Plasma-derived exosome-like vesicles are enriched in lyso-phospholipids and pass the blood-brain barrier.

Exosomes are vesicles involved in intercellular communication. Their membrane structure and core content is largely dependent on the cell of origin. Exosomes have been investigated both for their biological roles and their possible use as disease biomarkers and drug carriers. These potential technological applications require the rigorous characterization of exosomal blood brain barrier permeability and a description of their lipid bilayer composition. To achieve these goals, we have established a 3D static blood brain barrier system based on existing systems for liposomes and a complementary LC-MS/MS and 31P nuclear magnetic resonance methodology for the analysis of purified human plasma-derived exosome-like vesicles. Results show that the isolated vesicles pass the blood brain barrier and are taken up in endothelial cells. The compositional analysis revealed that the isolated vesicles are enriched in lyso phospholipids and do not contain phosphatidylserine. These findings deviate significantly from the composition of exosomes originating from cell culture, and may reflect active removal by macrophages that respond to exposed phosphahtidylserine.

A strategy to characterize chlorophyll protein interaction in LIL3.

BACKGROUND: The function of proteins is at large determined by cofactors selectively bound to protein structure. Without chlorophyll specifically bound to protein, light harvesting and photosynthesis would not be possible. The binding of chlorophyll to light harvesting proteins has been extensively studied in reconstitution assays using proteins expressed in vitro; however, the mechanism of the reconstitution reaction remained unclear. We have shown that membrane integral light-harvesting-like protein, LIL3, binds chlorophyll a with a Kd of 146 nM in vitro by thermophoresis. Here, reconstitution of chlorophyll binding to LIL3 has been characterized by four different methods. RESULTS: Structural changes in the reconstitution process have been investigated by light-scattering and differential Trp-fluorescence. For characterization of the chlorophyll binding site at LIL3, the analysis of LIL3 mutants has been conducted using native PAGE and thermophoresis. We find that the oxidized state of dithiothreitol is the essential component for reconstitution of chlorophyll binding to LIL3 in n-Dodecyl ß-d-maltoside micelles at RT. Chlorophyll increased the polydispersity of the micellar states while dithiothreitol maintained LIL3 in a partially unfolded state at RT. Dimerization of LIL3 was abolished if amino acids N174, R176, and E171 were mutated to Ala; while, chlorophyll binding to LIL3 was abolished in mutant N174A, but retained in E171A, and R176A albeit at an about six- and five-fold decreased dissociation constant. Results show that N174 of LIL3 is essential for binding chlorophyll a. CONCLUSIONS: Chlorophyll binding to LIL3 can be shown by thermophoresis, and native gel electrophoresis, while analysis of reconstitution conditions by dynamic light scattering and differential scanning fluorometry are of critical importance for method optimization.

Characterization of chlorophyll binding to LIL3.

The light harvesting like protein 3 (LIL 3) from higher plants, has been linked to functions in chlorophyll and tocopherol biosynthesis, photo-protection and chlorophyll transfer. However, the binding of chlorophyll to LIL3 is unclear. We present a reconstitution protocol for chlorophyll binding to LIL3 in DDM micelles. It is shown in the absence of lipids and carotenoids that reconstitution of chlorophyll binding to in vitro expressed LIL3 requires pre-incubation of reaction partners at room temperature. We show chlorophyll a but not chlorophyll b binding to LIL3 at a molar ratio of 1:1. Neither dynamic light scattering nor native PAGE, enabled a discrimination between binding of chlorophyll a and/or b to LIL3.

Lil3 dimerization and chlorophyll binding in Arabidopsis thaliana.

The two-helix light harvesting like (Lil) protein Lil3 belongs to the family of chlorophyll binding light harvesting proteins of photosynthetic membranes. A function in tetrapyrrol synthesis and stabilization of geranylgeraniol reductase has been shown. Lil proteins contain the chlorophyll a/b-binding motif; however, binding of chlorophyll has not been demonstrated. We find that Lil3.2 from Arabidopsis thaliana forms heterodimers with Lil3.1 and binds chlorophyll. Lil3.2 heterodimerization (25±7.8 nM) is favored relative to homodimerization (431±59 nM). Interaction of Lil3.2 with chlorophyll a (231±49 nM) suggests that heterodimerization precedes binding of chlorophyll in Arabidopsis thaliana.