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Cloning and expression of the recombinant mouse natural killer cell granzyme Met-ase-1
J M Kelly1, M D O'Connor, M D Hulett
1Cellular Cytotoxicity Laboratory, Austin Research Institute, Austin Hospital, Heidelberg, 3084 Victoria, Australia.
Abstract:
Met-ase-1 is a 30 000 Mr serine protease (granzyme) that was first isolated in the cytolytic granules of rat CD3(-) large granular lymphocytes. We screened a mouse genomic library with rat Met-ase-1 cDNA, and obtained bacteriophage clones that contained the mouse Met-ase-1 gene. The mouse Met-ase-1 gene comprises five exons spanning approximately 5.2 kilobases (kb) and exhibits a similar structural organization to its rat homologue and a family of neutrophil elastase-like serine proteases. Mouse Met-ase-1 mRNA was only detected in total cellular and poly A mRNA of mouse CD3(-) GM1(+) large granular lymphocytes derived from splenocytes stimulated with IL-2 and the mouse NK1.1(+) cell line 4 - 16. Spleen T-cell populations generated by Concanavalin A stimulation and a number of mouse pre-NK and T cell lines did not express mouse Met-ase-1 mRNA. The 5' flanking region of the mouse Met-ase-1 gene also shares considerable regions of identity with the 5' flanking region of the rat Met-ase-1 gene. A 3.3 kb segment of 5' sequence flanking the mouse Met-ase-1 gene was inserted upstream of the chloramphenicol acetyltransferase reporter gene and this construct transiently transfected into a variety of mouse and rat large granular lymphocyte leukemia and T-cell lines. The transcriptional activity of the mouse Met-ase-1 5' flanking region was significant in the RNK-16 large granular lymphocyte leukemia, strongest in the 4 - 16 mouse NK1.1(+) cell line, and weak in several mouse pre-NK cell lines. Reverse transcriptase polymerase chain reaction of mouse large granular lymphocyte mRNA was used to derive the full-length coding sequence for mouse Met-ase-1. The predicted hexapropeptide of mouse Met-ase-1 (Asn-6 to Gln-1), was deleted by polymerase chain reaction mutagenesis to enable expression of active mouse Met-ase-1 in mammalian COS-7 cells. Northern blot analysis and protease assays of transfected COS cell lysates against a panel of thiobenzyl ester substrates formally demonstrated that the mouse Met-ase-1 gene encodes a serine proteinase that hydrolyzes substrates containing a long narrow hydrophobic amino acids like methionine, norleucine, and leucine in the P1.
Insights
Researchers identified the mouse Met-ase-1 gene, a serine protease found in large granular lymphocytes. This gene encodes a proteinase specific for hydrophobic amino acids, crucial for immune cell function.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Met-ase-1, a serine protease (granzyme), was initially isolated from rat CD3(-) large granular lymphocytes (LGLs).
- Understanding the genetic basis and expression of Met-ase-1 in mice is crucial for comparative immunology and protease function studies.
Purpose of the Study:
- To isolate and characterize the mouse Met-ase-1 gene.
- To determine the expression pattern of mouse Met-ase-1 mRNA in different immune cell populations.
- To investigate the transcriptional regulation of the mouse Met-ase-1 gene.
Main Methods:
- Screening of a mouse genomic library using rat Met-ase-1 cDNA.
- Gene structure analysis (exons, introns) and sequence comparison with rat homologues.
- Detection of mRNA expression using Northern blot and RT-PCR.
- Reporter gene assays (chloramphenicol acetyltransferase) to study promoter activity.
- Site-directed mutagenesis to express active mouse Met-ase-1 in COS-7 cells.
- Protease activity assays using specific substrates.
Main Results:
- The mouse Met-ase-1 gene consists of five exons spanning approximately 5.2 kb, with structural similarity to its rat counterpart.
- Mouse Met-ase-1 mRNA expression is restricted to CD3(-) GM1(+) LGLs derived from IL-2-stimulated splenocytes and the NK1.1(+) cell line 4-16.
- The 5' flanking region of the mouse Met-ase-1 gene shows significant sequence identity with the rat gene and exhibits cell-specific transcriptional activity, strongest in the 4-16 cell line.
- The derived full-length coding sequence confirmed the gene's identity, and mutated Met-ase-1 (lacking the hexapeptide) expressed an active serine proteinase in COS-7 cells.
- The proteinase hydrolyzes substrates with long, narrow hydrophobic amino acids (methionine, norleucine, leucine) at the P1 position.
Conclusions:
- The mouse Met-ase-1 gene encodes a granzyme-like serine proteinase specifically expressed in cytotoxic LGLs.
- The 5' flanking region contains regulatory elements that confer cell-specific expression, particularly in NK1.1(+) cells.
- Mouse Met-ase-1 functions as a protease targeting specific hydrophobic residues, contributing to the cytotoxic functions of LGLs.