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Secretory granule proteases in rat mast cells. Cloning of 10 different serine proteases and a carboxypeptidase A from various rat mast cell populations.

Lützelschwab C, Pejler G, Aveskogh M, Hellman L - J. Exp. Med. (1997)

Bottom Line: In contrast to what has been reported for human MCs, no expression of cathepsin G or cathepsin G-like proteases was detected in any of the rat MC populations.To determine mRNA frequencies for the various proteases expressed by normal tissue MCs, an unamplified peritoneal MC cDNA library was screened with a panel of mono-specific cDNA probes.These results showed that peritoneal MCs are highly specialized effector cells with mRNA frequencies for the major proteases in the range of several percent of the total mRNA pool.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Immunology and Microbiology, University of Uppsala, Sweden.

ABSTRACT
Two of the major rat mast cell proteases, rat mast cell protease 1 (RMCP-1) and RMCP-2, have for many years served as important phenotypic markers for studies of various aspects of mast cell (MC) biology. However, except for these proteases only fragmentary information has been available on the structure and complexity of proteases expressed by different subpopulations of rat MCs. To address these questions, cDNA libraries were constructed from freshly isolated rat peritoneal MCs and from the rat mucosal MC line RBL-1. cDNA clones for 10 different serine proteases (RMCP-1-10), and the MC carboxypeptidase A were isolated and characterized. Six of these proteases have not been isolated previously. Based on their protease content, three separate subpopulations of MCs were identified. Connective tissue MCs (CTMCs) from the ear and peritoneum express the chymases RMCP-1 and -5, the tryptases RMCP-6, and -7 and the carboxypeptidase A. However, based on a large difference in the level of expression of RMCP-7, CTMCs of these two organs may be regarded as two separate subpopulations. RMCP-2 and the three closely related proteases of the RMCP-8 subfamily were identified as the major mucosal MC proteases in rat. In contrast to what has been reported for human MCs, no expression of cathepsin G or cathepsin G-like proteases was detected in any of the rat MC populations. To determine mRNA frequencies for the various proteases expressed by normal tissue MCs, an unamplified peritoneal MC cDNA library was screened with a panel of mono-specific cDNA probes. These results showed that peritoneal MCs are highly specialized effector cells with mRNA frequencies for the major proteases in the range of several percent of the total mRNA pool.

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Northern and Southern blot analysis. (A) Northern blot analysis of poly(A)+RNA isolated from the ears of Lewis, Brown Norway, Hooded  Lister, Dark Agouti, and Sprague Dawley rats, poly(A)+RNA from purified peritoneal MCs (PMC; from Sprague Dawley rats) and poly(A)+RNA isolated from rat liver (Sprague Dawley). These mRNAs were analyzed for the expression of RMCP-1 (R-I), RMCP-5 (R-V), RMCP-6 (R-VI), RMCP-7  (R-VII), RMCP-8 (R-VIII), carboxypeptidase A (CPA), and the α chain of the IgERI using the specific probes described in Materials and Methods. (B)  Northern blot analysis of poly(A)+RNA from: Sprague Dawley lung, intestine, liver, spleen, Lewis ears and from the RBL-1 cell line. The various mRNAs  were analyzed with probes for RMCP-1 (R-I), RMCP-2 (R-II), RMCP-3 (R-III), RMCP-4 (R-IV), RMCP-5 (R-V), RMCP-8 (R-VIII), carboxypeptidase A (CPA), or the α chain of the IgERI using the specific probes described in Materials and Methods. In both A and B, a β-actin probe was  used to normalise the amount of RNA loaded in each lane to facilitate a comparative analysis between different organs and cell lines. (C) Genomic Southern blot analysis of DNA from Sprague Dawley rats. The DNA was cleaved with three different restriction enzymes, BamHI, BglII, and EcoRI. Size  markers are indicated at the left side of C. The blot was hybridized with the entire insert from the full-length cDNA clone for RMCP-8 and the blot was  washed under medium stringency conditions (2 × SSC and 0.5% SDS at 65°C).
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Figure 9: Northern and Southern blot analysis. (A) Northern blot analysis of poly(A)+RNA isolated from the ears of Lewis, Brown Norway, Hooded Lister, Dark Agouti, and Sprague Dawley rats, poly(A)+RNA from purified peritoneal MCs (PMC; from Sprague Dawley rats) and poly(A)+RNA isolated from rat liver (Sprague Dawley). These mRNAs were analyzed for the expression of RMCP-1 (R-I), RMCP-5 (R-V), RMCP-6 (R-VI), RMCP-7 (R-VII), RMCP-8 (R-VIII), carboxypeptidase A (CPA), and the α chain of the IgERI using the specific probes described in Materials and Methods. (B) Northern blot analysis of poly(A)+RNA from: Sprague Dawley lung, intestine, liver, spleen, Lewis ears and from the RBL-1 cell line. The various mRNAs were analyzed with probes for RMCP-1 (R-I), RMCP-2 (R-II), RMCP-3 (R-III), RMCP-4 (R-IV), RMCP-5 (R-V), RMCP-8 (R-VIII), carboxypeptidase A (CPA), or the α chain of the IgERI using the specific probes described in Materials and Methods. In both A and B, a β-actin probe was used to normalise the amount of RNA loaded in each lane to facilitate a comparative analysis between different organs and cell lines. (C) Genomic Southern blot analysis of DNA from Sprague Dawley rats. The DNA was cleaved with three different restriction enzymes, BamHI, BglII, and EcoRI. Size markers are indicated at the left side of C. The blot was hybridized with the entire insert from the full-length cDNA clone for RMCP-8 and the blot was washed under medium stringency conditions (2 × SSC and 0.5% SDS at 65°C).

