C1q, the key component of the classical pathway of the Complement system, is known for its vast functional activity including clearance of apoptotic cells. The binding of C1q to apoptotic blebs occurs via an interaction with the phosphatidylserine externalized on the cell surface. In this study, we characterized the interaction between C1q and phosphatidylserine, with emphasis on the structure of the phosphatidylserine-binding site within the globular domains of C1q and the nature of binding of C1q with phosphatidylserine, using both in vitro and in silico methods. We established that all three globular fragments, forming one C1q globular domain, bound phosphatidylserine with the leading role of the phosphatidylserine-binding site pertaining to the A chain of the globular fragment of C1q. We also determined the closest-contact amino acids of C1q participating in the interaction with phosphatidylserine. An important role is suggested for the glycosylated Asn124 residue in the A chain of the globular fragment.

Human retinal pigment epithelial (RPE) cells express the transmembrane Ca²⁺-dependent Cl⁻ channel bestrophin-1 (hBest1) of the plasma membrane. Mutations in the hBest1 protein are associated with the development of distinct pathological conditions known as bestrophinopathies. The interactions between hBest1 and plasma membrane lipids (cholesterol (Chol), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and sphingomyelin (SM)) determine its lateral organization and surface dynamics, i.e., their miscibility or phase separation. Using the surface pressure/mean molecular area (π/A) isotherms, hysteresis and compressibility moduli (C_s⁻¹) of hBest1/POPC/Chol and hBest1/SM/Chol composite Langmuir monolayers, we established that the films are in an LE (liquid-expanded) or LE-LC (liquid-condensed) state, the components are well-mixed and the Ca²⁺ ions have a condensing effect on the surface molecular organization. Cholesterol causes a decrease in the elasticity of both films and a decrease in the ΔG_mix^π values (reduction of phase separation) of hBest1/POPC/Chol films. For the hBest1/SM/Chol monolayers, the negative values of ΔG_mix^π are retained and equalized with the values of ΔG_mix^π in the hBest1/POPC/Chol films. Shifts in phase separation/miscibility by cholesterol can lead to changes in the structure and localization of hBest1 in the lipid rafts and its channel functions.

Bestrophinopathies are a group of inherited retinal diseases caused by mutations in the BEST1 gene. The protein encoded by this gene, bestorphin-1 (hBest1), is a calcium-dependent transmembrane channel localized on the basolateral membrane of retinal pigment epithelial (RPE) cells. We have already demonstrated the surface behavior and organization of recombinant hBest1 and its interactions with membrane lipids such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingomyelin (SM) and cholesterol (Chol) in models of biological membranes, which affect the hBest1 structure–function relationship. The main aim of our current investigation is to integrate pure hBest1 protein into lipid bilayer nanostructures. We synthesized and characterized various hBest1-containing nanostructures based on 1,2-Dipalmitoylphosphatidylcholine (DPPC), SM, glycerol monooleate (GMO) and Chol in different ratios and determined their cytotoxicity and incorporation into cell membranes and/or cells by immunofluorescence staining. Our results show that these newly designed nanoparticles are not cytotoxic and that their incorporation into MDCK II cell membranes (used as a model system) may provide a mechanism that could be applied to RPE cells expressing mutated hBest1 in order to restore their ion transport functions, affected by mutated and malfunctioning hBest1 molecules.