title: Targeted knock-down of a structurally atypical zebrafish 12S-lipoxygenase leads to severe impairment of embryonic development.
abstract: Lipoxygenases (LO) are a class of dioxygenases, which form hydroperoxy, hydroxy, and epoxy derivatives of arachidonic acid with distinct positional and stereochemical configurations. In man, there are two known types of 12-LO that are distinguished by their expression patterns and catalytic properties. The platelet 12S-LO plays a role in platelet aggregation and 12R-LO seems to be important for normal skin function. Using BLAST searches of the zebrafish (zf) genome we identified one candidate zf12-LO gene with 43% identity with human 12R-LO at the mRNA level and the deduced primary sequence carried the so called "Coffa" structural determinant (Gly residue) for R stereoselectivity of LOs. However, incubations of recombinant, purified, zf12-LO with arachidonic acid revealed exclusive formation of 12(S)-hydroperoxy-eicosatetraenoic acid. Further studies with immunohistochemistry showed prominent expression of zf12-LO in the cell nuclei of skin epithelium, the epithelial lining of the stomodeum, and the pharyngeal pouches in zf embryos. To probe its function, zf12-LO was subjected to targeted knock-down in zf embryos, resulting in the development of a severe phenotype, characterized by abnormal development of the brain, the eyes, and the tail as well as pericardial and yolk sac edema. Hence, we have identified a unique vertebrate 12S-LO that breaks the current structure-function paradigms for S and R stereo-specificity and with critical roles in normal embryonic development.
authors.affiliation: Division of Chemistry 2, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Haas
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Raschperger
authors.forename: Mats
authors.initials: M
authors.lastname: Hamberg
authors.forename: Bengt
authors.initials: B
authors.lastname: Samuelsson
authors.forename: Karl
authors.initials: K
authors.lastname: Tryggvason
authors.forename: Jesper Z
authors.initials: JZ
authors.lastname: Haeggström
mesh_terms: D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000737:chemistry* / Q000235:genetics; D016718:Arachidonic Acid / Q000737:chemistry; D001792:Blood Platelets / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D003001:Cloning, Molecular; D015509:Developmental Biology / Q000379:methods; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D007150:Immunohistochemistry / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D010641:Phenotype; D013237:Stereoisomerism; D015027:Zebrafish
chemical_list: D016718:Arachidonic Acid; D001092:Arachidonate 12-Lipoxygenase