第二型去氧核醣核酸酶中各組胺酸殘基之功能

Abstract

第二型（乙型）去氧核醣核酸酶 (Deoxyribonuclease II, DNase II, EC 3.1.22.1)為一好酸性之非專一性核醣核酸內切酶，其最適作用的 pH 值為 4.7。目前認為第二型去氧核醣核酸酶可能參與水解外來 DNA，並有可能參與細胞凋亡。為研究其酵素催化活性，我們進行一系列之組胺酸定點突變，首先構築了豬脾臟第二型去氧核醣核酸酶 (DNase II) 之各組胺酸 (Histidine) 突變成白胺酸 (Leucine) 之突變株，將這些突變株分別轉染人類胚胎腎臟293T細胞。在轉染48小時之後，可在細胞培養液中得到突變株表現蛋白質，收集培養液之後再對醋酸緩衝液透析，濃縮，經由管柱層析得到純化蛋白質。分析這些蛋白質的比活性，發現H41L, H109L, H206L, H207L, H274L 及H322L 突變蛋白質的比活性分別為 223.2, 286.9, 218.8, 135.0, 178.2, 320.0 U/mg 而野生型DNase II的比活性為320.0 U/mg，由比活性的結果可知上述突變株對於DNase II的催化能力並無影響。 在H115及H297方面，經由圓二色偏光光譜分析發現 H115 及 H297 之白胺酸及丙胺酸突變株之二級結構與野生型並無太大差異；gel-retard 實驗則證實 H115 及 H297 之白胺酸及丙胺酸突變株之DNA結合能力與野生型相比並無不同，可見此二組胺酸之突變株之所以沒有活性，並非突變株改變了酵素結構所致，而是此二組胺酸直接參與了 DNase II 催化水解 DNA。接著我們以化學救法實驗證實 H115及 H297此二組胺酸直接參與第二型去氧核醣核酸酶之催化反應，H115A於外加imidazole 濃度達 100 mM時，DNase II活性回復倍率達 7 倍；而 H297A於外加imidazole 濃度達 100 mM時，DNase II 活性回復倍率達 11 倍，而在由pH 4.0 至 5.6 之間不同 pH 值之下進行化學救援法實驗，發現 H115A 在 pH 4.7 時有最大活性回復倍率，而H297A 則在 pH 4.95 時有最大活性回復倍率，相較於 H115A，H297A之較大活性回復倍率較偏鹼性，顯示偏鹼性之 imidazole 對 H297A 之活性回復較佳，而較偏酸性之imidazole 則對 H115A 之活性回復情形較佳，因此，H115在 DNase II 催化水解 DNA 時為催化酸而H297為催化鹼。在H132的方面，在轉染16小時後，可以在細胞內發現有H132L的表現，但在轉染24小時後， H132L的表現量卻急速下降，另外在細胞表現H132L時添加proteosome抑制劑（Z-Leu-Leu-Leu-al），則可在培養液中偵測到H132L的表現，但不具酵素活性。因此H132L為一不折疊 (unfolded)蛋白質，故會啟動細胞內不折疊蛋白質反應，被細胞內的降解機制所辨識，進而將之降解。另一突變株H132K的表現方面，經由分離細胞胞器實驗發覺 H132K 並沒有被送至溶酶體中而是分佈於內質網 (ER) 或是高基氏體 (Golgi body) 中，共軛焦顯微鏡搭配免疫染色等實驗證實，H132K在細胞內會與內質網標記蛋白質 calnexin之分佈情形相同，因此H132K的表現應是被侷限於內質網中，所以H132K應為錯誤折疊 (misfolded) 蛋白質，因細胞內蛋白質合成品管機制作用之故而無法傳送至細胞質中。另外經由比對七個物種九條DNase II的序列後，發現 H115，H297以及H132在每個DNase II序列中都是保留的，其他的組胺酸殘基則或多或少都有些微差異。可見此三個組胺酸殘基對DNase II而言的確相當重要。

Deoxyribonuclease II (DNase II) is an acid endonuclease that is involved in the degradation of exogenous DNA and is important for DNA fragmentation and degradation during cell death. In an effort to understand its catalytic mechanism, we constructed plasmids encoding nine different His-to-Leu mutants of porcine DNase II, and examined the enzyme properties of the mutant proteins. Of the nine mutant proteins, all but H132L were secreted into the growth media of H293Tcells. Six of the mutated DNase II proteins showed enzymatic activities (H41L, H109L, H206L, H207L, H274L and H322L with specific activities of 223.2, 286.9, 218.8, 135.0, 178.2, 320.0 U/mg, respectively), whereas the H115L, H132L and H297L mutant proteins exhibited very little activity. The H115L and H297L mutant proteins were found to undergo correct protein folding, but were inactive. To further examine these mutants, we expressed H115A and H297A DNase II; these mutant proteins were inactive, but their DNase activities could be rescued with imidazole. Addition of 100 mM imidazole increased the DNase II activity of H115A by approximately 7-fold and that of H297A by approximately 11-fold. We next assessed the chemical rescue of H115A and H297A in buffers with different pH values. The pH-activity profiles showed the optimum pH for the imidazole-based restoration of catalytic activity was more acidic for H115A versus H297A, indicating that H115 and H297 are likely to function as a general acid and a general base, respectively, in the catalytic center of the enzyme. In contrast to the secreted mutants, the H132L mutant protein was found in cell lysates within 16 h after transfection. This protein was inactive, improperly folded and was drastically degraded via the proteosomal pathway after 24 h. The polypeptide of another substitution for H132 with Lys resulted in the misfolded form and retained in endoplasmic reticulum. In conclusion, we identified the H115 is a general acid whereas H297 is a general base in the catalysis of DNase II. H132 is a key residue for DNase II folding, other His residues are dispensable. The results provide important new insights into the catalytic mechanism and protein folding in DNase II.