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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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mRNA frequencies for various MC proteins. The expression  levels of different proteins synthesized by rat serosal MCs were determined from the number of individual clones for these mRNAs in an unamplified rat peritoneal MC cDNA library. mRNA frequencies are expressed at the right side of each bar as positive hybridization signals per  100,000 plaques. (A) mRNA frequencies for the secretory granule proteins: RMCP-1, -2, -3, -4, -5, -6, -7, and -8, cathepsin G (Cath.G), carboxypeptidase A (CPA), and the heparin proteoglycan core protein (Heparin c.p.). (B) mRNA frequencies for a number of cell surface markers: the  α, β, and γ chains of the IgE high-affinity receptor, the IgE binding protein (Mac-2), and the stem cell factor receptor (c-kit). Glyceraldehyde3-phosphate dehydrogenase (GAPDH) and actin were used as controls.
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Figure 8: mRNA frequencies for various MC proteins. The expression levels of different proteins synthesized by rat serosal MCs were determined from the number of individual clones for these mRNAs in an unamplified rat peritoneal MC cDNA library. mRNA frequencies are expressed at the right side of each bar as positive hybridization signals per 100,000 plaques. (A) mRNA frequencies for the secretory granule proteins: RMCP-1, -2, -3, -4, -5, -6, -7, and -8, cathepsin G (Cath.G), carboxypeptidase A (CPA), and the heparin proteoglycan core protein (Heparin c.p.). (B) mRNA frequencies for a number of cell surface markers: the α, β, and γ chains of the IgE high-affinity receptor, the IgE binding protein (Mac-2), and the stem cell factor receptor (c-kit). Glyceraldehyde3-phosphate dehydrogenase (GAPDH) and actin were used as controls.

Mentions: The specific 3′ fragments for RMCP-1,-2,-3,-4,-6,-7, and the full-length cDNAs coding for RMCP-5, -8, R-CPA and the mouse heparin core protein (53, 54) were used as probes to determine the mRNA frequencies for these proteins by screening of the unamplified rat peritoneal MC cDNA library. In addition, nearly full-length cDNA probes for the rat α chain of the high-affinity receptor for IgE (IgERI-α), the mouse γ chain of the IgERI, the rat Mac-2 cell surface marker (the IgE binding protein), mouse lysozyme, a partial cDNA clone for the 3′ region of the rat c-kit receptor, a cDNA covering the entire coding region of mouse α2-microglobulin, and cDNA clones covering the entire coding regions of Mouse GATA-1 and mouse Pu.1 were used as probes in the screening of this cDNA library. These latter cDNAs have been cloned by PCR amplification or direct isolation from cDNA libraries from various mouse and rat hematopoietic cell lines. In addition to these cDNA probes we also used a few oligonucleotide probes, one directed against the 3′ terminal region of the rat IgE receptor β-chain, and one degenerate oligonucleotide directed against a conserved motif in the zinc finger region of members of the Krüppel-related zinc finger proteins. Positive signals from 4–8 filters were carefully counted and presented in Fig. 8 as number of positive signals in 100,000 phage plaques. The size of the cDNA library is ∼2.5 × 106 recombinants and the number of plaques screened with each probe was in the range of 100,000–200,000. Since the cDNA library was constructed with oligo-dT as primers the majority of all clones contain the 3′ ends of the mRNAs. A number of clones from each of the above described screenings and cloning of the various proteases have been analyzed and all clones were found to contain the 3′ end of the mRNAs. Several platings of the unamplified library were made with ∼25,000 recombinants/138-mm plate. One additional 10-fold dilution was made and a few plates were plated at this density to determine the actual number of plaques in the screening. Only filters with the high density were used for the screenings, and the filters were used only up to a maximum of four consecutive rounds of hybridizations. The results were reconfirmed by at least two independent experiments.


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)

mRNA frequencies for various MC proteins. The expression  levels of different proteins synthesized by rat serosal MCs were determined from the number of individual clones for these mRNAs in an unamplified rat peritoneal MC cDNA library. mRNA frequencies are expressed at the right side of each bar as positive hybridization signals per  100,000 plaques. (A) mRNA frequencies for the secretory granule proteins: RMCP-1, -2, -3, -4, -5, -6, -7, and -8, cathepsin G (Cath.G), carboxypeptidase A (CPA), and the heparin proteoglycan core protein (Heparin c.p.). (B) mRNA frequencies for a number of cell surface markers: the  α, β, and γ chains of the IgE high-affinity receptor, the IgE binding protein (Mac-2), and the stem cell factor receptor (c-kit). Glyceraldehyde3-phosphate dehydrogenase (GAPDH) and actin were used as controls.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: mRNA frequencies for various MC proteins. The expression levels of different proteins synthesized by rat serosal MCs were determined from the number of individual clones for these mRNAs in an unamplified rat peritoneal MC cDNA library. mRNA frequencies are expressed at the right side of each bar as positive hybridization signals per 100,000 plaques. (A) mRNA frequencies for the secretory granule proteins: RMCP-1, -2, -3, -4, -5, -6, -7, and -8, cathepsin G (Cath.G), carboxypeptidase A (CPA), and the heparin proteoglycan core protein (Heparin c.p.). (B) mRNA frequencies for a number of cell surface markers: the α, β, and γ chains of the IgE high-affinity receptor, the IgE binding protein (Mac-2), and the stem cell factor receptor (c-kit). Glyceraldehyde3-phosphate dehydrogenase (GAPDH) and actin were used as controls.
Mentions: The specific 3′ fragments for RMCP-1,-2,-3,-4,-6,-7, and the full-length cDNAs coding for RMCP-5, -8, R-CPA and the mouse heparin core protein (53, 54) were used as probes to determine the mRNA frequencies for these proteins by screening of the unamplified rat peritoneal MC cDNA library. In addition, nearly full-length cDNA probes for the rat α chain of the high-affinity receptor for IgE (IgERI-α), the mouse γ chain of the IgERI, the rat Mac-2 cell surface marker (the IgE binding protein), mouse lysozyme, a partial cDNA clone for the 3′ region of the rat c-kit receptor, a cDNA covering the entire coding region of mouse α2-microglobulin, and cDNA clones covering the entire coding regions of Mouse GATA-1 and mouse Pu.1 were used as probes in the screening of this cDNA library. These latter cDNAs have been cloned by PCR amplification or direct isolation from cDNA libraries from various mouse and rat hematopoietic cell lines. In addition to these cDNA probes we also used a few oligonucleotide probes, one directed against the 3′ terminal region of the rat IgE receptor β-chain, and one degenerate oligonucleotide directed against a conserved motif in the zinc finger region of members of the Krüppel-related zinc finger proteins. Positive signals from 4–8 filters were carefully counted and presented in Fig. 8 as number of positive signals in 100,000 phage plaques. The size of the cDNA library is ∼2.5 × 106 recombinants and the number of plaques screened with each probe was in the range of 100,000–200,000. Since the cDNA library was constructed with oligo-dT as primers the majority of all clones contain the 3′ ends of the mRNAs. A number of clones from each of the above described screenings and cloning of the various proteases have been analyzed and all clones were found to contain the 3′ end of the mRNAs. Several platings of the unamplified library were made with ∼25,000 recombinants/138-mm plate. One additional 10-fold dilution was made and a few plates were plated at this density to determine the actual number of plaques in the screening. Only filters with the high density were used for the screenings, and the filters were used only up to a maximum of four consecutive rounds of hybridizations. The results were reconfirmed by at least two independent experiments.

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