# Sensing of cardiolipin exposure on plasma membranes of apoptotic cells by EryA‐mCherry protein

## TL;DR

A protein from mushrooms can detect cardiolipin on cell membranes during early apoptosis, potentially serving as a new marker for this process.

## Contribution

EryA-mCherry is shown to specifically bind cardiolipin in artificial and mammalian membranes, offering a novel tool for apoptosis detection.

## Key findings

- EryA-mCherry binds specifically to cardiolipin in artificial lipid vesicles.
- EryA-mCherry selectively labels apoptotic cells by detecting surface-exposed cardiolipin.
- EryA-mCherry binding is less effective than annexin V-FITC but more specific to cardiolipin.

## Abstract

Erylysin A (EryA), an aegerolysin protein produced by the edible king oyster mushroom (Pleurotus eryngii), interacts strongly with an invertebrate‐specific membrane sphingolipid ceramide phosphoethanolamine. Recently, a fluorescently fused variant of EryA was shown to bind to artificial and bacterial lipid membranes containing cardiolipin (CL). This tetra‐acylated glycerophospholipid, present in bacteria and in inner mitochondrial membranes of eukaryotic cells, was shown to be externalized to the plasma membrane surface during the process of apoptosis. In this work, we evaluated the interaction of EryA‐mCherry with CL‐containing artificial lipid vesicles and with mammalian cells undergoing apoptosis and compared its binding affinity and specificity to that of the well‐established apoptosis marker, annexin V‐FITC. Our results show that, in contrast to annexin V‐FITC, which binds several negatively charged glycerophospholipids, EryA‐mCherry specifically recognizes and binds CL in artificial membrane systems. However, this binding of EryA‐mCherry to CL‐supplemented membranes is less effective (K
D = 4.7 ± 1.6 μm) than that of annexin V‐FITC, whose binding is observed at nanomolar concentrations. Experiments using mammalian cells showed the ability of EryA‐mCherry to selectively label the membranes of apoptotic cells, binding to the same membrane regions as anti‐CL antibodies and annexin V‐FITC. Our data suggest that EryA‐mCherry might be used as a marker of early apoptosis, as well as a marker of CL in biological and artificial lipid membranes.

An aegerolysin protein erylysin A (EryA) fused with mCherry binds to artificial lipid vesicles supplemented with cardiolipin (CL). This binding is much more specific than that of annexin V, which binds various negatively charged glycerophospholipids (PG, PS, PI). In mammalian cells, the CL exposition at the membrane surface represents an early hallmark of apoptosis. This surface‐exposed CL can be effectively sensed by EryA‐mCherry, proposing it as a marker of early apoptosis.