The Role of Annexin A5 in Phagocytosis of Photoreceptor Outer Segment Fragments by the Retinal Pigment Epithelium
Diurnal phagocytosis of spent photoreceptor outer segment fragments (POS) by the retinal pigment epithelium (RPE) is critical for vision. Recognition of POS by apical integrin αvβ5 receptors of the RPE triggers signaling that synchronizes POS engulfment in the RPE. Annexin A5 (ANXA5) is a Ca 2+ dependent phospholipid binding protein with the binding motif for integrin β5. However, the physiological function of such interaction remains unknown in the eye. The purpose of this study is to test if and how ANXA5 contributes to RPE phagocytosis. Here, I found that ANXA5 was expressed in both neural retina and eyecups, and was primarily localized in phagocytic surface of the RPE. Transient silencing and overexpressing experiments revealed that ANXA5 was required for the binding stage of phagocytosis but did not directly contribute to internalization or was not critical for digestion. The rescue experiments in ANXA5 KO mouse embryonic fibroblasts (MEFs) also supported this conserved role in phagocytic clearance. By contrast, either silencing or overexpressing ANXA5 in integrin β5 KO MEF or primary RPE cells, or overexpression of integrin β5 in ANXA5 KO MEF had no effect on particle binding, suggesting a functional interdependence of two proteins. Coimmunoprecipitation and immunostaining further revealed the interaction between them in eyecups and primary RPE cells. Moreover, silencing ANXA5 dramatically reduced the surface dimers of integrin αvβ5 in primary RPE cells. C-terminal truncated ANXA5 deleting the integrin β5 binding motif failed to increase the surface level of integrin αvβ5 and POS binding, but this truncation alone was insufficient to abolish the interaction with integrin αvβ5 completely. In addition, I explored RPE phagocytic activity and overall retinal function in ANXA5 KO mice. I found that ANXA5 KO RPE in situ lacked the peak of phagosome load that synchronizes with circadian photoreceptor shedding in WT mice but retained more phagosomes at the latest time although I did not detect distinct morphological alteration. Collectively, these results demonstrate a novel and essential role of intracellular ANXA5 in RPE phagocytosis.
Yu, Chen, "The Role of Annexin A5 in Phagocytosis of Photoreceptor Outer Segment Fragments by the Retinal Pigment Epithelium" (2017). ETD Collection for Fordham University. AAI10607598.