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, β-carotene, i.e. 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 holoforms, while its rather minimalistic mass (∼20 kDa) makes it an especially attractive antioxidant delivery module. In vitro, AstaP transfers different carotenoids to the 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.

Nanocontainer derived from silkworm carotenoprotein for carotenoid extraction and presentation in biotechnology and biomedical applications

“…5b,c), which is beneficial for constructing modular antioxidant delivery systems based on BmCBP in the future. Unlike reported for the microalgal AstaP carotenoprotein 5 , BmCBP did not display tight physical association in the holo-(Fig. 6c) and apoform (Fig.…”

“…To improve the yield of the BmCBP holoform with ZEA, we added BSA during cell lysis and additionally used chromatography on a hydroxyapatite column. Carotenoid content of the BmCBP holoforms was verified by acetone extraction and thin-layer chromatography 5,13 . Purified proteins were stored frozen at -80 °C.…”

“…Reconstitution of BmCBP holoforms with astaxanthin (AXT) and βcarotene (βCar) was accomplished by mixing 20 μl of 20 μM BmCBP apoprotein with either 2 μl buffer (negative control) or 2 μl of carotenoid solutions in DMSO (βCar 60 μM, CAN 62 μM, AXT 530 μM). CAN-bound BmCBP was obtained by adding CAN extract in DMSO, similar to that in the case of AXT and βCar, or by expression in CAN-producing E. coli cells 5 . Physical migration of carotenoids between proteins and membranes was studied by spectrochromatography.…”

Crystal structure and functional peculiarities of a primordial Orange Carotenoid Protein (OCPX)

“…All constructs were verified by DNA sequencing in Evrogen (Moscow, Russia) and transformed into C41(DE3) E.coli cells already carrying the pACCAR25ΔcrtXZcrtO plasmid (chloramphenicol resistance), which harbors the gene cluster including crtY, crtI, crtB, crtE and crtO sequences from Erwinia uredovora for ECH expression 25 . Alternatively, for making GlOCPX(CAN) protein, co-expression with the βcarotene ketolase crtW (ampicillin resistance) on the background of the pACCAR16ΔcrtX plasmid (synthesizing β-carotene with the gene cluster crtY, crtI, crtB, crtE; chloramphenicol resistance) was used 10,25 . IPTG-induced protein expression lasted overnight at 30 °C.…”

“…OCP is a unique photoreceptor whose photoactivity is determined by the carotenoid cofactor, a ketocarotenoid 3'-hydroxyechinenone (hECH), echinenone (ECH) or canthaxanthin (CAN) 5,8,9 . Although OCP can bind hydroxycarotenoids, only OCP complexes with ketocarotenoids are photoactive 9,10 . According to X-ray structures of OCP known since 2003 11 , such complexes are formed upon embedment of the ketocarotenoid between two structural domains, the N-terminal (NTD) and C-terminal (CTD) connected by a long interdomain linker [12][13][14] .…”