Cloning of the cDNA encoding a novel rat mast-cell proteinase, rMCP-3, and its expression in comparison with other rat mast-cell proteinases

摘要

pA 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./p