| First Author | Kelly JM | Year | 1996 |
| Journal | Immunogenetics | Volume | 44 |
| Issue | 5 | Pages | 340-50 |
| PubMed ID | 8781119 | Mgi Jnum | J:35546 |
| Mgi Id | MGI:82991 | Doi | 10.1007/BF02602778 |
| Citation | Kelly JM, et al. (1996) Cloning and expression of the recombinant mouse natural killer cell granzyme Met-ase-1. Immunogenetics 44(5):340-50 |
| abstractText | Met-ase-l is a 30000 M(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 rat Met-ase-l cDNA, and obtained bacteriophage clones that contained the mouse Met-ase-l gene. The mouse Met-ase-l 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-l mRNA was only detected in total cellular and poly A mRNA of mouse CD3- GM1(+) large granular lymphocytes derived from splenocytes stimulated with IL2 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 Mer-ase-l mRNA. The 5' flanking region of die mouse Met-ase-l gene also shares considerable regions of identity with the 5' flanking region of the rat Met-ase-l gene. A 3.3 kb segment of 5' sequence flanking the mouse Met-ase-l 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-l 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 fines. Reverse transcriptase polymerase chain reaction of mouse large granular lymphocyte mRNA was used to derive the full-length coding sequence for mouse Met-ase-l The predicted hexapropeptide of mouse Met-ase-l (Asn(-6) to Gln(-1)), was deleted by polymerase chain reaction mutagenesis to enable expression of active mouse Met-ase-l in mammalian COS7 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-l gene encodes a serine proteinase that hydrolyzes substrates containing a long narrow hydrophobic aminoacids like methionine, norleucine, and leucine in the P-1. |
