A New Protein Complex Identified on the Basolateral Surface of Human Retinal Pigment Epithelium Involving a Complement Regulatory Protein (CD46) and Integrin

Abstract

Abstract: : Purpose: Evidence is growing that the complement system may play a role in age-related macular degeneration (AMD). Complement proteins have been localized in drusen and it has been suggested that their formation involves complement activation and retinal pigment epithelial (RPE) pathology. Some complement proteins are present in the retina and RPE, but very little is known about their role. We have identified a new protein complex in RPE that includes a complement regulatory protein (CD46) and ß1 integrin and that may function in RPE adhesion. This study is to further characterize this complex and to identify other protein partners associated with CD46 and integrin. Methods: Freshly isolated and primary or first-passaged human RPE from donor eyes or ARPE-19 human cell lines have been used for immunoprecipitation or confocal immunohistochemical localization. For immunoprecipitation, cultures are lysed and the immune complex precipitated with the specific antibodies (to CD46 or ß1 integrin) and sepharose protein G and the gel-blots probed for co-precipitated proteins. For immunohistochemistry, cultures are aldehyde fixed and stained with specific fluorescent-labelde antibodies and examined in a laser scanning confocal microscope. Results: Both CD46 and ß1 integrin can be co-immunoprecipitated when either protein is used as the precipitating antibody. Confocal microscopy confirms that both CD46 and ß1 integrin have the same co-localization along the basolateral RPE surface. When anti-CD46 is used to co-precipitate other associated proteins, ezrin, an actin-binding protein and SAP97 (synapse-associated protein), co-immunoprecipitate with CD46. In addition, when anti-ß1 integrin is used as the precipitating protein, SAP97 is co-immunoprecipitated with integrin. When anti-SAP97 is used as the precipitating antibody, ß1 integrin is co-precipitated with SAP97. Confocal microscopy confirms that SAP97 is also co-localized at the basolateral RPE surface. Conclusions: The co-immunoprecipitation and basolateral localization of CD46 with ß1 integrin and also with ezrin and SAP97 suggests that this is a protein complex that may share a functional relationship with the integrin adhesion pathway. The co-immunoprecipitation of SAP97 with ß1 integrin may also suggest a functional relationship. SAP97 belongs to a family of proteins that cluster protein receptors at the cell surface and may serve in that capacity at the basolateral RPE membrane to tether CD46 and its partners with ß1 integrin.

Keywords: retinal pigment epithelium • protein structure/function • immunohistochemistry