A NOVEL FORM OF CYTOCHROME-P-450 FAMILY-4 IN HUMAN POLYMORPHONUCLEAR LEUKOCYTES - CDNA CLONING AND EXPRESSION OF LEUKOTRIENE-B4 OMEGA-HYDROXYLASE

Isolation of cDNA clones for human leukotriene B4 (LTB4) omega-hydroxy lase clearly demonstrates that the hydroxylase is a member of the cyto chrome P-450 (CYP) superfamily. cDNA clones isolated from a human leuk ocyte 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 K(m) value of 0.71 muM, and its activity was significantly inh ibited by carbon monoxide and by antisera against CYP4A4, consistent w ith the properties previously reported with LTB4 omega-hydroxylase in human polymorphonuclear leukocytes. The amino acid sequence of LTB4 om ega-hydroxylase (P-450LTBomega) shows 31-44% similarity to those of CY P4A, CYP4B, and CYP4C, whereas less than 25% similarity was observed w ith any of the other P-450 families. According to the systematic class ification of the P-450 superfamily, P-450LTBomega is classified into t he CYP4 family but does not belong to any of the known CYP4 subfamilie s. This P-450 composes a new subfamily of CYP4. RNA blot analysis indi cated that mRNA hybridized to the cDNA was expressed in the polymorpho nuclear leukocytes as well as leukocytes from four individuals. Isolat ion of the cDNA opens the way to investigate the physiological role an d to regulation of the hydroxylase in the inflammation process.