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- Aveskogh, Maria, et al.
(författare)
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Characterization of cDNA clones encoding mouse proteinase 3 (myeloblastine) and cathepsin G
- 1997
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Ingår i: Immunogenetics. - : Springer Science and Business Media LLC. - 0093-7711 .- 1432-1211. ; 46:3, s. 181-191
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Tidskriftsartikel (refereegranskat)abstract
- Serine proteases are the most abundant granule constituents of several major hematopoietic cell lineages. Due to their high abundance and their strict tissue specificity they have become important phenotypic cell markers used for studies of various aspects of hematopietic cell development. Using a polymerase chain reaction (PCR)-based strategy for the isolation of trypsin-related serine proteases, we were able to isolate cDNAs for two of the major neutrophil and monocyte serine proteases in the mouse, cathepsin G and mouse protease 3 (myeloblastin). The internal PCR fragments were used as probes to screen a mouse mast cell cDNA library and a cDNA library originating from a mouse monocytic cell line (WEHI-274.1). Full-length cDNAs for mouse cathepsin G and proteinase 3 were isolated and their complete sequences were determined. Northern blot analysis revealed expression of cathepsin G in immature cells of the monocyte macrophage lineage but also in the connective tissue mast cell line MTC. Proteinase 3 was expressed in several cell lines of myelo-monocytic origin and in one B-cell line, but not in any of the other cell lines tested. The isolation of cDNAs for mouse cathepsin G and mouse proteinase 3, together with the previous characterization of the gene for mouse N-elastase, and the entire or partial amino acid sequences for porcine azurocidine, equine N-elastase and proteinase 3, rat, dog, and rabbit cathepsin Gs in evolutionary relatively distantly related mammalian species, indicates that these four members of the serine protease family have been maintained for more than 100 million years of mammalian evolution. This latter finding indicates a strong evolutionary pressure to maintain specific immune functions associated with these neutrophil and monocyte proteases. All amino acid positions of major importance for the cleavage site selection have also been fully conserved between mouse and human proteinase 3 and a few minor changes have occurred between mouse and human cathepsin G.
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| 2. |
- Lützelschwab, Claudia, et al.
(författare)
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Characterization of mouse mast cell protease-8, the first member of a novelsubfamily of mouse mast cell serine proteases, distinct from both theclassical chymases and tryptases
- 1998
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Ingår i: European Journal of Immunology. - 0014-2980 .- 1521-4141. ; 28:3, s. 1022-1033
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Tidskriftsartikel (refereegranskat)abstract
- Using a recently developed PCR-based strategy, a cDNA encoding a novel mouse mast cell (MC) serine protease (MMCP-8) was isolated and characterized. The MMCP-8 mRNA contains an open reading frame of 247 amino acids (aa), divided into a signal sequence of 18 aa followed by a 2-aa activation peptide (Gly-Glu) and a mature protease of 227 aa. The mature protease has an M(r) of 25072, excluding post-translational modifications, a net positive charge of +12 and six potential N-glycosylation sites. MMCP-8 showed a high degree of homology with mouse granzyme B in the critical regions for determining substrate cleavage specificity, indicating that MMCP-8, similar to granzyme B, preferentially cleaves after Asp residues. A comparative analysis of the aa sequence of MMCP-8 with other hematopoietic serine proteases shows that it is more closely related to cathepsin G and T cell granzymes than to the MC chymases. We therefore conclude that MMCP-8 belongs to a novel subfamily of mouse MC proteases distinct from both the classical chymases and tryptases. Southern blot analysis of BALB/c genomic DNA indicated that only one MMCP-8 gene (or MMCP-8 like gene) is present in the mouse genome. Northern blot analysis of rodent hematopoietic cell lines revealed high levels of MMCP-8 mRNA in a mouse connective tissue MC-like tumor line. However, MMCP-8 mRNA could not be detected in mouse liver, intestine, lung or ears, indicating very low expression in normal tissues. Analysis of the expression of different MMCP in the tissues of Schistosoma mansoni-infected BALB/c mice showed a strong increase in MMCP-8 levels in the lungs but not in the intestines of infected animals, suggesting the presence of a novel subpopulation of MC in the lungs that expressed MMCP-8, either alone or in combination with MMCP-5 and carboxypeptidase A. The dramatic increase in MMCP-1 and MMCP-2 levels but not of MMCP-8 in the intestines of parasitized animals also shows that MMCP-8 is not expressed in mucosal MC in the mouse. This latter is in clear contrast to what has been observed in the rat where the MMCP-8 homologues, RMCP-8, -9 and -10, can be considered as true mucosal MC proteases.
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