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研究生:彭于綾
研究生(外文):Yu-Ling Peng
論文名稱:探討斑馬魚亞精胺/精胺乙醯轉移酶2異構基因之酵素活性及生理功能
論文名稱(外文):Characterization of the enzyme activity and physiological functions of zebrafish spermidine/spermine-N1-acetyltransferase 2 isogenes
指導教授:林翰佳
指導教授(外文):Han-Jia Lin
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:101
中文關鍵詞:亞精胺/精胺乙醯轉移酶2S-乙胺基-L-半胱胺酸5-氫氧基離胺酸
外文關鍵詞:spermidine/spermine-N1-acetyltransferase 2thialysine5-hydroxylysine
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亞精胺/精胺乙醯轉移酶 (spermidine /spermine acetyltransferase, Ssat),是屬於 GCN5 相關的 N-乙醯轉移酶 (GCN5-related N-acetyltransferase, GNAT) 家族蛋白,其催化機制是將乙醯輔酶A的乙醯基轉移至胺類基質而發生乙醯化作用。以人類為例,ssat有兩個異構基因,分別為 h_SSAT1 和 h_SSAT2。h_SSAT1 的反應基質為多胺類的亞精胺及精胺,主要負責調節多胺類的恆定。然而序列類似的h_SSAT2 卻不會對多胺類作用。雖然過去有報導 thialysine 可作為h_SSAT2的基質。然而 thialysine 在人體中含量極低且生理意義不明,因此連帶著 h_SSAT2 的真正生理功能也尚無定論。
本論文從演化與生化的角度探討 ssat 家族的功能演變,首先針對後口動物進行 ssat 基因搜尋,發現所有的脊椎動物都具有 ssat1及ssat2 基因,若繼續追溯較為原始的頭索動物卻只有存在 ssat-like 基因。屬於低等脊椎動物的斑馬魚,具有三條 ssat1 基因及兩條 ssat2 基因。我們嘗試以斑馬魚為模式,探討比較由原始物種到高等脊椎動物間 Ssat2 酵素特性的演進差異,進而探討 Ssat2 的生理功能。
我們利用 RT-PCR 分析斑馬魚 ssat2a 及 ssat2b 兩個異構基因在胚胎及組織中的表現情形,結果得知 ssat2a 只在部分組織中表現;而 ssat2b 則會在胚胎時期及各個組織中表現。以 thialysine 為基質進行酵素活性分析,結果只有 Ssat2b 具有酵素活性。進一步分析造成Ssat2a 沒有活性的原因,發現 Ssat2b 的 Ser81 在 Ssat2a 變為 Asn81,因此對酵素活性產生很大的影響。此外,Ssat2b 第 92 個胺基酸之前的序列也被發現與基質結合有關。使用 5-hydroxylysine 這類結構類似的其他基質時,結果仍只有 Ssat2b 具有酵素活性。過去文獻指出 Ssat 家族蛋白必須形成同型雙體才具有活性。實驗結果發現除了形成同形雙體之外,斑馬魚 Ssat2a 和 Ssat2b 之間會形成異構雙體;甚至 Ssat2b 也會和 Ssat1 的三種異構酶形成異構雙體,然而 Ssat2a 只能和 Ssat1c 形成異構雙體。
由本論文的實驗結果顯示,雖然從脊椎動物開始 ssat家族分成 ssat1 與 ssat2,但斑馬魚 Ssat2b 與其他更原始物種的 Ssat-like 相關酵素一樣都可作用 thialysine 以及結構類似之基質。顯示該酵素活性在這些物種中都具重要,至於沒有活性的 Ssat2a 則可能藉由蛋白質交互作用調控其他生理功能。


