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

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.