Purification and cloning of a novel serine protease, RNK-Tryp-2, from the granules of a rat NK cell leukemia.
We have biochemically purified a 27-kDa serine protease (designated RNK-Tryp-2) from the granules of the rat large granular lymphocyte leukemia cell line (RNK-16) which has tryptase activity. Utilizing molecular sieve chromatography and reverse-phase HPLC, we purified RNK-Tryp-2 to homogeneity and sequenced 33 NH2-terminal amino acids. Oligonucleotide primers were used in the PCR to generate a 528-bp cDNA clone encoding a novel serine protease from RNK-16 mRNA. This cDNA clone was used to isolate an 884-bp RNK-Tryp-2 cDNA from an RNK-16 lambda-gt11 library. The open reading frame predicts a mature protein of 233 amino acids which does not have potential sites for N-linked glycosylation. The cDNA encodes a leader peptide of at least 25 amino acids. The characteristic Ile-Ile-Gly-Gly amino acids of the N-terminus, and the His, Asp, and Ser amino acids that form the catalytic triad of serine proteases, are conserved. The amino acid sequence has less than 45% identity with any other member of the serine protease family, indicating that RNK-Tryp-2 is distinct protease. Southern blot analysis suggests the existence 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 nonparenchymal 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.
Sayers
T J
TJ
Biological Carcinogenesis and Development Program, PRI/DynCorp, Frederick, MD 21702.
Wiltrout
T A
TA
Smyth
M J
MJ
Ottaway
K S
KS
Pilaro
A M
AM
Sowder
R
R
Henderson
L E
LE
Sprenger
H
H
Lloyd
A R
AR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D053818:Chymases; D003001:Cloning, Molecular; D003594:Cytoplasmic Granules / Q000201:enzymology; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D007694:Killer Cells, Natural / Q000201:enzymology*; D007942:Leukemia, Experimental / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification*; D053802:Tryptases; D014407:Tumor Cells, Cultured / Q000201:enzymology
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; C509223:Tpsab1 protein, rat; C509221:Tpsb2 protein, rat; D053802:Tryptases