Gold fluorescent annexin A5 as a novel apoptosis detection tool

Abstract

We describe a golden fluorescent apoptosis detection tool, which we generated by a fusion of golden fluorescent protein (GdFP) with human annexin A5 (anxA5). GdFP was obtained by replacement of tryptophan at position 66 with 4-aminotryptophan in the chromophore of enhanced cyan fluorescent protein. The GdFP-anxA5 construct combines highly desirable features originating from both fusion partners. These include (i) strong binding to membrane phosphatidylserine patches of apoptotic cells in the presence of Ca 21 which is brought about by anxA5, (ii) the stable and homogeneous monomeric state, (iii) as well as the red-shifted fluorescence maximum at 574 nm originating from GdFP. We found that GdFP-anxA5 is equally well applicable for apoptosis studies as a routinely used fluorescein 5 0 -isothiocyanate-annexin A5 conjugate. Golden fluorescent annexin A5 represents a new, stable, and homogeneous red-shifted optical probe for the efficient detection of apoptosis by fluorescence microscopy or by flow cytometry. ' International Society for Advancement of CytometryKey terms apoptosis; annexin; gold fluorescent protein; flow cytometry APOPTOSIS, or programmed cell death, is characterized by a series of biochemical events and cell morphological changes eventually leading to the demise of the cell (1,2). During apoptosis, the phospholipid asymmetry of the plasma membrane is lost which results in the redistribution of phosphatidylserine (PS) from its ordinary location in the inner leaflet of the plasma membrane to the outer leaflet (3). PS exposed on the surface of apoptotic cells can be detected by its affinity for annexin A5 (anxA5), a phospholipid binding protein (4). Annexin A5 interacts strongly and specifically with PS residues in the presence of calcium (5-7). Fluorescently labeled anxA5 derivatives have extensively been used to detect and analyze apoptotic cells by flow cytometry (FCM) or fluorescence microscopy (8-11).Commercially available fluorescent anxA5 conjugates are prepared by chemical coupling of, e.g., lysine side chains with amine-reactive fluorophores such as Fluorescein 5 0 -isothiocyanate (FITC)-, Oregon-Green-, or Alexa Fluor 488 succinimidyl esters. Though easy and reliable, chemical coupling always yields heterogeneous mixtures of labeled anxA5 molecules because the number and position of bound fluorophores cannot be controlled. Moreover, many freely accessible lysine residues of anxA5 reside in or near the PS-binding domain, i.e., the concave side of the molecule (see Fig. 1A). This can result in serious fluorescence quenching upon binding to the PS exposed on the cell surface (12,13). Ernst and coworkers first elucidated the shortcomings of chemical labeling by the recombinant fusion of anxA5 with green fluorescent protein (GFP-anxA5). Genetically encoded GFP-anxA5 was labeled homogeneously and did not change its fluorescence properties upon binding to membrane phospholipids (13). The ratio of PS-binding function to fluorophore is 1:1 in GFP-anxA5, which renders the quantitation of apopt...

Quantitative analysis of annexin V–membrane interaction by flow cytometry

“…Moreover, the amine-directed chemical modification of anxV will reduce its membrane-binding activity (Tait et al 2006). The final products are heterogeneous mixtures of differentially labeled proteins, while fusion proteins such as anxVenhanced green fluorescent protein (EGFP) are brighter, more homogeneous, and photostable (Ernst et al 1998;Kurschus et al 2009;Stocker et al 2008;Tait et al 2006). Nowadays, at least 18 companies can provide EGFPlabeled anxV for applications.…”

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

“…This coupling also supports the large Stokes shift and necessarily reduces the fluorescence quantum yield. In comparison to other red-shifted FPs, GdFP even exhibits improved protein stability and a low tendency for aggregation 33 61 62 . It not only differs in color from other FP variants but also exhibits a substantially increased thermostability and enhanced cooperative folding 33 .…”

Improved method to retain cytosolic reporter protein fluorescence while staining for nuclear proteins

“…Thereafter, antibody‐bound beads were incubated with either recombinant EGFP [rEGFP; prepared similarly as previously described (Ref. )] or with 100 µL of supernatants of buffer‐treated cells in a total volume of 200 µL for 2 h at 4°C.…”