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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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A comparative analysis based  on the amino acid sequence identities between a panel of mammalian hematopoietic serine proteases. MC serine proteases  are shown in black. The rat MC serine  proteases are indicated by asterisks. The  percent of divergence among the different  proteases, as indicated by the branch  points, is depicted at the bottom of the  figure.
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Figure 6: A comparative analysis based on the amino acid sequence identities between a panel of mammalian hematopoietic serine proteases. MC serine proteases are shown in black. The rat MC serine proteases are indicated by asterisks. The percent of divergence among the different proteases, as indicated by the branch points, is depicted at the bottom of the figure.

Mentions: Mucosal MCs are difficult to isolate as a pure population. Therefore, we decided to use the mucosal MC line RBL-1 as a source of mRNA for the isolation of rat mucosal MC proteases. The RBL-1 cell line has been widely used as an important in vitro model for studies of rodent MC biology and is considered to be a good representative of rat MMC (59). A RBL-1 cDNA library of ∼50,000 independent recombinants was constructed. Only one rat MMC protease, RMCP-2 (the rat homologue to MMCP-1) has previously been identified. Except for MMCP-1, the only additional MMC protease isolated in mouse, MMCP-2, could not be used as probe because of strong cross-hybridization between the different MC chymases. We therefore decided to use a novel technique based on the use of degenerate PCR primers directed against conserved regions surrounding the histidine and serine residues of the catalytic triad characteristic for all proteolytically active members of the large multigene family of trypsin-related serine proteases. Using this technique, five novel proteases, designated RMCP-3, -4, -8, -9, and -10 were isolated from the RBL-1 cell line. Based on a high degree of sequence identity with previously isolated rat MC chymases, RMCP-3 and -4 were classified as chymases. RMCP-8, -9, and 10 are all highly homologous and may be regarded as a new subfamily of MC proteases, more closely related to the T cell granzymes than to the previously characterised MC proteases (see Fig. 6). The cleavage specificities of RMCP-8,-9, and -10 have not yet been determined. To obtain the complete sequences of these proteases, the ∼500-bp insert fragments were used as probes in screenings of the RBL-1 cDNA library. Together with a full-length cDNA copy of the previously published RMCP-2, full-length or nearly full-length cDNA clones were isolated for RMCP-3, -4, -8, and -9. No full-length clone was obtained for RMCP-10 (see below). The nucleotide sequences of the RMCP-3 and -4 clones are depicted in Fig. 2 A, and the sequences of the clones encoding RMCP-8,-9, and -10 are shown in Fig. 2 B. The deduced aa sequences of these clones are depicted in Figs. 5, A and B. Fig. 6 shows a comparative analysis of the rat MC serine proteases with other rat, mouse, and human serine proteases. The characteristics of the clones, encoding the five different serine proteases isolated from the RBL-1 cDNA library, and some of the biochemical properties of these proteases are listed below. A summary of their biochemical characteristics is presented in Table 1.


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)

A comparative analysis based  on the amino acid sequence identities between a panel of mammalian hematopoietic serine proteases. MC serine proteases  are shown in black. The rat MC serine  proteases are indicated by asterisks. The  percent of divergence among the different  proteases, as indicated by the branch  points, is depicted at the bottom of the  figure.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196094&req=5

Figure 6: A comparative analysis based on the amino acid sequence identities between a panel of mammalian hematopoietic serine proteases. MC serine proteases are shown in black. The rat MC serine proteases are indicated by asterisks. The percent of divergence among the different proteases, as indicated by the branch points, is depicted at the bottom of the figure.
Mentions: Mucosal MCs are difficult to isolate as a pure population. Therefore, we decided to use the mucosal MC line RBL-1 as a source of mRNA for the isolation of rat mucosal MC proteases. The RBL-1 cell line has been widely used as an important in vitro model for studies of rodent MC biology and is considered to be a good representative of rat MMC (59). A RBL-1 cDNA library of ∼50,000 independent recombinants was constructed. Only one rat MMC protease, RMCP-2 (the rat homologue to MMCP-1) has previously been identified. Except for MMCP-1, the only additional MMC protease isolated in mouse, MMCP-2, could not be used as probe because of strong cross-hybridization between the different MC chymases. We therefore decided to use a novel technique based on the use of degenerate PCR primers directed against conserved regions surrounding the histidine and serine residues of the catalytic triad characteristic for all proteolytically active members of the large multigene family of trypsin-related serine proteases. Using this technique, five novel proteases, designated RMCP-3, -4, -8, -9, and -10 were isolated from the RBL-1 cell line. Based on a high degree of sequence identity with previously isolated rat MC chymases, RMCP-3 and -4 were classified as chymases. RMCP-8, -9, and 10 are all highly homologous and may be regarded as a new subfamily of MC proteases, more closely related to the T cell granzymes than to the previously characterised MC proteases (see Fig. 6). The cleavage specificities of RMCP-8,-9, and -10 have not yet been determined. To obtain the complete sequences of these proteases, the ∼500-bp insert fragments were used as probes in screenings of the RBL-1 cDNA library. Together with a full-length cDNA copy of the previously published RMCP-2, full-length or nearly full-length cDNA clones were isolated for RMCP-3, -4, -8, and -9. No full-length clone was obtained for RMCP-10 (see below). The nucleotide sequences of the RMCP-3 and -4 clones are depicted in Fig. 2 A, and the sequences of the clones encoding RMCP-8,-9, and -10 are shown in Fig. 2 B. The deduced aa sequences of these clones are depicted in Figs. 5, A and B. Fig. 6 shows a comparative analysis of the rat MC serine proteases with other rat, mouse, and human serine proteases. The characteristics of the clones, encoding the five different serine proteases isolated from the RBL-1 cDNA library, and some of the biochemical properties of these proteases are listed below. A summary of their biochemical characteristics is presented in Table 1.

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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