Engineered annexin A5 variants have impaired cell entry for molecular imaging of apoptosis using pretargeting strategies

Abstract

Phosphatidylserine (PS) on apoptotic cells is a target for diagnosis and therapy using annexin A5 (anxA5). Pretargeting is a strategy developed to improve signal to background ratio for molecular imaging and to minimize undesired side effects of pharmacological and radiotherapy. Pretargeting relies on accessibility of the target finder on the surface of the target cell. anxA5 binds PS and crystallizes in a two-dimensional network covering the PS-expressing cell surface. Two-dimensional crystallization is the driving force for anxA5 internalization by PS-expressing cells. Here, we report structure/function analysis of anxA5 internalization. Guided by structural bioinformatics including protein-protein docking, we revealed that the amino acids Arg(63), Lys(70), Lys(101), Glu(138), Asp(139), and Asn(160) engage in intermolecular salt bridges within the anxA5 trimer, which is the basic building block of the two-dimensional network. Disruption of the salt bridges by site-directed mutagenesis does not affect PS binding but inhibits trimer formation and cell entry of surface-bound anxA5. The anxA5 variants with impaired internalization are superior molecular imaging agents in pretargeting strategies as compared with wild-type anxA5.

FIGURE 1.

Three-dimensional presentation of the trimer composed of three monomers of anxA5 as elucidated previously (14). The six amino acids, which are candidates for stabilizing the trimer by forming intermolecular salt bridges, are highlighted. On the right panel, the trimer (above) is projected on a phospholipid surface showing its bended conformation (below).

FIGURE 2.

Ca²⁺-dependent binding of anxA5 to a bilayer of PS/PC. A, binding isotherm measured with ellipsometry. Time points of anxA5 (final concentration, 1 μg·ml⁻¹) and EDTA (final concentration, 5 mm) addition are indicated by arrows (21). B, table summarizing the key characteristics of binding of anxA5 variants (final concentration, 10 μg·ml⁻¹) to a bilayer of PS/PC as determined with ellipsometry. C, dot plot of a flow cytometric measurement of binding of anxA5-F (final concentration, 1 μg·ml⁻¹) to apoptotic Jurkat cells (10⁶ cells·ml⁻¹). R1 events comprised the Jurkat cells with viable forward and side scatter that bind anxA5-F. These are the early apoptotic cells. D, histograms of R1 gated events of flow cytometric measurements of apoptotic Jurkat cells and anxA5 variants. E, dose response of anxA5-F binding to apoptotic Jurkat cells (10⁶ cells·ml⁻¹). Mean fluorescence intensity (MFI) was determined with statistics of the R1 events. F, Ca²⁺-dependence of anxA5-F binding (final concentration, 6 μg·ml⁻¹) to apoptotic Jurkat cells (10⁶ cells·ml⁻¹). Mean fluorescence intensity was determined with statistics of R1-gated events. J-anx, J-anxA5.

FIGURE 3.

A, spectra of MALDI TOF/TOF analyses of 2D1–6, 2D1–6-fluorescein, and 2D1–6-Alexa Fluor 568. B, representative excitation scans with emission at 600 nm of PS/PC LUVs and anxA5-F and anxA5-A in the presence of EDTA (black line) and Ca²⁺ (gray line). Subtraction of the 492 nm values of the two spectra delivers the FRET signal. C, FRET signals generated by mixtures of anxA5 variants and 20 PS/80 PC LUVs in the presence of 1 and 3 mm Ca²⁺ (n = 9). D, FRET signals generated by mixtures of anxA5 variants and apoptotic Jurkat cells in the presence of 1 and 3 mm Ca²⁺ (n = 9). FRET signals generated on Jurkat cells were determined by flow cytometry as described under “Experimental Procedures.” E, 1024 × 1024 pixels digital image of a negatively stained J-anxA5 two-dimensional crystal. The resolution of the image is 0.39 nm/pixel; the image size is 3994.5 × 3994.5 nm. The inset shows a Fourier transform of the image. The first Thon (dark) ring (contrast transfer function minimum) is centered at 2.0 nm, which limits the resolution for the reconstruction to 2.0 nm without applying contrast transfer function (CTF) correction to the original digital image. The (0, 1) and (1, 0) diffraction peaks on the a* and b* axis, respectively, have been manually indexed, and the lattice refinement was carried out with the CRISP two-dimensional crystallography software. F, projection density map of J-anxA5 (J-anx) two-dimensional crystals onto a dioleoyl-PS:dioleoyl-PC monolayer limited at a 2.0 nm resolution. The protein density is in white. P3 symmetry has been imposed. The unit cell dimensions are a = b = 11 nm, and the angle − γ = 120°. The phase residual is 11.6°.

FIGURE 4.

Confocal laser scanning microscopy of apoptotic Jurkat cells that were incubated with anxA5 variant-F (green) during the execution of apoptosis and with anxA5-A (red) at the end of the time period of apoptosis execution. A1, J-anxA5 (J-anx); A2, 2D1–5; A3, 2D2–6; A4, 2D1–6; A5, anxA1; A6, lactadherin. B, the amount of anxA5-fluorescein internalized by Jurkat cells during the execution of apoptosis (n = 9). AU, arbitrary units. C, pretargeting capacity of anxA5 variants was determined using the biotin-streptavidin couple. Pretargeting capacity is determined for various periods of time of internalization (n = 4).