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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.

SUBMITTER: Haas U

PROVIDER: S-EPMC3251132 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Targeted knock-down of a structurally atypical zebrafish 12S-lipoxygenase leads to severe impairment of embryonic development.

Haas Ulrike U Raschperger Elisabeth E Hamberg Mats M Samuelsson Bengt B Tryggvason Karl K Haeggström Jesper Z JZ

Proceedings of the National Academy of Sciences of the United States of America 20111205 51

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 zf ...[more]

PMID: 22143766

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Project description:BackgroundProtein-tyrosine kinases (PTKs) regulate virtually all biological processes. PTKs phosphorylate substrates in a sequence-specific manner and relatively short peptide sequences determine selectivity. Here, we developed new technology to determine PTK activity profiles using peptide arrays. The zebrafish is an excellent model system to investigate signaling in the whole organism, given its wealth of genetic tools, including morpholino-mediated knock down technology. We used zebrafish embryo lysates to determine PTK activity profiles, thus providing the unique opportunity to directly compare the effect of protein knock downs on PTK activity profiles on the one hand and phenotypic changes on the other.MethodologyWe used multiplex arrays of 144 distinct peptides, spotted on a porous substrate, allowing the sample to be pumped up and down, optimizing reaction kinetics. Kinase reactions were performed using complex zebrafish embryo lysates or purified kinases. Peptide phosphorylation was detected by fluorescent anti-phosphotyrosine antibody binding and the porous chips allowed semi-continuous recording of the signal. We used morpholinos to knock down protein expression in the zebrafish embryos and subsequently, we determined the effects on the PTK activity profiles.Results and conclusionReproducible PTK activity profiles were derived from one-day-old zebrafiish embryos. Morpholino-mediated knock downs of the Src family kinases, Fyn and Yes, induced characteristic phenotypes and distinct changes in the PTK activity profiles. Interestingly, the peptide substrates that were less phosphorylated upon Fyn and Yes knock down were preferential substrates of purified Fyn and Yes. Previously, we demonstrated that Wnt11 knock down phenocopied Fyn/Yes knock down. Interestingly, Wnt11 knock down induced similar changes in the PTK activity profile as Fyn/Yes knock down. The control Nacre/Mitfa knock down did not affect the PTK activity profile significantly. Our results indicate that the novel peptide chip technology can be used to unravel kinase signaling pathways in vivo.

Dataset Information

Targeted knock-down of a structurally atypical zebrafish 12S-lipoxygenase leads to severe impairment of embryonic development.

Publications

Targeted knock-down of a structurally atypical zebrafish 12S-lipoxygenase leads to severe impairment of embryonic development.

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