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Cloning of the cDNA encoding a novel rat mast-cell proteinase, rMCP-3, and its expression in comparison with other
Abstract:
A cDNA encoding a novel rat mast-cell proteinase (MCP) named rMCP-3 was successfully cloned and sequenced from the peritoneal cells of Lewis rats infected with the intestinal nematode Nippostrongylus brasiliensis by using the combination of reverse transcription-PCR and rapid-amplification-of-cDNA-ends ('RACE') methods. The cDNA was 979 bp long and included a 741 bp open reading frame. When the deduced amino acid sequence was compared with those of other known mast-cell proteinases, rMCP-3 was considered to be translated as a preproenzyme with a 19-amino-acid signal peptide, a two-amino-acid activation peptide and a 226-amino-acid mature enzyme. The amino acid identity in the mature enzyme was 52.9% and 55.1% with rMCP-1 and rMCP-2 respectively. The rMCP-3 mRNA was not detected in the peritoneal cells of mast-cell-deficient Ws/Ws rats, though it was strongly detected in those of littermate +/+ and Lewis rats, indicating the mast-cell origin of rMCP-3 In addition to being present in peritoneal mast cells, the rMCP-3 mRNA was strongly detected in the skin, tongue, and RBL2H3 rat basophilic leukaemia cells and weakly in the jejunum of N. brasiliensis-infected rats by RNA blot analysis using a rMCP-3 gene-specific probe. By reverse transcription-PCR, the rMCP-3 mRNA was also detected in the lung. While the expression of rMCP-1 and rMCP-2 are clearly restricted in connective-tissue mast cells and mucosal mast cells respectively, rMCP-3 was widely expressed in both types of mast cells with a predominance in connective-tissue mast cells.
Insights
Researchers identified a novel rat mast-cell proteinase, rMCP-3, crucial for immune responses. This proteinase is expressed in various tissues, indicating its broad role in mast cell function.
Area of Science:
- Immunology
- Molecular Biology
- Parasitology
Background:
- Mast-cell proteinases (MCPs) play key roles in inflammatory and immune responses.
- Understanding the diversity and expression patterns of MCPs is crucial for elucidating their specific functions.
Purpose of the Study:
- To clone and characterize a novel rat mast-cell proteinase, designated rMCP-3.
- To investigate the expression pattern and cellular localization of rMCP-3 in rats infected with Nippostrongylus brasiliensis.
Main Methods:
- cDNA cloning and sequencing using reverse transcription-PCR and RACE.
- Sequence analysis and comparison with known MCPs.
- RNA blot analysis and reverse transcription-PCR to determine mRNA expression.
Main Results:
- A novel rat MCP cDNA, rMCP-3, was successfully cloned and sequenced.
- rMCP-3 mRNA was detected in peritoneal mast cells, skin, tongue, RBL2H3 cells, jejunum, and lung of infected rats.
- rMCP-3 is expressed in both connective-tissue and mucosal mast cells, with a predominance in connective-tissue mast cells.
Conclusions:
- rMCP-3 is a novel mast-cell proteinase originating from mast cells.
- rMCP-3 exhibits a distinct expression pattern compared to rMCP-1 and rMCP-2, suggesting specialized roles.
- The widespread expression of rMCP-3 indicates its involvement in diverse physiological and pathological processes mediated by mast cells.
