乳脂肪小球表皮生長因子8 (MFG-E8)變異型之基因選殖、蛋白表現及分析

Abstract

背景 (Background)：乳脂肪小球表皮生長因子8 (milk-fat globule EGF factor 8, MFG-E8)最早是發現於泌乳時乳脂肪小球中的分泌型醣蛋白。其蛋白主要表現於乳腺細胞及免疫調節器官中的細胞，如：淋巴結或脾臟中的著色體巨噬細胞 (tingible-body MΦ)。蛋白結構帶有表皮生長因子相似功能區 (EGF-like domain)與第五及第八凝血因子相似功能區(FV/FVIII type C-like domain)，可分別與吞噬細胞表面的integrin αvβ3/5及凋亡細胞面的磷脂絲胺酸 (phosphatidylserine, PS)結合。近期的研究指出，MFG-E8的功能主要是在吞噬細胞 (如：巨噬細胞macrophage及樹突細胞dendritic cell)清除凋亡細胞的過程中扮演架橋分子的角色。在MFG-E8基因剔除的小鼠模型中發現，吞噬細胞吞吃凋亡細胞的能力下降，凋亡細胞無法被有效的清除，並且在40週大的雌鼠體內觀察到肝脾腫大及腎絲球腎炎的現象，同時偵測到高量的自體抗體；這些症狀都與人類自體免疫疾病—全身性紅斑性狼瘡 (systemic lupus erythematosus, SLE)的特徵非常相似，顯示缺少MFG-E8造成的凋亡細胞清除缺失與SLE的致病原因可能有關聯性。我們先前研究比較SLE病患和健康對照組間MFG-E8基因單點核苷酸多型性 (single nucleotide polymorphism, SNP)的差異，結果發現MFG-E8第76位點變異型76Met與SLE的罹病有顯著正相關。該點及附近的核苷酸序列恰好符合真核細胞轉譯起始所需的序列consensus Kozac sequence (A/G-3NNATGG+4)，對此我們提出一個假設—可能在76Met位點出現替代性轉譯 (alternative translation)的現象，並產生短片段的MFG-E8蛋白。 方法 (Methods)：為了研究替代性轉譯是否有發生，本研究中 1. 選殖MFG-E8 cDNA並建立以大腸桿菌製備重組MFG-E8蛋白方法，供後續研究。2. 在轉染不同MFG-E8變異型的293T細胞及帶不同MFG-E8變異型的人類周邊血液單核球獲致的人類巨噬細胞中觀察MFG-E8-76Met變異型是否發生替代性轉譯。3. 偵測SLE病患血清中MFG-E8蛋白的表現量，了解MFG-E8第76位點基因型的變異與血清中MFG-E8蛋白量的變化是否有相關性。 結果 (Results)：1. 完成MFG-E8基因選殖並成功在大腸桿菌中誘導蛋白表現，但但白表現量低，即使調整誘導條件蛋白產量仍過低，無法進行後續蛋白純化。2. 轉染MFG-E8 (Δ1-75)短片段序列之293T細胞中確實可表現MFG-E8 Δ1-75短片段蛋白，但其表現效率遠低於全長MFG-E8序列。3. 在MFG-E8變異型76Met轉染細胞中未明顯觀察到短片段MFG-E8蛋白。4. MFG-E8變異型為76Met的人類巨噬細胞中未觀察到短片段MFG-E8蛋白，不過發現偵測到的MFG-E8蛋白較預期分子量小，且MFG-E8表現量在不同MFG-E8-76變異型之巨噬細胞中無規律性。5. SLE病患血清中MFG-E8的表現量分布顯著高於健康對照組，但SLE病患血清中MFG-E8升高與MFG-E8第76位點變異型之間沒有顯著的相關性。 結論 (Conclusions)：1. MFG-E8第76位點ATG密碼子並非有效的替代性轉譯起始位。2. 部份SLE病患血清中發現高於健康對照組之MFG-E8表現，但與MFG-E8第76位點變異型無顯著相關。3. 巨噬細胞中MFG-E8表現量與MFG-E8-76變異型無相關性，但偵得分子量低於預期，暗示可能由於巨噬細胞中特有的處理方式所造成。MFG-E8再吞噬細胞中的處理及表現情形有待進一步的探討。