Mentions: To study the distribution of the different MC proteases in various rat tissues, mRNA was prepared from different organs of Sprague Dawley rats, from ears of several additional rat strains and from purified peritoneal MCs. The purified peritoneal MCs represent an essentially pure population of CTMCs. In the ear, the majority of the MCs present are of the connective tissue subtype, although it should be emphasized that the percentage of MCs in this tissue is relatively low. In mRNA isolated from both ears and from peritoneal MCs, Northern blot analysis showed high expression levels for the chymases RMCP-1 and -5, the tryptase RMCP-6 and the MC carboxypeptidase A (Fig. 9 A). In mRNA isolated from the ears of several different strains of rats we detected relatively high levels of RMCP-7 (Fig. 9 A). In contrast, peritoneal MCs expressed only low levels of this protease compared with the expression levels for RMCP-1, -5, -6, and CPA. These results are thus in agreement with the results from the mRNA frequency analysis described above (Fig. 8). With respect to their RMCP-7 content, CTMCs of ears and peritoneum may therefore be regarded as two distinct subpopulations of MCs. When comparing different strains of rats, most MC components showed similar patterns of expression. However, the amounts of mRNA for the various MC proteins were different among the various strains, indicating that the number of MCs may differ.


Secretory granule proteases in rat mast cells. Cloning of 10 different serine proteases and a carboxypeptidase A from various rat mast cell populations.

Lützelschwab C, Pejler G, Aveskogh M, Hellman L - J. Exp. Med. (1997)

Northern and Southern blot analysis. (A) Northern blot analysis of poly(A)+RNA isolated from the ears of Lewis, Brown Norway, Hooded  Lister, Dark Agouti, and Sprague Dawley rats, poly(A)+RNA from purified peritoneal MCs (PMC; from Sprague Dawley rats) and poly(A)+RNA isolated from rat liver (Sprague Dawley). These mRNAs were analyzed for the expression of RMCP-1 (R-I), RMCP-5 (R-V), RMCP-6 (R-VI), RMCP-7  (R-VII), RMCP-8 (R-VIII), carboxypeptidase A (CPA), and the α chain of the IgERI using the specific probes described in Materials and Methods. (B)  Northern blot analysis of poly(A)+RNA from: Sprague Dawley lung, intestine, liver, spleen, Lewis ears and from the RBL-1 cell line. The various mRNAs  were analyzed with probes for RMCP-1 (R-I), RMCP-2 (R-II), RMCP-3 (R-III), RMCP-4 (R-IV), RMCP-5 (R-V), RMCP-8 (R-VIII), carboxypeptidase A (CPA), or the α chain of the IgERI using the specific probes described in Materials and Methods. In both A and B, a β-actin probe was  used to normalise the amount of RNA loaded in each lane to facilitate a comparative analysis between different organs and cell lines. (C) Genomic Southern blot analysis of DNA from Sprague Dawley rats. The DNA was cleaved with three different restriction enzymes, BamHI, BglII, and EcoRI. Size  markers are indicated at the left side of C. The blot was hybridized with the entire insert from the full-length cDNA clone for RMCP-8 and the blot was  washed under medium stringency conditions (2 × SSC and 0.5% SDS at 65°C).
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Related In: Results  -  Collection

