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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.

L├╝tzelschwab 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 sequences for rat MC proteases. The cleavage sites for the signal sequence and the activation peptide are marked by arrows. The  positions of the three aa of the catalytic triad are shown within boxes. The polyadenylation signal is underlined. Nucleotide numbers per line are depicted  at the right of the figure. Schematic drawings of the different cDNA clones are shown at the top of the figure where the coding regions for the mature  proteins are indicated by grey boxes, and the signal sequences and the activation peptides are indicated by black and white boxes, respectively. (A) Comparative analysis of the complete nucleotide sequences coding for RMCP-1, -2, -3, -4, and -5 and the deduced amino acid sequence for RMCP-1. (B)  Comparative analysis of RMCP-8, -9, and -10 and the deduced aa sequence for RMCP-8.
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Figure 2: Nucleotide sequences for rat MC proteases. The cleavage sites for the signal sequence and the activation peptide are marked by arrows. The positions of the three aa of the catalytic triad are shown within boxes. The polyadenylation signal is underlined. Nucleotide numbers per line are depicted at the right of the figure. Schematic drawings of the different cDNA clones are shown at the top of the figure where the coding regions for the mature proteins are indicated by grey boxes, and the signal sequences and the activation peptides are indicated by black and white boxes, respectively. (A) Comparative analysis of the complete nucleotide sequences coding for RMCP-1, -2, -3, -4, and -5 and the deduced amino acid sequence for RMCP-1. (B) Comparative analysis of RMCP-8, -9, and -10 and the deduced aa sequence for RMCP-8.

Mentions: A cDNA clone encoding the entire coding region of the major rat MC chymase RMCP-1 was isolated (Fig. 2 A, see also Fig. 5 A). A comparison was made with the previously published partial sequence determined by PCR amplification (23) and no differences between the nucleotide sequences were found (23). RMCP-1 is translated as a 260-aa primary polypeptide with an 18-aa-long signal sequence, a 2-aa-long NH2-terminal propeptide (Glu-Glu), and a 13-aa-long COOH-terminal propeptide. The processed mature protease of 227 aa has a molecular mass of 25,193 (excluding potential carbohydrate additions), a net charge of +19.1 (Arg + Lys = 34; Asp + Glu = 16) and contains no potential N-glycosylation sites. (The nucleotide sequence is deposited in GenBank under the accession number U67915.)


Secretory granule proteases in rat mast cells. Cloning of 10 different serine proteases and a carboxypeptidase A from various rat mast cell populations.

L├╝tzelschwab C, Pejler G, Aveskogh M, Hellman L - J. Exp. Med. (1997)

Nucleotide sequences for rat MC proteases. The cleavage sites for the signal sequence and the activation peptide are marked by arrows. The  positions of the three aa of the catalytic triad are shown within boxes. The polyadenylation signal is underlined. Nucleotide numbers per line are depicted  at the right of the figure. Schematic drawings of the different cDNA clones are shown at the top of the figure where the coding regions for the mature  proteins are indicated by grey boxes, and the signal sequences and the activation peptides are indicated by black and white boxes, respectively. (A) Comparative analysis of the complete nucleotide sequences coding for RMCP-1, -2, -3, -4, and -5 and the deduced amino acid sequence for RMCP-1. (B)  Comparative analysis of RMCP-8, -9, and -10 and the deduced aa sequence for RMCP-8.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196094&req=5

Figure 2: Nucleotide sequences for rat MC proteases. The cleavage sites for the signal sequence and the activation peptide are marked by arrows. The positions of the three aa of the catalytic triad are shown within boxes. The polyadenylation signal is underlined. Nucleotide numbers per line are depicted at the right of the figure. Schematic drawings of the different cDNA clones are shown at the top of the figure where the coding regions for the mature proteins are indicated by grey boxes, and the signal sequences and the activation peptides are indicated by black and white boxes, respectively. (A) Comparative analysis of the complete nucleotide sequences coding for RMCP-1, -2, -3, -4, and -5 and the deduced amino acid sequence for RMCP-1. (B) Comparative analysis of RMCP-8, -9, and -10 and the deduced aa sequence for RMCP-8.
Mentions: A cDNA clone encoding the entire coding region of the major rat MC chymase RMCP-1 was isolated (Fig. 2 A, see also Fig. 5 A). A comparison was made with the previously published partial sequence determined by PCR amplification (23) and no differences between the nucleotide sequences were found (23). RMCP-1 is translated as a 260-aa primary polypeptide with an 18-aa-long signal sequence, a 2-aa-long NH2-terminal propeptide (Glu-Glu), and a 13-aa-long COOH-terminal propeptide. The processed mature protease of 227 aa has a molecular mass of 25,193 (excluding potential carbohydrate additions), a net charge of +19.1 (Arg + Lys = 34; Asp + Glu = 16) and contains no potential N-glycosylation sites. (The nucleotide sequence is deposited in GenBank under the accession number U67915.)

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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