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Title: Cloning and expression of the recombinant mouse natural killer cell granzyme Met-ase-1.
Austin Authors: Kelly, J M;O'Connor, M D;Hulett, M D;Thia, K Y;Smyth, Mark J
Affiliation: Cellular Cytotoxicity Laboratory, Austin Research Institute, Austin Hospital, Heidelberg, 3084 Victoria, Australia
Issue Date: 16-May-1996
Publication information: Immunogenetics; 44(5): 340-50
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.
Gov't Doc #: 8781119
Type: Journal Article
Subjects: Amino Acid Sequence
Base Sequence
Cell Line, Transformed
Cercopithecus aethiops
Cloning, Molecular
Killer Cells, Natural.enzymology
Lymphocyte Activation
Mice, Inbred BALB C
Molecular Sequence Data
Polymerase Chain Reaction
Recombinant Fusion Proteins.genetics
Sequence Alignment
Sequence Deletion
Sequence Homology, Amino Acid
Serine Endopeptidases.genetics
Substrate Specificity
Appears in Collections:Journal articles

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