CDNA and protein sequence for canine arachidonate 5-lipoxygenase: stuctural modelling and species differences

Abstract

5-lipoxygense is an important enzyme in the genesis of leukotrienes through arachidonic acid metabolism to 5-hydroxyecosatetranoic acid (5)-HETE. Not only is this enzyme important in producing the chemotactic leukotrienes, but it may also be important in producing autocrine lipid signaling molecules (5-oxo-ETE, LTB4) for cellular proliferation. mRNA was isolated in our laboratory from canine leukocytes, and transitional carcinoma cells using the Qiagen whole blood mRNA extraction kit. An internal cDNA fragment was cloned using primers designed according to homologous regions in human and rat 5-LOX. Once an internal sequence was determined both 5′ and 3’ RACE reactions were performed according to the manufacturer's suggestions using the Ambion RACE kit. The 5′ region just upstream of the ATG start site to 300 bp beyond the terminal codon was sequenced. Protein structural modeling was performed through threading of the presumed sequence onto the known rabbit 15-LOX using Swiss PDB viewer. Based on the known human sequence and structural modeling there are three areas of interest in the canine structure. (i) In the catalytic domain there are a number of charged amino acid differences in an external alpha helix at amino acids 645-660, which may confer structural differences between human and canine 5-LOX. (ii) An important phosphorylation site at ser 532 is actually a threonine in canine 5-LOX. (iii) All mammalian 5-lipoxygense enzymes are between 673–674 amino acids; however, the canine 5-LOX sequence contains two additional amino acids, a leucine and valine at residues 379 and 380 respectively, making canine 5-LOX 676 amino acids rather than the 674 identified in human, mouse and bovine.