Spermidine/spermine acetyltransferase (Ssat), which belongs to GCN5-related N-acetyltransferase (GNAT) superfamily, catalyzes an acetyl-transferring reaction from acetyl-CoA to primary amine substrates. Two ssat isogenes, SSAT1 and SSAT2, were found in human. The substrates of human SSAT1 are spermidine and spermine, thus it regulates polyamine homeostasis. On the other hand, human SSAT2 cannot catalyze polyamine catabolism. Previous reports have indicated that thialysine can be a substrate of SSAT2. However, thialysine is rarely found in human body and its function is still unclear. Therefore, the real function of human SSAT2 is still a mystery.
Studying the evolutionary path of the properties of ssat gene family may provide some clues to elucidate the function of Ssat2. By searching the genomic database from 10 deuterostomia, we found that all vertebrates contain both ssat1 and ssat2 but invertebrates only contain ssat-like genes. Zebrafish, a lower vertebrate, contains 3 ssat1 and 2 ssat2 isogenes, which could be used as an example to trace and compare the functional evolution of Ssat2 from unicellular organism to higher animals.
We performed RT-PCR to characterize the expression pattern of ssat2a and ssat2b isogenes. ssat2a was only expressed in certain tissues but ssat2b was widely expressed in all tissues from adult fish and every stages during embryo development. When thialysine was used as a substrate, the enzyme activity was only detected from Ssat2b but not Ssat2a. Further analysis revealed that the substitution of Ser81 to Asn81 in Ssat2a was the cause of enzyme activity lost. In addition, the first 92 residues of Ssat2b were also related to the substrate binding. When 5-hydroxylysine, a structurally analog of thialysine, was used as a substrate, Ssat2b is still the only isoenzyme possessed activity. It has been reported that all members of Ssat family possess a homodimer configuration as their active form. Our results demonstrated that, in addition to homodimer, Ssat2a and Ssat2b were able to form a heterodimer with each other. Moreover, we found that Ssat2b could also form a heterodimer with all zebrafish Ssat1 isoenzymes, but Ssat2a could only form a heterodimer with zebrafish Ssat1c.
Notwithstanding the divergency of ssat2 and ssat1 from ssat ancestor gene, however, zebrafish Ssat2b preserved the activity to catalyze thialysine and its structural analogs as other Ssat-like enzyme from lower species. Therefore, such enzyme activity might be important in these species. Although Ssat2a does not show any enzyme activity, it might be able to regulate other physiological functions through protein-protein interactions.

謝誌 ………..………………………………………………………………………….…I
摘要……………………………… III
Abstract….. V
目錄……………………………………………………………………………………VII
圖目錄…… IX
表目錄…… IX
壹、 緒論 1
1.1 GNAT 家族 1
1.2 亞精胺/精胺乙醯轉移酶家族 3
1.3 各物種 Ssat2 的研究 4
1.4 Ssat2 作用基質的探討 5
1.5 Ssat2 酵素重要活性區域探討 8
1.6 Ssat2 其他可能的生理功能探討 10
1.7 研究動機 11
貳、 材料與方法 16
2.1實驗材料 16
2.2 分子生物學方法 19
2.2.1 斑馬魚 RNA 萃取 19
2.2.2核醣核酸洋菜膠體電泳 21
2.2.3斑馬魚 cDNA 製備 22
2.2.4基因選殖引子設計 23
2.2.5聚合酶鏈鎖反應 (PCR) 25
2.2.6 PCR 產物磷酸化 26
2.2.7 Chimeric PCR 27
2.2.8 小量及中量質體抽取 31
2.2.9 重組質體製備 34
2.2.10 質體轉型 36
2.2.11 Site-directed mutagenesis method 37
2.3蛋白質實驗方法 39
2.3.1 誘導重組蛋白於大腸桿菌表現 39
2.3.2 帶有 GST 與His-tag 的重組蛋白之親和層析純化 41
2.3.3 蛋白質溶液濃縮 44
2.3.4 蛋白質濃度測定 44
2.3.5 SDS-聚丙烯醯胺膠體電泳 45
2.3.6 Coomassie blue 染色 47
2.3.7 GST 重組蛋白 pull-down 分析 48
2.3.8 西方點墨法 49
2.3.9 SSAT2 蛋白活性分析 52
2.4 細胞實驗方法 54
2.4.1 人類 HEK293T 細胞株的培養 54
2.4.2 細胞之磷酸鈣轉染 (calcium phosphate transfection) 55
2.4.3 細胞存活率實驗 (Cell viability XTT method ) 57
参、 結果 60
3.1斑馬魚ssat2 異構基因序列分析及其他物種間的序列比較 60
3.2斑馬魚 ssat2 異構基因 mRNA 在胚胎發育過程及各個組織的表現情形 ………………………………………………………………………………...60
3.3斑馬魚 Ssat2 異構酶的酵素活性特徵 62
3.3.1基因選殖與重組蛋白表現 62
3.3.2斑馬魚 Ssat2 異構酶的酵素活性測試 62
3.3.3斑馬魚 Ssat2 異構酶蛋白質序列與酵素活性的關連性探討 63
3.3.4斑馬魚 Ssat2 異構酶酵素動力測試 64
3.3.5 找尋其他斑馬魚 Ssat2 異構酶的可能基質 66
3.4斑馬魚 Ssat2 異構酶的蛋白質交互作用 68
肆、 討論 83
4-1 以生物資訊搜尋 ssat 相關基因探討 83
4-2 斑馬魚 ssat2 異構基因 mRNA 在胚胎發育過程及各個組織的表現情形之探討 84
4-3 斑馬魚 Ssat2 異構酶之間的酵素活性探討 84
4-4斑馬魚 Ssat2 異構酶的蛋白質交互作用 87
4-5 以演化角度探討斑馬魚 ssat2 的生理功能 88
4-6 未來展望 89
參考文獻……………………………………………………………………………….94
附錄一…………….. 97
附錄二………….. 98
附錄三…………… 99
附錄四………….. 100
附錄五....... 101

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