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XIUPING CHEN, Feiye Guo, Ying Ding, Akhalesh Kumar Shakya, Wei Li; Characterization of Mesdc2 as a novel RPE phagocytosis ligand. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2399.
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© ARVO (1962-2015); The Authors (2016-present)
Mesoderm development candidate 2 (Mesdc2) is an endoplasmic reticulum (ER) chaperone for the low-density lipoprotein receptor (LDLR) family. Mesdc2 was reported to bind LDLR-related protein 6 (LRP6) on cell surface. LRPs are known for their role in endocytosis, and LRP1 is a well-characterized phagocytic receptor. The purpose of this study is to investigate if Mesdc2 is capable of binding to retinal pigment epithelial (RPE) cell and promoting its phagocytosis.
Recombinant Mesdc2 was expressed as the fusion protein of maltose-binding protein in bacteria and affinity purified using amylose columns. POS vesicles were prepared from fresh bovine retinas, labeled with pHrodo and used for phagocytosis assay in D407 RPE cells, primary RPE cells and RPE cups in the presence or absence of purified Mesdc2. Moreover, plasmid membrane vesicles were prepared from HEK293 cells with or without expression of recombinant Mesdc2 and labeled with fluorescence for phagocytosis assay. Phagocytosed POSs and plasma membrane vesicles were analyzed and quantified by confocal microscopy. Immunocytochemistry was performed with anti-Rab7 antibody. Mesdc2-FLAG binding to shed POSs and RPE cells was analyzed by flow cytometry and confocal microscopy.
Phagocytosis assay showed that Mesdc2 stimulated RPE phagocytosis of pHrodo-labeled POSs. Plasma membrane vesicles prepared from Mesdc2-expressing cells were preferentially phagocytosed over control membrane vesicles. Quantitative analysis of phagocytosed fluorescence signals indicated that Mesdc2 significantly promoted RPE phagocytosis. Immunocytochemistry revealed that phagocytosed fluorescent cargos were co-localized with phagosome marker Rab7, suggesting that Mesdc2 stimulated internalization of POSs through phagocytosis pathway. Flow cytometry and confocal analysis showed that Mesdc2 preferentially bound to POS vesicles and apoptotic cells over healthy cells, suggesting that Mesdc2 selectively recognizes phagocytosis cargos. Moreover, Mesdc2 was verified for its binding to RPE cells.
These results suggest that Mesdc2 is a genuine phagocytosis ligand that functions as a bridging molecule by simultaneously binding to phagocytosis cargos and RPE and link them for phagocytosis. Similar to heat shock proteins (Hsps), Mesdc2 as a molecular chaperone is capable of stimulating RPE phagocytosis.
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