Donald F. Gibson

Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma

MicroRNAs (miRNAs) circulate in the bloodstream in a highly stable, extracellular form and are being developed as blood-based biomarkers for cancer and other diseases. However, the mechanism underlying their remarkable stability in the RNase-rich environment of blood is not well understood. The current model in the literature posits that circulating miRNAs are protected by encapsulation in membrane-bound vesicles such as exosomes, but this has not been systematically studied. We used differential centrifugation and size-exclusion chromatography as orthogonal approaches to characterize circulating miRNA complexes in human plasma and serum. We found, surprisingly, that the majority of circulating miRNAs cofractionated with protein complexes rather than with vesicles. miRNAs were also sensitive to protease treatment of plasma, indicating that protein complexes protect circulating miRNAs from plasma RNases. Further characterization revealed that Argonaute2 (Ago2), the key effector protein of miRNA-mediated silencing, was present in human plasma and eluted with plasma miRNAs in size-exclusion chromatography. Furthermore, immunoprecipitation of Ago2 from plasma readily recovered non–vesicle-associated plasma miRNAs. The majority of miRNAs studied copurified with the Ago2 ribonucleoprotein complex, but a minority of specific miRNAs associated predominantly with vesicles. Our results reveal two populations of circulating miRNAs and suggest that circulating Ago2 complexes are a mechanism responsible for the stability of plasma miRNAs. Our study has important implications for the development of biomarker approaches based on capture and analysis of circulating miRNAs. In addition, identification of extracellular Ago2–miRNA complexes in plasma raises the possibility that cells release a functional miRNA-induced silencing complex into the circulation.

Phospholipid binding of annexin V: Effects of calcium and membrane phosphatidylserine content☆

We studied the binding of fluorescein-labeled annexin V (placental anticoagulant protein I) to small unilamellar phospholipid vesicles at 0.15 M ionic strength as a function of calcium concentration and membrane phosphatidylserine (PS) content. As the mole percentage of PS in the membrane increased from 10 to 50%, the stoichiometry of binding decreased hyperbolically from 1100 mol phospholipid/mol annexin V to a limiting value of 84 mol/mol for measurements made at 1.2 mM CaCl2. Over the same range of PS content, Kd remained approximately constant at 0.036 +/- 0.011 nM. A similar hyperbolic decrease in stoichiometry was observed with vesicles containing 10 or 20% PS when the calcium concentration was increased from 0.4 to 10 mM. Thus, the density of membrane binding sites is strongly dependent on the membrane PS content and calcium concentration. The effect of calcium on annexin V-membrane binding is proposed to be due to the formation of phospholipid-calcium complexes, to which the protein binds, rather than to an allosteric effect of calcium on protein-phospholipid affinity.

Entropic and Enthalpic Contributions to Annexin V-Membrane Binding A COMPREHENSIVE QUANTITATIVE MODEL

Annexin V binds to membranes with very high affinity, but the factors responsible remain to be quantitatively elucidated. Analysis by isothermal microcalorimetry and calcium titration under conditions of low membrane occupancy showed that there was a strongly positive entropy change upon binding. For vesicles containing 25% phosphatidylserine at 0.15 m ionic strength, the free energy of binding was –53 kcal/mol protein, whereas the enthalpy of binding was –38 kcal/mol. Addition of 4 m urea decreased the free energy of binding by about 30% without denaturing the protein, suggesting that hydrophobic forces make a significant contribution to binding affinity. This was confirmed by mutagenesis studies that showed that binding affinity was modulated by the hydrophobicity of surface residues that are likely to enter the interfacial region upon protein-membrane binding. The change in free energy was quantitatively consistent with predictions from the Wimley-White scale of interfacial hydrophobicity. In contrast, binding affinity was not increased by making the protein surface more positively charged, nor decreased by making it more negatively charged, ruling out general ionic interactions as major contributors to binding affinity. The affinity of annexin V was the same regardless of the head group present on the anionic phospholipids tested (phosphatidylserine, phosphatidylglycerol, phosphatidylmethanol, and cardiolipin), ruling out specific interactions between the protein and non-phosphate moieties of the head group as a significant contributor to binding affinity. Analysis by fluorescence resonance energy transfer showed that multimers did not form on phosphatidylserine membranes at low occupancy, indicating that annexin-annexin interactions did not contribute to binding affinity. In summary, binding of annexin V to membranes is driven by both enthalpic and entropic forces. Dehydration of hydrophobic regions of the protein surface as they enter the interfacial region makes an important contribution to overall binding affinity, supplementing the role of protein-calcium-phosphate chelates.

Transmembrane voltage regulates binding of annexin V and lactadherin to cells with exposed phosphatidylserine

BackgroundCells expose phosphatidylserine during apoptosis. The voltage across the plasma membrane also decreases or disappears during apoptosis, but the physiological significance of this is unknown.ResultsHere we show that transmembrane potential regulates membrane binding of two unrelated proteins that recognize exposed phosphatidylserine on apoptotic cells. In Jurkat T leukemia cells and K562 promyelocytic leukemia cells undergoing apoptosis, extracellular binding of annexin V was increased by decreasing membrane potential in a dose-dependent manner. Studies with phospholipid vesicles showed that the effect was mediated via an increase in binding affinity. The effect was independent of the apoptotic stimulus. The same phenomenon occurred with lactadherin, a structurally unrelated protein that also binds to apoptotic cells via phosphatidylserine and is essential for in vivo clearance of dying cells.ConclusionAlterations in membrane potential regulate the binding of annexin V and lactadherin to cell membranes, and may also influence the membrane binding of other classes of phosphatidylserine-binding proteins.

Characterization of a Recombinant Form of Annexin VI for Detection of Apoptosis

We developed a recombinant form of human annexin VI called annexin VI-601 (M(r) 76,224) with the N-terminal extension of Ala-Gly-Gly-Cys-Gly-His to allow ready attachment of fluorescent or radioactive labels. The protein was produced by expression in E. coli and was purified by calcium-dependent membrane binding, anion-exchange chromatography, and heparin-Sepharose affinity chromatography. The protein could be readily labeled with iodoacetamidofluorescein and with (99m)Tc. The protein bound with high affinity to PS-containing phospholipid vesicles and to erythrocytes with exposed phosphatidylserine. Fluorescent annexin VI-601 readily detected apoptosis of Jurkat cells by flow cytometry at much lower calcium concentrations than those required for equivalent detection by annexin V. In vivo administration of radiolabeled protein showed that blood clearance was much slower than annexin V. In conclusion, annexin VI may have advantages over annexin V in certain situations for both in vitro and in vivo detection of apoptosis and therapeutic targeting of PS due to its lower calcium requirement for membrane binding and its higher molecular weight.