Study Of Structure And Function Of AnnexinⅤ As Probe Of Apoptosis Detection

Human annexin Ⅴ can combine negatively charged phospholipids on the membrance through calcium ion. When apoptosis occurs, phosphatidyl serine (phosphatidylserine, PS) flips from the inner side of membrance to the lateral. Fluorescence or radioactivity marked Annexin Ⅴ specifically combines PS, which can be used to detect apoptosis together with chromatin fluorescent dye propidium iodide (PI).When apoptosis occurs, cell membrane keeps integrity and PI can not enter cell, but labeled Annexin V has membrane-bound characteristics, which is different from the double positive results during cell necrosis. In addition, radioactive Annexin V can be used for diagnosis and treatment assessment of atherosclerosis, cancer and other diseases in vivo.Annexin Ⅴ has the molecular weight of36.5kDa, contains320amino acid residues in one single-chain. Annexin Ⅴ is composited by the N-terminal leader sequence and the C-terminal core area, which contains four repeat sequences and each repeat contains about70amino acid residues, consisting of five alpha helix and four loops.The AB and DE loops play important roles in combination of Ca2+. Four repeats forma disc-shaped structure, where calcium binding sites is located in the convex surface and two terminals are located in the concave side. Convex side bounds negatively charged phospholipids in the presence of calcium ion. The wild-type Annexin Ⅴ is commonly used for early detection of apoptosis, this study was designed to investigate the structure and function relationship of Annexin Ⅴ with the aim to develop a better apoptotic detector.In the current research, four mutants were constructed based on bio informatics analysis:variant Ⅰ-remove the N-terminal10amino acids; variantⅡ-remove the entire N-terminal leader sequence; variantⅢ-remove the third repeat with minimal homology; variantⅣ-remove the last repeat. Four target proteins were expressed by induction,isolated and purified. Flow cytometric analysis showed that variant Ⅰ remains similar to wild-type Annexin Ⅴ, variants Ⅱ and Ⅲ did not show apoptotic detection peak during double labeling with Annexin Ⅴ and PI, but displayed specific apoptotic peak when using Annexin Ⅴ alone. Mean while variant Ⅳ gave specific but declined detections compared to wild-type. All four variants could detected apoptotic cells observed under the fluorescent microscope.