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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 sequences for MC tryptases. The  figure shows a comparative analysis of the nucleotide sequences  for the cDNAs encoding the  mouse and rat MC tryptases:  RMCP-6, MMCP-6, RMCP-7,  and MMCP-7, and the deduced  aa sequence for RMCP-6. 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. A schematic drawing of the RMCP-6  and -7 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 3: Nucleotide sequences for MC tryptases. The figure shows a comparative analysis of the nucleotide sequences for the cDNAs encoding the mouse and rat MC tryptases: RMCP-6, MMCP-6, RMCP-7, and MMCP-7, and the deduced aa sequence for RMCP-6. 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. A schematic drawing of the RMCP-6 and -7 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 cDNA clone containing the entire coding region for the rat tryptase RMCP-6 was isolated. This clone contains an open reading frame of 824 bp encoding a signal sequence of 19 aa, a 10-aa activation peptide (AlaPro-Cys-Pro-Val-Lys-Gln-Arg-Val-Gly) and the entire coding region of the mature protein (Figs. 3 and 5 C). The coding region is followed by a 3′ untranslated region of 246 bp with the non-canonical ATTAAA polyadenylation signal located at position 1061. The mature protein of 245 aa has a molecular mass of 27,464 and contains two putative N-glycosylation sites at positions 75 and 102 of the mature protein. The processed proteolytically active RMCP-6 is an acidic protein with a net charge of −4.6 (Arg + Lys = 19; Asp + Glu = 25). (The nucleotide sequence is deposited in GenBank under the accession number U67909.) The sequence differs in five positions from the previously published sequence of the rat tryptase (A pos. 399, C pos. 432, C pos. 748, T pos. 946, and one base pair deleted at position 1024) (24).


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 sequences for MC tryptases. The  figure shows a comparative analysis of the nucleotide sequences  for the cDNAs encoding the  mouse and rat MC tryptases:  RMCP-6, MMCP-6, RMCP-7,  and MMCP-7, and the deduced  aa sequence for RMCP-6. 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. A schematic drawing of the RMCP-6  and -7 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

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

Figure 3: Nucleotide sequences for MC tryptases. The figure shows a comparative analysis of the nucleotide sequences for the cDNAs encoding the mouse and rat MC tryptases: RMCP-6, MMCP-6, RMCP-7, and MMCP-7, and the deduced aa sequence for RMCP-6. 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. A schematic drawing of the RMCP-6 and -7 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 cDNA clone containing the entire coding region for the rat tryptase RMCP-6 was isolated. This clone contains an open reading frame of 824 bp encoding a signal sequence of 19 aa, a 10-aa activation peptide (AlaPro-Cys-Pro-Val-Lys-Gln-Arg-Val-Gly) and the entire coding region of the mature protein (Figs. 3 and 5 C). The coding region is followed by a 3′ untranslated region of 246 bp with the non-canonical ATTAAA polyadenylation signal located at position 1061. The mature protein of 245 aa has a molecular mass of 27,464 and contains two putative N-glycosylation sites at positions 75 and 102 of the mature protein. The processed proteolytically active RMCP-6 is an acidic protein with a net charge of −4.6 (Arg + Lys = 19; Asp + Glu = 25). (The nucleotide sequence is deposited in GenBank under the accession number U67909.) The sequence differs in five positions from the previously published sequence of the rat tryptase (A pos. 399, C pos. 432, C pos. 748, T pos. 946, and one base pair deleted at position 1024) (24).

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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