Abstract
Purpose :
Age-related macular degeneration (AMD) is a devastating disease characterized by the ectopic accumulation of calcified protein-lipid deposits. Understanding the molecular basis for deposit formation is key for developing diagnostic, preventive, and therapeutic approaches. Using biophysical methods with purified proteins and lipids, we characterized the role of vitronectin (Vn) in mediating hydroxyapatite (HAP) mineralization on lipid surfaces. Vn is a calcium-binding protein and a major component of the AMD proteome with a potential role in HAP deposition; Vn is known to interact with the complement system and variants have been identified as AMD risk factors.
Methods :
Using mammalian and E. coli expression systems, we purified full-length Vn and a shorter construct encompassing its hemopexin domain (Vn-HX). The purified proteins were labeled with fluorescent tags for microscopy and characterized structurally by NMR and crystallography. Their interactions with lipids and HAP were determined using thermal shift assays. To examine the effects of Vn on deposit formation, HAP mineralization was initiated from chemically-defined solutions of calcium and phosphate ions, and monitored in the presence of lipids by fluorescence techniques with HAP-specific dyes. A two-tailed Student’s t-test was used for statistical analysis.
Results :
Vn and Vn-HX bind HAP, with HAP association significantly increasing their melting temperatures by ~6 °C (Vn p<0.01; Vn-HX p<0.01). Both promoted HAP mineralization on lipid vesicles leading to the formation of concentric lipid-HAP-protein micro-spheres, ranging in size from 3-10 µm and similar to those observed in AMD pathology. Surface coating of Vn and Vn-HX could be visualized with fluorescently labeled proteins. Addition of Vn (p<0.01) and Vn-HX (p<0.01) significantly increased HAP mineralization measured by fluorescence with the HAP-specific stains Xylenol Orange, OsteoSense and Alizarin Red S.
Conclusions :
Our results demonstrate that Vn can strongly interact with HAP using the calcium-binding site in its hemopexin domain. The Vn-dependent, HAP-specific fluorescence enhancement suggests that Vn plays a role in regulating HAP deposition on lipid surfaces. Our in vitro assays identify Vn as a promising target for AMD diagnosis and treatment, and future work will focus on translation to cellular and in vivo models.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.