Abstract
Two types of 12-lipoxygenase that catalyze the transformation of arachidonic acid to 12(S)-hydroperoxyeicosatetraenoic acid (12-HPETE) have been previously classified into platelet-type and leukocyte-type categories. Here, we document, for the first time, a molecular characterization of both forms within the same species. The amino acid sequence of the murine platelet 12-lipoxygenase deduced from its cDNA is 58% identical to the murine spleen/leukocyte 12-lipoxygenase. Expression constructs carrying the cDNAs for the two 12-lipoxygenase forms were introduced into human embryonic kidney 293 cells. The platelet-type enzyme metabolized arachidonic acid exclusively to 12-HPETE, whereas the leukocyte-type enzyme formed both 12-HPETE and 15-hydro(pero)xyeicosatetraenoic acid in a ratio of approximately 3:1. Linoleic acid was metabolized to a similar extent by the latter enzyme to 13-hydro(pero)xyoctadecadienoic acid but not by the platelet enzyme. Mutagenesis and deletion of the highly conserved lipoxygenase C-terminal isoleucine (Ile663), a residue believed to be involved in the non-heme iron atom coordination of all lipoxygenases, was performed. Deletion of Ile663 and substitution with most amino acids abolished enzyme activity. Only a valine substitution retained significant activity. These findings would tend to indicate a stringent requirement for the proper spatial alignment and folding of the C-terminal chain back into the core of the enzyme to interact with the iron atom by analogy with the recently determined crystal structure of a soybean lipoxygenase (Boyington, J. C., Gaffney, B. J., and Amzel, L. M. (1993) Science 260, 1482-1486). The platelet-type and leukocyte-type 12-lipoxygenase genes were cloned from a murine 129 Sv genomic library. Both genes are divided into a similar 14-exon/13-intron format, with the platelet-type gene being approximately twice the size of the leukocyte-type gene (13 versus 7.5 kilobases). A segment of a third gene was also isolated and probably represents a pseudogene derivative of either of these 12-lipoxygenase genes. All three genes were mapped to the central region of mouse chromosome 11 in a region of homology with human chromosome 17. Antibodies prepared against the two forms of 12-lipoxygenase revealed the differential distribution of the two enzymes throughout the mouse.
Highlights
Cosatetraenoic acid (12-HPETE) have been previously classified into platelet-type and leukocyte-type categories
The amino acid sequence of the murine platelet 1 2 4 - kingdoms, which stereospecifically incorporate molecular oxypoxygenase deduced from its cDNA is 58% identical to the murine spleedeukocyte 12-lipoxygenase.Expression constructs carrying the cDNAs for the two 1 2 4
Total RNA was prepared from the platelets, leukocytes, and spleen tissubey the methodof Chomzcynskiand Sacchi [20]A. liquots (1pg for platelets and 10 pg for otherso)f total RNA served as templates for cDNA synthesis with murine Moloney leukemia virus reverse transcriptase and random primers as described [21].Polymerase chain reaction was carried outfor 40 cycles in a Perkin-Elmer thermaclycler with Taq polymerase at 94 "C for 45 s, 48-50"C for 45 s, and 72 "C for 1 min 30 s
Summary
The methodof Chomzcynskiand Sacchi [20]A. liquots (1pg for platelets and 10 pg for otherso)f total RNA served as templates for cDNA synthesis with murine Moloney leukemia virus reverse transcriptase and random primers as described [21].Polymerase chain reaction was carried outfor 40 cycles in a Perkin-Elmer thermaclycler with Taq polymerase at 94 "C for 45 s, 48-50"C for 45 s, and 72 "C for 1 min 30 s. Primers were designedfrom conserved leukocyte-type12-lipoxygenase kocyte-type 12-lipoxygenase was isolatedin similarfashion ussequences [22,23,24] and from the murine genomic 12-lipoxygenase sequence (for platelet 12-lipoxygenase). The two forms of murine 12-lipoxygenase are 58%identical at the amino acid level and, interestingly, the platelet enzyme lacks 1of the 5 conserved histidine lipoxygenases were constructed in pCMV5 [25] and pcDNA-1 (Invitro- residues found in all plant and animal lipoxygenases, in the gen), respectively, by ligation of various PCR fragments.Details of central region of the molecule (Fig. 1).Clearly, themurine construction are available upon request. Human embryonic kidney (HEK) 2c9e3lls were transfected with the (93%identity) and about related to 15-lipoxygenases purified DNAs by the calcium phosphate method [26]; 48 h later the and leukocyte-type 12-lipoxygenases from other species Linoleic acid and subjected to HPLC analysis [27]
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