Microalgal protein AstaP is a potent carotenoid solubilizer and delivery module with a broad carotenoid binding repertoire

Abstract

Carotenoids are lipophilic substances with many biological functions, from coloration to photoprotection. Being potent antioxidants, carotenoids have multiple biomedical applications, including the treatment of neurodegenerative disorders and retina degeneration. Nevertheless, the delivery of carotenoids is substantially limited by their poor solubility in the aqueous phase. Natural water-soluble carotenoproteins can facilitate this task, necessitating studies on their ability to uptake and deliver carotenoids. One such promising carotenoprotein, AstaP (astaxanthin-binding protein), was recently identified in eukaryotic microalgae, but its structure and functional properties remained largely uncharacterized. By using a correctly folded recombinant protein, here we show that AstaP is an efficient carotenoid solubilizer that can stably bind not only astaxanthin but also zeaxanthin, canthaxanthin, and, to a lesser extent, b-carotene, that is, carotenoids especially valuable to human health. AstaP accepts carotenoids provided as acetone solutions or embedded in membranes, forming carotenoid-protein complexes with an apparent stoichiometry of 1:1. We successfully produced AstaP holoproteins in specific carotenoid-producing strains of Escherichia coli, proving it is amenable to cost-efficient biotechnology processes. Regardless of the carotenoid type, AstaP remains monomeric in both apo-and holoform, while its rather minimalistic mass (~20 kDa) makes it an especially attractive antioxidant delivery module. In vitro, AstaP transfers different carotenoids to liposomes and to unrelated proteins from cyanobacteria, which can modulate their photoactivity and/or oligomerization. These findings expand the toolkit of the characterized carotenoid binding proteins and outline the perspective of the use of AstaP as a unique monomeric antioxidant nanocarrier with an extensive carotenoid binding repertoire.

Structural basis for the ligand promiscuity of the neofunctionalized, carotenoid-binding fasciclin domain protein AstaP

“…Considering the presumable domain structure of this protein (Fig. 1b ) and assuming almost equivalent binding of xanthophylls AXT, CAN, and ZEA 15 , we prepared AstaPo1 mutants lacking either the N-terminal region, or the C-terminal region, or both, and selected to produce them in ZEA-synthesizing E. coli cells, which were known to yield stable ZEA-bound non-truncated AstaPo1 15 . Analytical size-exclusion chromatography (SEC) with continuous absorbance spectrum detection confirmed that truncations lead to a stepwise reduction of the protein monomer size (Fig.…”

“…While the preprotein contained a predicted signal peptide for secretion, it was accumulated on the cell periphery in response to hyperinsolation and osmotic stress (often associated with drought conditions), to perform a photoprotective role, presumably, via direct quenching of reactive oxygen species (ROS) and also via high-energy light filtering effects 13 . Unlike the cyanobacterial photosensory Orange Carotenoid Protein (OCP) 14 , AstaP did not undergo a photo-induced transformation even after intense illumination 15 . Several orthologs of AstaP have been isolated from Scenedesmus sp.…”

“…However, focusing on the first AstaP described, from Coelastrella astaxanthina sp. Ki-4 (AstaP-orange1, or AstaPo1 for short) 13 , 16 , we have recently demonstrated that the recombinant AstaPo1 can bind a uniquely broad repertoire of carotenoids, which includes not only xanthophylls such as canthaxanthin (CAN) and zeaxanthin (ZEA), but also non-oxygenated β-carotene (βCar) 15 . AstaPo1 holoforms could be efficiently reconstituted in vitro by mixing with pure carotenoids or by expression in special carotenoid-synthesizing E. coli cells and could deliver the bound carotenoid to the apoforms of unrelated carotenoproteins or to biological membrane models 15 .…”

“…Ki-4 (AstaP-orange1, or AstaPo1 for short) 13 , 16 , we have recently demonstrated that the recombinant AstaPo1 can bind a uniquely broad repertoire of carotenoids, which includes not only xanthophylls such as canthaxanthin (CAN) and zeaxanthin (ZEA), but also non-oxygenated β-carotene (βCar) 15 . AstaPo1 holoforms could be efficiently reconstituted in vitro by mixing with pure carotenoids or by expression in special carotenoid-synthesizing E. coli cells and could deliver the bound carotenoid to the apoforms of unrelated carotenoproteins or to biological membrane models 15 . While these results expanded the toolkit of known carotenoproteins for carotenoid solubilization and targeted delivery, the structural basis for the carotenoid binding mechanism by AstaP, and potentially other FAS1 proteins, remained enigmatic.…”

Modulation of Membrane Microviscosity by Protein-Mediated Carotenoid Delivery as Revealed by Time-Resolved Fluorescence Anisotropy

“…PCC 7120 (hereinafter AnaCTDH protein) can uptake carotenoids (echinenone and canthaxanthin) from membranes of carotenoid-producing E. coli strains [ 21 , 22 ] and serve as an effective machinery for delivery of ketocarotenoids into cell membranes [ 23 ]. Exploitation of carotenoid-producing strains allowed to reconstitute holoforms of the ~20 kDa microalgal Astaxanthin-binding Protein (AstaP), which can accommodate several carotenoid types (astaxanthin, canthaxanthin, zeaxanthin, lutein and β-carotene) and transfer them to liposomes and apoforms of unrelated carotenoproteins from cyanobacteria [ 24 ]. In addition, it is known that the color of cocoons in silkworm Bombyx mori originates from carotenoids that are delivered by the intracellular ~30 kDa Carotenoid-Binding Protein (CBP) [ 25 ].…”