RGD-containing Ankyrin May Be Externalized To The Cell Surface And Trigger α_Vβ₃ Integrin-Mediated Erythrophagocytosis

Cell adhesion is fundamental to the function of multi-cellular organisms. Proteins that contain the Arg-Gly-Asp (RGD) attachment site, together with the integrins that serve as receptors for them, constitute a major recognition system for cell adhesion. A large number of extracellular matrix proteins contain the RGD motif as the cell attachment site in cell-matrix interaction. There are also proteins on cell surface that contain the RGD motif recognized by integrins in cell-cell interaction.The life span of erythrocytes amounts to some 100-120 days for humans and 40-50 days for mice. That is, more than 2 x 1011 human erythrocytes need to be replenished each day. Thus, the mechanisms by which macrophages sequester aged or diseased erythrocytes must be very robust. However, it is not known whetherα_vβ₃ integrin expressing macrophages may also utilize the RGD-integrin recognition system in the process of erythrophagocytosis to clear aged or damaged erythrocytes from circulation.There are about a dozen major membrane skeletal proteins that interact to form basic repeating units. These units in turn interconnect to construct a thin layer of"spoked"hexagonal network underneath the lipid bilayer, providing the elasticity and durability for erythrocytes. Among them,αspectrin, ankyrin and protein 4.2 are RGD-containing proteins in humans. The heterodimer ofα(²40 kDa) andβspectrin (²20 kDa) is the largest and longest molecule in erythrocytes that forms the lattice of the network. Protein 4.2 (⁷2 kDa) is a pseudo-transglutaminase positioned in the suspension complex (SC), which functions to"suspend"the network to the lipid bilayer. Other SC associated proteins include transmembrane protein band 3 (⁹5 kDa), which functions as an anion exchanger (mainly between Cl- and H2CO3-), and peripheral protein ankyrin (²05 kDa) which belongs to the cyclin family. Each of the three ankyrins (i.e., erythrocyte, brain and kidney ankyrins) in humans is encoded by a distinct gene, each contains an RGD motif.Integrin-mediated adhesion regulates cell migration, growth, differentiation, and apoptosis, and we noticed that several erythrocyte membrane skeletal proteins bear a RGD motif. Therefore, we wish to investigate whether any of these interior proteins may be recognized by integrins, externalized, and participate in the adhesion of erythrocytes to macrophages, and consequently leading to erythrophagocytosis.Here we report that the RGD motif of ankyrin, but not others, is recognized by the α_vβ₃ integrin receptor. In addition, the RGD motif of ankyrin, a peripheral membrane protein, can be externalized onto the cell surface when erythrocytes are incubated with calcium and sheared both at physiological levels. Furthermore, the erythrocyte-macrophage adhesion can be specifically inhibited by ankyrin and/orα_vβ₃. Thus, externalization of ankyrin followed by RGD/integrin recognition may be a novel mechanism by which erythrocytes adhere to macrophages preceding phagocytosis.We also have hypothesized that increased Ca2+ activated calpain, which cleavage in the regulatory domain of ankyrin, and the calpain fragment that may externalized on the cell. To clarify this issue, the normoblastosis mouse (nb/nb) erythrocytes were used to investigate the interaction of ankyrin andα_vβ₃ integrin. Protein 4.2-deficient erythrocytes and band 3-deficient erythrocytes were used as the comparison.In this report, we indeed found that ankyrin externalization is more significant in nb/nb erythrocyte comparing with +/+ erythrocyte at the same condition, shows that ankyrin without regulatory domain is easier to externalize. However, it's hard to capture the ankyrin externalized signal in band 3 (-/-) erythrocytes, which means the existence of band 3 is essential for ankyrin externalization.The proposed steps of ankyrin externalization described below are based on published literatures and our results: (1) shearing may increase Ca2+ influx across the erythrocyte membrane and extend the membrane skeletal network; (2) the submembrane may accumulate above the threshold (10μM) and activate calpain (a cysteine protease) and other enzymes; (3) calpain cleavage in the regulatory domain may loosen ankyrin's bindings with cdb3 and/or detach ankyrin from spectrin; and (4) elastic 24 ankyrin repeats may eject part of the ankyrin's spectrin-binding domain through the lipid bilayer (5 nm) onto the cell surface, exposing RGD.