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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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SDS-PAGE analysis  of MC extracts. Purified peritoneal MCs (∼2 × 106 cells) were  solubilized and mixed with 3HDFP (see Materials and Methods). Lane A shows a Coomassie  blue stained gel separation of the  MC extract. Lane B shows the  same lane after fluorography  which visualises proteins that covalently bind 3H-DFP.
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Figure 1: SDS-PAGE analysis of MC extracts. Purified peritoneal MCs (∼2 × 106 cells) were solubilized and mixed with 3HDFP (see Materials and Methods). Lane A shows a Coomassie blue stained gel separation of the MC extract. Lane B shows the same lane after fluorography which visualises proteins that covalently bind 3H-DFP.

Mentions: Cellular extracts from peritoneal MCs were analyzed by SDS-PAGE (Fig. 1, lane A). A dominant band corresponding to an apparent molecular mass of ∼29 kD was detected along with other major bands of ∼32 and 17 kD. In addition, a ∼38-kD band and several faint bands of ∼28, ∼31 and ∼9-13 kD could be distinguished. Our previous studies have shown that the 29-kD protein gives an NH2-terminal sequence identical to that of mature RMCP-1 (57). Attempts were made to identify additional bands by NH2terminal aa sequence analysis. Most of the bands yielded mixtures of several sequences, or the NH2-terminals appeared to be blocked. However, the NH2-terminal sequence: Asn-Phe-Tyr-Ser-Asn-Leu-His-Asp-Ile-Met-Leu was obtained for a 13-kD band. This sequence corresponds to an internal portion of RMCP-1 starting at Asn82 (numbering according to Le Trong et al., 1987[21]), indicating a cleavage at the Tyr81-Asn82 bond. Since cleavage after aromatic aa residues is a characteristic feature of MC chymases (29, 58); it appears likely that the cleavage is catalysed by either RMCP-1 (auto catalysis) or by RMCP-5, the other major chymase of rat CTMCs. A ∼12-kD band gave the NH2-terminal sequence Ile-Ile-Gly-Gly-Val-Glu, indicating that it is an RMCP-1 fragment containing the intact NH2-terminal. When the MC extracts were radiolabeled with 3H-DFP, major incorporation of radioactivity was observed for the ∼32- and ∼29-kD bands, indicating that these bands correspond to serine proteases (Fig. 1, lane B). Minor incorporation of label was also observed for a 17-kD polypeptide as well as into polypeptides of ∼15 and ∼10 kD. Since the latter DFP-binding proteins appear to be too small to represent intact serine proteases it is likely that they correspond to various proteolytic fragments.


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)

SDS-PAGE analysis  of MC extracts. Purified peritoneal MCs (∼2 × 106 cells) were  solubilized and mixed with 3HDFP (see Materials and Methods). Lane A shows a Coomassie  blue stained gel separation of the  MC extract. Lane B shows the  same lane after fluorography  which visualises proteins that covalently bind 3H-DFP.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196094&req=5

Figure 1: SDS-PAGE analysis of MC extracts. Purified peritoneal MCs (∼2 × 106 cells) were solubilized and mixed with 3HDFP (see Materials and Methods). Lane A shows a Coomassie blue stained gel separation of the MC extract. Lane B shows the same lane after fluorography which visualises proteins that covalently bind 3H-DFP.
Mentions: Cellular extracts from peritoneal MCs were analyzed by SDS-PAGE (Fig. 1, lane A). A dominant band corresponding to an apparent molecular mass of ∼29 kD was detected along with other major bands of ∼32 and 17 kD. In addition, a ∼38-kD band and several faint bands of ∼28, ∼31 and ∼9-13 kD could be distinguished. Our previous studies have shown that the 29-kD protein gives an NH2-terminal sequence identical to that of mature RMCP-1 (57). Attempts were made to identify additional bands by NH2terminal aa sequence analysis. Most of the bands yielded mixtures of several sequences, or the NH2-terminals appeared to be blocked. However, the NH2-terminal sequence: Asn-Phe-Tyr-Ser-Asn-Leu-His-Asp-Ile-Met-Leu was obtained for a 13-kD band. This sequence corresponds to an internal portion of RMCP-1 starting at Asn82 (numbering according to Le Trong et al., 1987[21]), indicating a cleavage at the Tyr81-Asn82 bond. Since cleavage after aromatic aa residues is a characteristic feature of MC chymases (29, 58); it appears likely that the cleavage is catalysed by either RMCP-1 (auto catalysis) or by RMCP-5, the other major chymase of rat CTMCs. A ∼12-kD band gave the NH2-terminal sequence Ile-Ile-Gly-Gly-Val-Glu, indicating that it is an RMCP-1 fragment containing the intact NH2-terminal. When the MC extracts were radiolabeled with 3H-DFP, major incorporation of radioactivity was observed for the ∼32- and ∼29-kD bands, indicating that these bands correspond to serine proteases (Fig. 1, lane B). Minor incorporation of label was also observed for a 17-kD polypeptide as well as into polypeptides of ∼15 and ∼10 kD. Since the latter DFP-binding proteins appear to be too small to represent intact serine proteases it is likely that they correspond to various proteolytic fragments.

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.

Show MeSH
Related in: MedlinePlus