Cloning and analysis of cDNA encoding rat bleomycin hydrolase, a DNA-binding cysteine protease.
We isolated and characterized almost the entire cDNA encoding BLM hydrolase from rat spleen cDNA libraries. The cDNA encoded a polypeptide composed of 454 amino acids, that had a slightly larger molecular mass than that was previously estimated by SDS-PAGE for purified BLM hydrolase subunit. Amino acid sequence alignments showed that rat BLM hydrolase is homologous to that of rabbit (94% identity of partial amino acid sequence), yeast cysteine protease (39% identity), and the pepC gene products of three bacteria (34-40% identity). In addition, it contained the three regions that are conserved in other cysteine proteases and thought to constitute the catalytic center. These results indicated that rat BLM hydrolase is a member of the papain superfamily of cysteine proteases. Sequencing revealed several putative sites phosphorylated by different types of protein kinases, but no signal sequence, transmembrane domain, N-linked glycosylation site or DNA-binding motif. The yeast homolog is a DNA-binding cysteine protease [Xu et al. (1994) J. Biol. Chem, 269, 21177-21183]. We demonstrated that rat BLM hydrolase also binds the single-stranded form of the Ga14 DNA-binding site oligonucleotide with high affinity compared with that of the double-stranded form. Northern blots revealed that the level of BLM hydrolase mRNA expression was very low in the rat skin, lung, and skeletal muscle. Furthermore, BLM hydrolase mRNA was ubiquitously expressed in the human cell lines, HeLa, SKG-IIIa, FL, KB, HEp-2, U373 GM, P3HR-1, Raji, THP-1, Jurkat, and Molt-4. These results suggested that BLM hydrolase plays important physiological roles, including the metabolism of antibiotics.
Takeda
A
A
Department of Clinical Pathology, Showa University Fujigaoka Hospital, Yokohama.
Masuda
Y
Y
Yamamoto
T
T
Hirabayashi
T
T
Nakamura
Y
Y
Nakaya
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D004247:DNA; D003546:Cysteine Endopeptidases; C031262:bleomycin hydrolase