應用不朽化山羊乳腺上皮細胞研究醣基化對原核溶葡萄球菌酶之影響與真核Rad21基因於細胞存活路徑之角色

Abstract

隨著雌性動物懷孕，乳腺上皮細胞經歷細胞增生與分化，故其為一研究細胞生長、凋亡、移動與表現有價值重組蛋白之良好模式。然而，初級培養乳腺上皮細胞之應用受其有限生命周期之限制。本研究中，藉由表現人類端粒反轉錄酶(human telomerase reverse transcriptiase; hTERT) 與第16型人類乳突病毒E7基因建立不朽山羊乳腺上皮細胞株(CMEC-08-D)。 為確認CMEC-08-D可應用於表現具生物活性之外源性重組蛋白，將自Staphylococcus simulans選殖出之溶葡萄球菌酶(lysostaphin) 表現於CMEC-08-D，並分析其蛋白質運送路徑與醣基化位置。藉由表現帶有乳腺專一乳蛋白山羊beta-酪蛋白、乳鐵蛋白或原核訊息胜肽之成熟溶葡萄球菌酶重組蛋白於CMEC-08-D，證實兩種真核訊息胜肽皆能成功的引導重組後的成熟溶葡萄球菌酶經內質網路徑分泌至培養液中。經由突變其可能醣基化位置試驗，結果證實當溶葡萄球菌酶蛋白表現於CMEC-08-D中時，僅其第125個胺基酸-天冬醯胺酸（asparagine; Asn）的位置會發生N-鏈結醣基化作用，而第232個胺基酸-天冬醯胺的位置則否。此外，當利用點突變的方式將天冬醯胺以穀胺醯胺基酸(glutamine; Gln)取代，結果顯示突變後的N125Q與 N125Q/N232Q-溶葡萄球菌酶重組蛋白具裂解金黃色葡萄球菌能力，然而，突變的N232Q-與野生型溶葡萄球菌酶則不具裂菌活性。更進一步檢測醣基化對溶葡萄球菌酶重組蛋白與金黃色葡萄球菌結合能力之影響，結果顯示處理組間結合能力皆無差異。綜上結果顯示，第125個胺基酸上之醣基化會明顯減弱溶葡萄球菌酶之裂菌能力而非其與金黃色葡萄球菌之結合能力。 在先前的試驗中顯示，連結姊妹染色分體之黏結蛋白(cohesin) 次單位Rad21基因差異表現於小鼠乳腺的退乳時期與泌乳時期。因此，CMEC-08-D亦被用作為研究Rad21是否參與乳腺退化之研究平台。目前已知，當誘導細胞凋亡時，全長的Rad21會裂解形成N端-與C端-Rad21裂解產物。由於細胞凋亡與移動皆為乳腺退化過程之重要事件。故利用自動化即時偵測細胞行為系統（Electric Cell-substrate Impedance Sensing，ECIS）與傷口癒合分析(wound healing assay)了解Rad21與細胞移動之關係。結果顯示，外源性穩定表現全長Rad21可促進CMEC-08-D移動能力。更進一步利用紫外光(ultraviolet light)誘導細胞凋亡進而研究全長、N端-與C端-Rad21裂解產物與細胞凋亡之關係。經由細胞核染色檢測DNA片斷化情形與Akt蛋白活化情形，結果顯示外源性表現Rad21降低紫外光所誘導之細胞凋亡比例，此外，在紫外光誘導細胞凋亡後，穩定表現有Rad21的細胞株皆有較高之Akt蛋白總量。以上結果顯示，Rad21可能在乳腺退化時期維持細胞存活。 綜合以上結果，不朽化CMEC-08-D細胞株為一有價值研究模式於外源性表現具生物活性之重組蛋白。定義確切溶葡萄球菌酶之醣基化位置與醣基化對其功能之影響將有助於未來利用轉基因動物方式表現溶葡萄球菌酶之預防乳房炎之研究。此外，利用CMEC-08-D細胞株為平台亦了解Rad21可能於乳腺退化時期扮演多功能角色。

The mammary epithelial cells undergo proliferation and differentiation during repeat pregnancy, is an ideal model for study cell growth, apoptosis, migration, and expressed valuable recombinant proteins. However, a finite life span of primary cells limits their application. Here, human telomerase reverse transcriptase (hTERT) and human papilloma virus 16 E7 genes were used to generate an immortalized caprine mammary epithelial cell line (CMEC-08-D). To examine whether the CMEC-08-D can apply to express prokaryotic recombinant protein with biological function, an anti-staphylococcal agent lysostaphin from Staphylococcus simulans that had been used to cure Staphylococcus aureus mastitis was expressed in CMEC-08-D to study the sorting pathway and site-specific glycosylation. Recombinant lysostaphin fused with the mammary specific milk protein signal peptides of goat beta-casein, lactoferrin or prokaryotic were separately ectopic expressed in CMEC-08-D, both eukaryotic signaling peptides successfully led recombinant lysostaphins secreted into media through ER secretory pathway. Results from site-directed mutagenesis show that Asn125 but not Asn232 is the exact glycosylation site of lysostaphin expressed in CMEC-08-D. In addition, the effect of glycosylation of lysostaphin on its staphylolytic activity was identified through bacterial plate assay. The data indicated that wild type and mutated N232Q-lysostaphin (Asn232 to Gln232 substitution) lacked staphylolytic activity. In contrast, mutated N125Q (Asn125 to Gln125 substitution) and N125Q/N232Q-lysostaphin possessed staphylolytic activity. On the other hand, all mutated lysostaphin showed no change in binding ability to S. aureus. This reveals that N-glycosylation at Asn125 of lysostaphin expressed in a eukaryotic system greatly decreases lysostaphin bacteriolytic activity but does not affect its binding ability to S. aureus. In the previous study, the Rad21, one of the major cohesion subunits that hold chromatids together has highly differential expression between involution and lactation stages of the murine mammary gland. Thus, the CMEC-08-D was also used as a platform to examine whether Rad21 involved in the involution events. Rad21 is cleaved to form carboxy-terminal and amino-terminal products during apoptotic stimuli. As the cell migration and apoptosis are the important events that involved in mammary gland involution. The cell migration ability was evaluated in stable expressed exogenous Rad21 CMEC-08-D cells. The results suggested that full-length Rad21 enhances cell migration by wound healing assay and electrical cell substrate impedance-sensing system (ECIS) migration assay. To examine the effect of different truncated form Rad21 on apoptosis, ultraviolet light (UV) was used to induce cell apoptosis. The apoptosis and its pathway were evaluated by DNA fragmentation using Hoechst 33258 and Akt activation. Rad21 protected cells undergoing apoptosis induced by UV damage. Moreover, the expression of Akt was increased in Rad21 stably expressed cell lines compared with CMEC-08-D cells after UV-induced apoptosis. These data suggest that the Rad21 may maintain cell viability during mammary gland involution. Together these data, it concluded that the immortalized CMEC-08-D cells provided a valuable model for studying the biological characteristics of recombinant protein which will be expressed in mammary gland. Definition of the specific glycosylation site and the role of N-linked glycosylation on lysostaphin may provide the information to improve the efficiency of the antibacterial activity of lysostaphin in future transgenic approach. In addition, the immortalized CMEC-08-D also serving as a platform to understand Rad21 may play multifunction during mammary gland involution.