Abstract
Met-ase-1 is a 30000M r serine protease (granzyme) that was first isolated in the cytolytic granules of rat CD3− large granular lymphocytes. We screened a mouse genomic library with ratMet-ase-1 cDNA, and obtained bacteriophage clones that contained the mouseMet-ase-1 gene. The mouseMet-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. MouseMet-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 mouseMet-ase-1 mRNA. The 5′ flanking region of the mouseMet-ase-1 gene also shares considerable regions of identity with the 5′ flanking region of the ratMet-ase-1 gene. A 3.3 kb segment of 5′ sequence flanking the mouseMet-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 mouseMet-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 mouseMet-ase-1. The predicted hexapropeptide of mouseMet-ase-1 (Asn−6 to Gln−1), was deleted by polymerase chain reaction mutagenesis to enable expression of active mouseMet-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 mouseMet-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.
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The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number L76741.
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Kelly, J.M., O'Connor, M.D., Hulett, M.D. et al. Cloning and expression of the recombinant mouse natural killer cell granzymeMet-ase-1 . Immunogenetics 44, 340–350 (1996). https://doi.org/10.1007/BF02602778
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DOI: https://doi.org/10.1007/BF02602778