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Cloning and expression of the recombinant mouse natural killer cell granzymeMet-ase-1

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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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  1. Mark J. Smyth

    Present address: Cellular Cytotoxicity Laboratory, Austin, Research Institute, Austin Hospital, 3084, Heidelberg, Victoria, Australia

Authors and Affiliations

  1. Cellular Cytotoxicity Laboratory, Austin, Research Institute, Austin Hospital, 3084, Heidelberg, Victoria, Australia

    Janice M. Kelly, Michael D. O'Connor, Mark D. Hulett & Kevin Y. T. Thia

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  1. Janice M. Kelly
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  2. Michael D. O'Connor
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  3. Mark D. Hulett
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  4. Kevin Y. T. Thia
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  5. Mark J. Smyth
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Additional information

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

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