PURIFICATION AND CLONING OF A NOVEL SERINE-PROTEASE, RNK-TRYP-2, FROMTHE GRANULES OF A RAT NK CELL LEUKEMIA

We have biochemically purified a 27-kDa serine protease (designated RN K-Tryp-2) from the granules of the rat large granular lymphocyte leuke mia cell line (RNK-16) which has tryptase activity. Utilizing molecula r sieve chromatography and reverse-phase HPLC, we purified RNK-Tryp-2 to homogeneity and sequenced 33 NH2 terminal amino acids. Oligonucleot ide primers were used in the PCR to generate a 528-bp cDNA clone encod ing a novel serine protease from RNK-16 mRNA. This cDNA clone was used to isolate an 884-bp RN K-Tryp-2 cDNA from an RNK-16 lambda-gt11 libr ary. The open reading frame predicts a mature protein of 233 amino aci ds which does not have potential sites for N-linked glycosylation. The cDNA encodes a leader peptide of at least 25 amino acids. The charact eristic Ile-Ile-Gly-Gly amino acids of the N-terminus, and the His, As p, and Ser amino acids that form the catalytic triad of serine proteas es, are conserved. The amino acid sequence has less than 45% identity with any other member of the serine protease family, indicating that R NK-Tryp-2 is distinct protease. Southern blot analysis suggests the ex istence of one or more related genes. A single 1.3-kb mRNA transcript was detected by Northern blot analysis of total cellular RNA from the in vivo passaged RNK-16, rat splenocytes, lung and liver nonparenchyma l cells, as well as in highly purified rat LGL and T cells. RNK-Tryp-2 is a novel serine protease that is expressed in the granules of large granular lymphocytes.