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

固醇酰基轉移酶 (Sterol O-acyltransferase，Soat)為一種將長鏈脂肪酸與膽固醇酯化形成膽固醇酯的細胞內酵素。在哺乳類動物中發現 Soat 有兩種同功酶(isoenzyme)： Soat1 與 Soat2 。 哺乳類動物中， Soat1 廣泛存在於組織中(包括小腸、腎臟、心臟、肝臟、巨噬細胞等)，而 Soat2 只存在於小腸和肝臟。 根據基因表現位置，Soat1 在生理上被認為是將過多游離膽固醇形成膽固醇酯後，以脂肪滴形式儲存於細胞內以維持細胞內膽固醇平衡； Soat2 則被認為是將飲食中攝取中的膽固醇酯化後，在內質網將膽固醇酯聚集到脂蛋白中以利體內運輸膽固醇。 Soat 在家禽胚胎發育過程中應該扮演重要角色，但此酵素確切功能仍待釐清。 為探討 Soat 在胚胎發育中扮演的角色，我們利用斑馬魚與禽類同為卵生動物作為動物模式，選殖斑馬魚 Soat 基因並分析蛋白質序列，結果顯示其與雞、小鼠、大鼠、人類有高度同源性。 RT-PCR 檢測基因表現後，發現 soat1 在胚胎發育一開始即表現，顯示可能為母性基因；而 soat2 則在發育後12小時才被偵測到，隨後不斷增加基因表現至 48 小時。在成魚組織中， soat1 表現於各個器官而 soat2 在成年斑馬魚中在肝臟、小腸、腦及睪丸表現，不同於哺乳類動物中 soat2 只在肝和小腸表現。 利用原位雜合試驗 (whole-mount in situ hybridization) 觀察 soats 在胚胎發育過程中表現位置，發現 soats 在卵黃周圍、腦部、孵化腺 (hatching gland) 均有表現，暗示 Soats 對於卵黃利用及這些部位發育的重要。從基因表現時間與位置的不同，推測 Soat1 與 Soat2 在斑馬魚生理上可能扮演著不同的角色。 為了證實斑馬魚 Soats 具有膽固醇酯化功能，我們將斑馬魚 soat1 與 soat2 基因轉染到 HEK-293 細胞後，利用具有螢光之膽固醇 (NBD-cholesterol) 追蹤細胞內膽固醇位置及膽固醇累積情形，發現 Soat2 表現細胞所顯示的螢光較控制組與 Soat1 表現細胞多。 此外，利用 Oil red-O 觀察細胞內脂肪滴累積情形也有相同的結果，顯示 Soat2 的膽固醇酯化能力比 Soat1 高。 利用 Soats 抑制劑則發現 Avasimibe 並不能降低斑馬魚 Soat1 及 Soat2 活性，而 Pyripyropene A (PPPA) 則可專一性抑制斑馬魚 Soat2 活性。 此外，將 Soat 抑制劑注射到斑馬魚胚胎後，發現 PPPA 能減緩卵黃的消耗速率，而 Avasimibe 則無影響，顯示胚胎發育時期 Soat2 對卵黃的吸收運輸有所影響。 為更深入探討 Soats 在胚胎發育中扮演的角色，我們使用 TALEN (Transcription activator-like effector nucleases) 技術，在魚胚中注射合成 TALEN mRNA 並成功誘導 Soats 標的基因突變。 綜合上述，Soats 在不同模式動物間，具有高度保留性。 而斑馬魚 Soat1 及 Soat2 除了基因表現位置不同外，對於脂質儲存的能力也不同，顯示著兩者間在斑馬魚生理功能上有所差異。 此外，利用 Soats 抑制劑可降低斑馬魚 Soats 活性且PPPA 抑制劑能降低胚胎對卵黃的利用，顯示 Soat2 在胚胎發育中對卵黃利用有所影響。 此外，應用 TALEN 基因剔除技術已成功誘導 Soats 基因突變之斑馬魚，未來將可更進一步探討 Soats 在卵生動物胚胎發育中扮演的功能及角色。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.致謝 i 中文摘要 ii Abstract iv CONTENTS vii LIST OF FIGURES ix Introduction 1 1. Physiological roles of Sterol O-acyltransferase (Soat) 1 2. Sources and functions of cholesterol 2 2.1.1 Sources of cholesterol 2 2.1.2 Functions of cholesterol 4 2.1.3 Disorder due to cholesterol deficiency during embryonic development- Smith-Lemli-Opitz Syndrome (SLOS) 5 3. Soat and disease 6 3.1.1 Atherosclerosis 6 3.1.2 Alzheimer’s disease 8 3.1.3 Tumor cell proliferation 9 4. Lipoprotein synthesis and cholesterol esterification during embryonic development 9 4.1.1 Defective lipoprotein synthesis leads to developmental abnormalities during embryogenesis 10 4.1.2 Cholesterol esterification in the yolk sac membrane during embryogenesis 11 5. Zebrafish as an experimental model for lipid metabolism 12 Specific aims 15 Materials and methods 17 Zebrafish 17 Total RNA extraction 17 Polymerase chain reaction 18 Rapid Amplification of cDNA Ends (5'' RACE-PCR and 3'' RACE-PCR) 18 PCR purification and plasmid DNA extraction 19 Construction of soat expression vector 19 Digoxigenin – labeled riboprobe synthesis and whole-mount in situ hybridization 20 Frozen section in situ hybridization 21 Fluorescent Soat activity assay 22 Establishing Soats expressing cell lines 22 Oil red O staining and quantification of lipid accumulation 23 Soats inhibitors in vitro studies 24 Soats inhibitors in vivo studies 24 Targeted mutagenesis of soat1 and soat2 using transcription activator-like effector nucleases (TALEN) 25 Statistical Analysis 25 Results 27 Genomic structure, sequence and phylogenetic analysis of soat1 and soat2 27 Expression pattern of Soat1 and Soat2 in zebrafish 29 Functional analysis of zebrafish Soats 30 The effect of Soats inhibitors on zebrafish embryos yolk utilization 32 Design of TALENs for the deletion of zebrafish soat genes 33 Discussion 35 Future work 41 Reference 42 Table 1 56 Figures 5