Molecular Cloning and Characterization of Sterol O-acyltransferases in Zebrafish Embryos

Sterol O-acyltransferase (Soat) is an intracellular enzyme that esterify long-chain fatty acyl-CoA and cholesterol to form cholesterol esters. Two isozymes of Soat had been identified in most animals, Soat1 and Soat2. In mammals, soat1 is expressed ubiquitously in various tissues (including kidney, liver, intestine, heart, macrophages, etc.), while soat2 is restrictively distributed only in the liver and intestine. There are two distinct roles of Soat in animal physiology: 1) formation of cholesterol esters in intracellular as lipid droplets for storage and 2) assembly of cholesterol esters into apoB-containing lipoproteins in the endoplasmic reticulum (ER) lumen for lipid transport. Soat also plays a role in bird embryonic development, but its exact role in embryogenesis remains to be further elucidated. Since Soat is expressed at high levels in the yolk sac membrane and cholesterol esters are synthesized extensively during chicken embryonic development, it is reasonable to hypothesize that Soat mediates the absorption of free fatty acids and yolk cholesterol in the yolk sac membrane and is responsible for the absorption and transportation of yolk lipids to the developing embryos.
To investigate the roles of Soat during embryonic development, zebrafish Soats were cloned and the soat1 and soat2 mRNA encoded proteins of 554 and 534 amino acids, respectively. Soat1 was detected as early as 0 hpf indicating the existence of maternal soat1 message and it was expressed ubiquitously in adult zebrafish tissues. Soat2 mRNA was detected from 12 hpf and it was expressed in liver, intestine, brain and testis in adult zebrafish. Whole mount in situ hybridization demonstrated that both soat1 and soat2 expressed around the yolk, brain and hatching gland, indicating that soats play an important role in these regions. The different gene expression pattern of soat1 and soat2, suggested that Soat1 and Soat2 may play different physiology roles in zebrafish. To investigate zebrafish Soat1 and Soat2 enzyme activity, we incubated Soat1 and Soat2-expressing cells with their substrates. Results suggested that Soat2-expressing cells have a higher activity of cholesterol esterification and thereby more intracellular lipids than Soat1-expressing cells and wild type cells, indicating that Soat2 activity is higher than Soat1. To test Soats inhibitors reduce zebrafish Soats activity, Avasimibe and Pyripyropene A (PPPA) were used for in vitro and in vivo studies. In vitro studies showed that Avasimibe had no effect on zebrafish Soat1 and Soat2, whereas PPPA could only inhibited zebrafish Soat2 activity. In addition, after injecting AVA into zebrafish embryos (3hpf), there was no significant difference between AVA and DMSO groups in yolk decreasing, whereas PPPA treatment leaded to slower rate of yolk decreasing, indicated that Soat2 plays a role in yolk utilization. To further investigate the roles of Soats during zebrafish embryogenesis, we used transcription activator-like effector nucleases (TALENs) to create the soat1 and soat2 knockouts in zebrafish. Results showed that the TALEN-mediated mutations of Soats were successful induced and the toxicity was moderate.
In conclusion, comparisons of soat gene sequences indicated that zebrafish soat1 and soat2 are highly conserved throughout evolution. In the in vitro study, we found there were differences between zebrafish Soat1 and Soat2, such as different gene expression profiles and different cholesterol-esterifying activity, indicating the distinct physiology roles in zebrafish. In Soats inhibitors studies, Avasimibe, were found to have no inhibitory activity toward zebrafish Soat1 and Soat2, whereas PPPA was identified that specifically inhibits Soat2. In vivo studies demonstrated that Soat2 could be responsible for the yolk utilization. To further investigate the roles of Soats in vivo, we have successfully generated the Soat1 and Soat2-f0 mutant fish by using TALEN system. The function of soats during zebrafish embryogenesis will be further demonstrated by the knockout study.