title: Cloning and expression of the recombinant mouse natural killer cell granzyme Met-ase-1.
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.
authors.affiliation: Cellular Cytotoxicity Laboratory, Austin Research Institute, Austin Hospital, Heidelberg, 3084 Victoria, Australia.
authors.forename: J M
authors.initials: JM
authors.lastname: Kelly
authors.forename: M D
authors.initials: MD
authors.lastname: O'Connor
authors.forename: M D
authors.initials: MD
authors.lastname: Hulett
authors.forename: K Y
authors.initials: KY
authors.lastname: Thia
authors.forename: M J
authors.initials: MJ
authors.lastname: Smyth
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D005796:Genes; D007694:Killer Cells, Natural / Q000201:enzymology*; D008213:Lymphocyte Activation; D051379:Mice / Q000235:genetics*; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics*; D013154:Spleen / Q000166:cytology; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; C084504:Hu-Met-1; D012697:Serine Endopeptidases