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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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Nucleotide and  amino acid sequences for MC  carboxypeptidases. The figure  shows a comparative analysis of  the nucleotide sequences encoding rat (R-CPA), mouse (MCPA), and human (H-CPA) MC  carboxypeptidase A, and the deduced aa sequence for R-CPA.  The cleavage sites for the signal  sequence and the activation peptide are marked by arrows. The  polyadenylation signal is underlined. Nucleotide numbers per  line are depicted at the right of  the figure. A schematic drawing  of the different cDNA clones is  shown at the top of the figure  where the coding region for the  mature protein is indicated by a  grey box, and the signal sequence and the activation peptide are indicated by black and  white boxes, respectively.
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Figure 4: Nucleotide and amino acid sequences for MC carboxypeptidases. The figure shows a comparative analysis of the nucleotide sequences encoding rat (R-CPA), mouse (MCPA), and human (H-CPA) MC carboxypeptidase A, and the deduced aa sequence for R-CPA. The cleavage sites for the signal sequence and the activation peptide are marked by arrows. The polyadenylation signal is underlined. Nucleotide numbers per line are depicted at the right of the figure. A schematic drawing of the different cDNA clones is shown at the top of the figure where the coding region for the mature protein is indicated by a grey box, and the signal sequence and the activation peptide are indicated by black and white boxes, respectively.

Mentions: A nearly full-length cDNA clone for rat MC carboxypeptidase A (R-CPA) was isolated (Figs. 4 and 5 D). This clone contains an open reading frame of 1,254 bp encoding approximately twothirds of the signal sequence, a 94-aa activation peptide and the entire coding region of the mature protein, but lacks the initiation codon and the NH2-terminal end of the signal sequence. The coding region is followed by a 3′ untranslated region of 178 bp with the canonical AATAAA polyadenylation signal located at position 1399. The mature protein of 308 aa has an molecular mass of 35701 and two putative N-glycosylation sites at positions 127 and 133. The processed proteolytically active R-CPA is a basic protein with a net charge of +18.1 (Arg + Lys = 46; Asp + Glu = 29). (The nucleotide sequence is deposited in GenBank under the accession number U67914.)


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)

Nucleotide and  amino acid sequences for MC  carboxypeptidases. The figure  shows a comparative analysis of  the nucleotide sequences encoding rat (R-CPA), mouse (MCPA), and human (H-CPA) MC  carboxypeptidase A, and the deduced aa sequence for R-CPA.  The cleavage sites for the signal  sequence and the activation peptide are marked by arrows. The  polyadenylation signal is underlined. Nucleotide numbers per  line are depicted at the right of  the figure. A schematic drawing  of the different cDNA clones is  shown at the top of the figure  where the coding region for the  mature protein is indicated by a  grey box, and the signal sequence and the activation peptide are indicated by black and  white boxes, respectively.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196094&req=5

Figure 4: Nucleotide and amino acid sequences for MC carboxypeptidases. The figure shows a comparative analysis of the nucleotide sequences encoding rat (R-CPA), mouse (MCPA), and human (H-CPA) MC carboxypeptidase A, and the deduced aa sequence for R-CPA. The cleavage sites for the signal sequence and the activation peptide are marked by arrows. The polyadenylation signal is underlined. Nucleotide numbers per line are depicted at the right of the figure. A schematic drawing of the different cDNA clones is shown at the top of the figure where the coding region for the mature protein is indicated by a grey box, and the signal sequence and the activation peptide are indicated by black and white boxes, respectively.
Mentions: A nearly full-length cDNA clone for rat MC carboxypeptidase A (R-CPA) was isolated (Figs. 4 and 5 D). This clone contains an open reading frame of 1,254 bp encoding approximately twothirds of the signal sequence, a 94-aa activation peptide and the entire coding region of the mature protein, but lacks the initiation codon and the NH2-terminal end of the signal sequence. The coding region is followed by a 3′ untranslated region of 178 bp with the canonical AATAAA polyadenylation signal located at position 1399. The mature protein of 308 aa has an molecular mass of 35701 and two putative N-glycosylation sites at positions 127 and 133. The processed proteolytically active R-CPA is a basic protein with a net charge of +18.1 (Arg + Lys = 46; Asp + Glu = 29). (The nucleotide sequence is deposited in GenBank under the accession number U67914.)

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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