Background: Milk-fat globule EGF factor 8 (MFG-E8) is a secreted glycoprotein found in milk-fat globule derived from lactating mammary. MFG-E8 proteins express mainly in the mammary cells and immuno-modulating cells such as tingible-body macrophages in spleen and lymph node. MFG-E8 contains EGF domains (Epidermal growth factor-like) and C1/C2 domains (Coagulation factor V and VIII type C-like) for integrin αvβ3/5 on (MΦ) binding and phosphatidylserine (PS)(on apoptotic cell) recognition, respectively. The importance of MFG-E8 function as a bridging molecule during apoptotic cell clearance has been emphasized in recent years. Decreased phagocytic clearance and accumulation of apoptotic cells was noted in MFG-E8-deficient mice. Intriguingly, the 40-week-old female MFG-E8-deficient mice present higher titer of autoantibodies and suffered splenomegaly/glomerulonephritis more prevalently than the male mice, which were characterized in human systemic lupus erythematosus (SLE). These results indicate the correlation between MFG-E8 deficiency-related apoptotic cell clearance defect and SLE pathogenesis. Our previous studies revealed that the variant on MFG-E8-76th residue (76Met) was a predisposing factor for human SLE. Interestingly, a concensus Kozac sequence (A/G-3NNATGG+4) resides around residue 76Met, which may potentially act as an alternative translation initiation site and subsequently produce short form MFG-E8-Δ1-75 in cells of MFG-E8-76Met variant type. Methods: Several approaches were applied to test the hypothesis whether there is alternative translation - 1. Molecular cloning of MFG-E8 cDNA and express recombinant MFG-E8 protein in E. coli. 2. Examine whether MFG-E8-76Met variant form perform alternative translation in different MFG-E8-76 variant forms-transfected 293T cell lines and PBMC-derived human macrophages with different MFG-E8-76 variant forms. 3. Detect serum MFG-E8 expression level in SLE cohort to realize the correlation between MFG-E8-76 variations and serum MFG-E8 level. Results: 1. MFG-E8 cDNA was successfully cloned and then expressed in E. coli. However, the protein expression levels are considered low even after optimizing the protein induction conditions. Further protein expression was hampered due to low expression level. 2. Short form MFG-E8 protein was produced in MFG-E8 (Δ1-75)-transfected 293T cell lines but the expression efficiency were considerably lower than that found in 293T cell transfected with full length MFG-E8. 3. No short form MFG-E8 protein was observed in MFG-E8-76Met-transfected 293T cell lines. 4. No short form MFG-E8 protein were observed in peripheral blood monocytic cells-derived macrophage of MFG-E8-76Met variant form. Interestingly, the MFG-E8 detected in human macrophage was of smaller size than the expected. The protein expression level differed independently of MFG-E8-76 variant forms. 5. Serum MFG-E8 expression levels in SLE cohort were significantly higher than that of non-lupus controls but the elevated MFG-E8 level did not show significant correlation with MFG-E8-76 variant forms. Conclusions: 1. MFG-E8-76th codon ATG does not show to be an appropriate alternative translational start site. 2. Serum MFG-E8 levels were higher in part of SLE patients than in healthy controls but the elevated MFG-E8 level did not correlation with the MFG-E8-76 variant forms. 3. MFG-E8 expression levels had no correlation with the MFG-E8-76 variant forms in human macrophage. The proteins determined in human macrophage were smaller than estimated size suggested that there have distinct processes in human macrophage. The differential processing and expression of MFG-E8 in the phagocytes await further investigation.