Abstract
Isolation of cDNA clones for human leukotriene B4 (LTB4) omega-hydroxylase clearly demonstrates that the hydroxylase is a member of the cytochrome P-450 (CYP) superfamily. cDNA clones isolated from a human leukocyte cDNA library with CYP4A4 cDNA as a probe encode a protein of 520 amino acids with a molecular weight of 59,805. The deduced amino acid sequence contains an invariant cysteine in the conserved heme-binding domain near the C terminus, characteristic of the P-450 superfamily. The microsomes from yeast cells transfected with an expression vector pAAH5 carrying isolated cDNA catalyzed the omega-hydroxylation of LTB4 with a Km value of 0.71 microM, and its activity was significantly inhibited by carbon monoxide and by antisera against CYP4A4, consistent with the properties previously reported with LTB4 omega-hydroxylase in human polymorphonuclear leukocytes. The amino acid sequence of LTB4 omega-hydroxylase (P-450LTB omega) shows 31-44% similarity to those of CYP4A, CYP4B, and CYP4C, whereas less than 25% similarity was observed with any of the other P-450 families. According to the systematic classification of the P-450 superfamily, P-450LTB omega is classified into the CYP4 family but does not belong to any of the known CYP4 subfamilies. This P-450 composes a new subfamily of CYP4. RNA blot analysis indicated that mRNA hybridized to the cDNA was expressed in the polymorphonuclear leukocytes as well as leukocytes from four individuals. Isolation of the cDNA opens the way to investigate the physiological role and to regulation of the omega-hydroxylase in the inflammation process.
Highlights
From the $Department ofFood Science and Technology, Faculty of Engineering, Fukuyama Uniuersity, Fukuyama, Hiroshima 729-02, Japan, SToneyama Institute for Tuberculosis Research, Osaka City University Medical School, Toyonaka, Osak5a60, Japan, and the
Two clones, termed F-22 and A-4, which contained inserts about 1.2 and 1.5 kilobase pairs in size, were obtained (Fig. 1).As the two cDNAs together did not seem t o cover the full length of the open reading frame of P-450, the cDNA library was synthesized from human leukocyte mRNA by the primer extension method using the F-22-specific primer
Thosetwo clones, termed M-6 and S-8, contained an insert about 1.2 kilobase pairs in size (Fig. 1). three cDNA clones, F-22, A-4, and "6, overlapped with each otherby restriction enzyme mapping (Fig. l), the clone S-8 was different from the others because of the lack of the Pstl site observed with clones A-4 and M-6
Summary
Isolation of P-450 cDNA Clones-Approximately 1 X lo plaques from a human leukocyte cDNA librarywere screened with rabbit P-450,.2 cDNA [11]used as a probe. Two clones, termed F-22 and A-4, which contained inserts about 1.2 and 1.5 kilobase pairs in size, were obtained (Fig. 1).As the two cDNAs together did not seem t o cover the full length of the open reading frame of P-450, the cDNA library was synthesized from human leukocyte mRNA by the primer extension method using the F-22-specific primer. Thosetwo clones, termed M-6 and S-8, contained an insert about 1.2 kilobase pairs in size (Fig. 1). three cDNA clones, F-22, A-4, and "6, overlapped with each otherby restriction enzyme mapping (Fig. l), the clone S-8 was different from the others because of the lack of the Pstl site observed with clones A-4 and M-6. The cDNA clone S-8, contained 1185 nucleotides and exhibited almost the samseequence as M-6cDNA This cDNA had four base replacements comparedwith the othercDNA clones in its coding sequence (Fig. 2). Expression of p - 4 5 0 ~mR~N~A~in Human PMN-Blot analysis of the total RNA from human PMNwas performed by using a cDNA fragment in the 3"untranslated region of PV SHpiESTpPAspSPav
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