D008969:Molecular Sequence Data; D010802:Phylogeny; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000145:classification; D013379:Substrate Specificity; D053802:Tryptases / Q000737:chemistry* / Q000145:classification / Q000235:genetics*
D011994:Recombinant Proteins; C083294:chymase 2; D053818:Chymases; C512639:Mcpt8 protein, mouse; C512640:Mcpt8 protein, rat; D053802:Tryptases