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Characterization of Annexin V Fusion with the Superfolder GFP in Liposomes Binding and Apoptosis Detection.

ABSTRACT: Programed cell death is a critical and unavoidable part of life. One of the most widely used markers for dying cells, by apoptosis or pyroptosis, is the redistribution of phosphatidylserine (PS) from the inner to the outer plasma membrane leaflet. Annexin V protein is a sensitive and specific probe to mark this event because of its high affinity to the exposed PS. Beyond that, annexin V can bind to any PS-containing phospholipid bilayer of almost all tiny forms of membranous vesicles like blood platelets, exosomes, or even nanostructured liposomes. In this work, recombinant human annexin V was produced as a fusion with a highly fluorescent superfolder derivative of the green fluorescent protein (sfGFP) in Escherichia coli. The fusion protein(sfGFP-ANXV, 64 kDa), annexin V (ANXV, 40 kDa), and sfGFP (27 kDa) were separately produced after cloning their encoding genes in pRSET plasmid, and all proteins were expressed in a soluble form, then purified in high yields because of their N-terminal 6× His tag (~150 mg of pure protein per 1 L culture). Superiority of this fluorescent fusion protein over fluorescein-conjugated annexin V was demonstrated in binding to phospholipids (and their liposomes), prepared from natural sources (soya bean and egg yolk) that have different content of PS, by using different methods including ELISA, dot-blotting, surface plasmon resonance, and flow cytometry. We also applied fluorescent annexin V in the detection of apoptotic cells by flow cytometry and fluorescent microscopy. Interestingly, sfGFP-ANXV fusion was more sensitive to early apoptotic stressed HeLa cells than fluorescein-conjugated-ANXV. This highly expressed and functional sfGFP-ANXV fusion protein provides a promising ready-to-use molecular tool for quantifying liposomes (or similarly exosomes) and detecting apoptosis in cells.

SUBMITTER: Abbady AQ

PROVIDER: S-EPMC5437369 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Characterization of Annexin V Fusion with the Superfolder GFP in Liposomes Binding and Apoptosis Detection.

Abbady Abdul Qader AQ Twair Aya A Ali Bouthaina B Murad Hossam H

Frontiers in physiology 20170519

Programed cell death is a critical and unavoidable part of life. One of the most widely used markers for dying cells, by apoptosis or pyroptosis, is the redistribution of phosphatidylserine (PS) from the inner to the outer plasma membrane leaflet. Annexin V protein is a sensitive and specific probe to mark this event because of its high affinity to the exposed PS. Beyond that, annexin V can bind to any PS-containing phospholipid bilayer of almost all tiny forms of membranous vesicles like blood ...[more]

PMID: 28579961

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Project description:UnlabelledImaging of apoptosis can allow noninvasive assessment of disease states and response to therapeutic intervention for a variety of diseases. The purpose of this study was to develop and evaluate a multimodal nanoplatform for the detection of apoptosis.MethodsTo modulate the pharmacokinetics of annexin A5, a 36-kDa protein that binds specifically with phosphatidylserine, annexin A5 was conjugated to polyethylene glycol-coated, core-cross-linked polymeric micelles (CCPMs) dually labeled with near-infrared fluorescence fluorophores and a radioisotope ((111)In). To evaluate the specificity of the binding of annexin A5-CCPM to apoptotic cells, both fluorescence microscopy and cell-binding studies were performed in vitro. Pharmacokinetics, biodistribution, dual nuclear and optical imaging, and immunohistochemical studies were performed in 2 xenografted tumor models to evaluate the potential applications of annexin A5-CCPM.ResultsIn cell-based studies, annexin A5-CCPM exhibited strongly specific binding to apoptotic tumor cells. This binding could be efficiently blocked by annexin A5. In mice, annexin A5-CCPM displayed a mean elimination half-life of 12.5 h. The mean initial concentration in blood was 22.4% of the injected dose/mL, and annexin A5-CCPM was mainly distributed in the central blood compartment. In mice bearing EL4 lymphoma treated with cyclophosphamide and etoposide and in mice bearing MDA-MB-468 breast tumors treated with poly(L-glutamic acid)-paclitaxel and cetuximab (IMC-C225) anti-epidermal growth factor receptor antibody, the tumor apoptosis was clearly visualized by both SPECT and fluorescence molecular tomography. In contrast, there was little accumulation of this nanoradiotracer in the tumors of untreated mice. The biodistribution data were consistent with the imaging data, with tumor-to-muscle and tumor-to-blood ratios of 38.8 and 4.1, respectively, in treated mice, and 14.8 and 2.2, respectively, in untreated mice bearing EL4 lymphoma. Moreover, further studies demonstrated that the conventional (99m)Tc-labeled hydrazinonicotinamide annexin A5 and the plain CCPM control exhibited significantly lower uptake in the tumors of the treated mice than annexin A5-CCPM. Immunohistochemistry staining study showed that radioactivity count correlated with fluorescence signal from the nanoparticles, and both signals colocalized with the region of tumor apoptosis.ConclusionAnnexin A5-CCPM allowed visualization of tumor apoptosis by both nuclear and optical techniques. The complementary information acquired with multiple imaging techniques should be advantageous in assessing and validating early response to therapy.

Dataset Information

Characterization of Annexin V Fusion with the Superfolder GFP in Liposomes Binding and Apoptosis Detection.

Publications

Characterization of Annexin V Fusion with the Superfolder GFP in Liposomes Binding and Apoptosis Detection.

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