We have purified a 30-kDa
serine protease (designated RNK-Met-1) from the granules of the rat
large granular lymphocyte leukemia cell line (RNK-16) that hydrolytically cleaves model
peptide substrates after
methionine,
leucine, and
norleucine (Met-ase activity). Utilizing molecular sieve chromatography,
heparin-agarose, chromatography, and reverse-phase high pressure liquid chromatography,
RNK-Met-1 was purified to homogeneity and 25 NH2-terminal
amino acids were sequenced. By using the polymerase chain reaction,
oligonucleotide primers derived from
amino acids at position 14-25 and from a downstream active site conserved in other
serine protease genes were used to generate a 534-base pair
cDNA clone encoding a novel
serine protease from RNK-16
mRNA. This
cDNA clone was used to isolate a full-length 867-base pair
RNK-Met-1 cDNA from an RNK-16 lambda-gt11 library. The open reading frame predicts a mature
protein of 238
amino acids with two potential sites for N-linked glycosylation. The
cDNA also encodes a
leader peptide of at least 20
amino acids. The characteristic Ile-Ile-Gly-Gly
amino acids of the NH2 terminus and the His, Asp, and Ser residues that form the catalytic triad of
serine proteases were both conserved. The amino acid sequence has less than 45% identity with any other member of the
serine protease family, indicating that
RNK-Met-1 is distinct and may itself represent a new subfamily of
serine proteases. Northern blot analysis of total cellular
RNA detected a single 0.9-kilobase
mRNA in the in vitro and in vivo variants of RNK-16 and in spleen-derived
plastic-adherent rat lymphokine-activated killer cells.
RNK-Met-1 mRNA was not detectable in freshly isolated rat splenocytes, thymocytes, brain, colon, and liver or activated nonadherent rat splenocytes and thymocytes. These data indicate that
RNK-Met-1 is a
serine protease with unique activity that is expressed in the granules of large granular lymphocytes.