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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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Dot blot analysis of probes for rat MC proteases. The probes  for RMCP-1 (R-I), RMCP-2 (R-II), RMCP-3 (R-III), RMCP-4 (R-IV),  RMCP-6 (R-VI), and RMCP-7 (R-VII) were 160–220-bp fragments  originating from the 3′ non-coding regions of the respective cDNA  clones (see Materials and Methods). The probes for RMCP-5 (R-V),  RMCP-8 (R-VIII), and carboxypeptidase A (CPA) are the entire inserts  of the corresponding full-length cDNA clones. Cross hybridization between RMCP-8, -9, and -10 was seen when using the R-VIII probe.
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Figure 7: Dot blot analysis of probes for rat MC proteases. The probes for RMCP-1 (R-I), RMCP-2 (R-II), RMCP-3 (R-III), RMCP-4 (R-IV), RMCP-6 (R-VI), and RMCP-7 (R-VII) were 160–220-bp fragments originating from the 3′ non-coding regions of the respective cDNA clones (see Materials and Methods). The probes for RMCP-5 (R-V), RMCP-8 (R-VIII), and carboxypeptidase A (CPA) are the entire inserts of the corresponding full-length cDNA clones. Cross hybridization between RMCP-8, -9, and -10 was seen when using the R-VIII probe.

Mentions: Several of the MC serine proteases show a high degree of sequence identity, making it difficult to use the entire inserts of the clones as probes in Northern blot, Southern blot, or plaque hybridizations. To solve this problem, the sequences for the different proteases were compared and regions with maximal divergence were selected as regions of interest for the construction of specific probes. Such regions were found in the 3′ non-coding regions of all the different mRNAs. Fragments located within these regions were excised from the cDNA clones and subcloned into plasmid vectors. The probes for RMCP-1, -2, -3, -4, -6, and -7 were all in the range of 165–220 nucleotides in length. For RMCP-5, -8, and CPA the entire inserts of the original cDNA clones were used as probes. To study the specificity of these probes, the insert fragments were purified, labeled and used in a dot blot analysis. All probes, except for the RMCP-8 probe, were found to be monospecific (Fig. 7). Because of the high degree of homology shared by RMCP-8, -9, and -10 it has not been possible to excise any fragment of a size large enough to label by random priming, and still maintain mono-specificity (Figs. 2 B and 7). Consequently, in all hybridizations performed with the RMCP-8 probe, we detect all three members of this subfamily of serine proteases.


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)

Dot blot analysis of probes for rat MC proteases. The probes  for RMCP-1 (R-I), RMCP-2 (R-II), RMCP-3 (R-III), RMCP-4 (R-IV),  RMCP-6 (R-VI), and RMCP-7 (R-VII) were 160–220-bp fragments  originating from the 3′ non-coding regions of the respective cDNA  clones (see Materials and Methods). The probes for RMCP-5 (R-V),  RMCP-8 (R-VIII), and carboxypeptidase A (CPA) are the entire inserts  of the corresponding full-length cDNA clones. Cross hybridization between RMCP-8, -9, and -10 was seen when using the R-VIII probe.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196094&req=5

Figure 7: Dot blot analysis of probes for rat MC proteases. The probes for RMCP-1 (R-I), RMCP-2 (R-II), RMCP-3 (R-III), RMCP-4 (R-IV), RMCP-6 (R-VI), and RMCP-7 (R-VII) were 160–220-bp fragments originating from the 3′ non-coding regions of the respective cDNA clones (see Materials and Methods). The probes for RMCP-5 (R-V), RMCP-8 (R-VIII), and carboxypeptidase A (CPA) are the entire inserts of the corresponding full-length cDNA clones. Cross hybridization between RMCP-8, -9, and -10 was seen when using the R-VIII probe.
Mentions: Several of the MC serine proteases show a high degree of sequence identity, making it difficult to use the entire inserts of the clones as probes in Northern blot, Southern blot, or plaque hybridizations. To solve this problem, the sequences for the different proteases were compared and regions with maximal divergence were selected as regions of interest for the construction of specific probes. Such regions were found in the 3′ non-coding regions of all the different mRNAs. Fragments located within these regions were excised from the cDNA clones and subcloned into plasmid vectors. The probes for RMCP-1, -2, -3, -4, -6, and -7 were all in the range of 165–220 nucleotides in length. For RMCP-5, -8, and CPA the entire inserts of the original cDNA clones were used as probes. To study the specificity of these probes, the insert fragments were purified, labeled and used in a dot blot analysis. All probes, except for the RMCP-8 probe, were found to be monospecific (Fig. 7). Because of the high degree of homology shared by RMCP-8, -9, and -10 it has not been possible to excise any fragment of a size large enough to label by random priming, and still maintain mono-specificity (Figs. 2 B and 7). Consequently, in all hybridizations performed with the RMCP-8 probe, we detect all three members of this subfamily of serine proteases.

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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