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Figure 9: Northern and Southern blot analysis. (A) Northern blot analysis of poly(A)+RNA isolated from the ears of Lewis, Brown Norway, Hooded Lister, Dark Agouti, and Sprague Dawley rats, poly(A)+RNA from purified peritoneal MCs (PMC; from Sprague Dawley rats) and poly(A)+RNA isolated from rat liver (Sprague Dawley). These mRNAs were analyzed for the expression of RMCP-1 (R-I), RMCP-5 (R-V), RMCP-6 (R-VI), RMCP-7 (R-VII), RMCP-8 (R-VIII), carboxypeptidase A (CPA), and the α chain of the IgERI using the specific probes described in Materials and Methods. (B) Northern blot analysis of poly(A)+RNA from: Sprague Dawley lung, intestine, liver, spleen, Lewis ears and from the RBL-1 cell line. The various mRNAs were analyzed with probes for RMCP-1 (R-I), RMCP-2 (R-II), RMCP-3 (R-III), RMCP-4 (R-IV), RMCP-5 (R-V), RMCP-8 (R-VIII), carboxypeptidase A (CPA), or the α chain of the IgERI using the specific probes described in Materials and Methods. In both A and B, a β-actin probe was used to normalise the amount of RNA loaded in each lane to facilitate a comparative analysis between different organs and cell lines. (C) Genomic Southern blot analysis of DNA from Sprague Dawley rats. The DNA was cleaved with three different restriction enzymes, BamHI, BglII, and EcoRI. Size markers are indicated at the left side of C. The blot was hybridized with the entire insert from the full-length cDNA clone for RMCP-8 and the blot was washed under medium stringency conditions (2 × SSC and 0.5% SDS at 65°C).
Mentions: To study the distribution of the different MC proteases in various rat tissues, mRNA was prepared from different organs of Sprague Dawley rats, from ears of several additional rat strains and from purified peritoneal MCs. The purified peritoneal MCs represent an essentially pure population of CTMCs. In the ear, the majority of the MCs present are of the connective tissue subtype, although it should be emphasized that the percentage of MCs in this tissue is relatively low. In mRNA isolated from both ears and from peritoneal MCs, Northern blot analysis showed high expression levels for the chymases RMCP-1 and -5, the tryptase RMCP-6 and the MC carboxypeptidase A (Fig. 9 A). In mRNA isolated from the ears of several different strains of rats we detected relatively high levels of RMCP-7 (Fig. 9 A). In contrast, peritoneal MCs expressed only low levels of this protease compared with the expression levels for RMCP-1, -5, -6, and CPA. These results are thus in agreement with the results from the mRNA frequency analysis described above (Fig. 8). With respect to their RMCP-7 content, CTMCs of ears and peritoneum may therefore be regarded as two distinct subpopulations of MCs. When comparing different strains of rats, most MC components showed similar patterns of expression. However, the amounts of mRNA for the various MC proteins were different among the various strains, indicating that the number of MCs may differ.

Bottom Line: In contrast to what has been reported for human MCs, no expression of cathepsin G or cathepsin G-like proteases was detected in any of the rat MC populations.To determine mRNA frequencies for the various proteases expressed by normal tissue MCs, an unamplified peritoneal MC cDNA library was screened with a panel of mono-specific cDNA probes.These results showed that peritoneal MCs are highly specialized effector cells with mRNA frequencies for the major proteases in the range of several percent of the total mRNA pool.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Immunology and Microbiology, University of Uppsala, Sweden.

ABSTRACT
Two of the major rat mast cell proteases, rat mast cell protease 1 (RMCP-1) and RMCP-2, have for many years served as important phenotypic markers for studies of various aspects of mast cell (MC) biology. However, except for these proteases only fragmentary information has been available on the structure and complexity of proteases expressed by different subpopulations of rat MCs. To address these questions, cDNA libraries were constructed from freshly isolated rat peritoneal MCs and from the rat mucosal MC line RBL-1. cDNA clones for 10 different serine proteases (RMCP-1-10), and the MC carboxypeptidase A were isolated and characterized. Six of these proteases have not been isolated previously. Based on their protease content, three separate subpopulations of MCs were identified. Connective tissue MCs (CTMCs) from the ear and peritoneum express the chymases RMCP-1 and -5, the tryptases RMCP-6, and -7 and the carboxypeptidase A. However, based on a large difference in the level of expression of RMCP-7, CTMCs of these two organs may be regarded as two separate subpopulations. RMCP-2 and the three closely related proteases of the RMCP-8 subfamily were identified as the major mucosal MC proteases in rat. In contrast to what has been reported for human MCs, no expression of cathepsin G or cathepsin G-like proteases was detected in any of the rat MC populations. To determine mRNA frequencies for the various proteases expressed by normal tissue MCs, an unamplified peritoneal MC cDNA library was screened with a panel of mono-specific cDNA probes. These results showed that peritoneal MCs are highly specialized effector cells with mRNA frequencies for the major proteases in the range of several percent of the total mRNA pool.

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