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

Proc Natl Acad Sci U S A. 2011 Dec 20;108(51):20479-84. doi: 10.1073/pnas.1117094108. Epub 2011 Dec 5.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arachidonate 12-Lipoxygenase / chemistry*
  • Arachidonate 12-Lipoxygenase / genetics
  • Arachidonic Acid / chemistry
  • Blood Platelets / metabolism
  • Chromatography, High Pressure Liquid / methods
  • Chromatography, Liquid / methods
  • Cloning, Molecular
  • Developmental Biology / methods
  • Gene Expression Profiling
  • Gene Expression Regulation, Developmental*
  • Immunohistochemistry / methods
  • Mass Spectrometry / methods
  • Microscopy, Fluorescence / methods
  • Phenotype
  • Stereoisomerism
  • Zebrafish

Substances

  • Arachidonic Acid
  • Arachidonate 12-Lipoxygenase