Carotenoid-binding Proteins Research Articles

Carotenoid metabolic profiling and transcriptome‐genome mining reveal functional equivalence among blue‐pigmented copepods and appendicularia

The tropical oligotrophic oceanic areas are characterized by high water transparency and annual solar radiation. Under these conditions, a large number of phylogenetically diverse mesozooplankton species living in the surface waters (neuston) are found to be blue pigmented. In the present study, we focused on understanding the metabolic and genetic basis of the observed blue phenotype functional equivalence between the blue-pigmented organisms from the phylum Arthropoda, subclass Copepoda (Acartia fossae) and the phylum Chordata, class Appendicularia (Oikopleura dioica) in the Red Sea. Previous studies have shown that carotenoid-protein complexes are responsible for blue coloration in crustaceans. Therefore, we performed carotenoid metabolic profiling using both targeted and nontargeted (high-resolution mass spectrometry) approaches in four different blue-pigmented genera of copepods and one blue-pigmented species of appendicularia. Astaxanthin was found to be the principal carotenoid in all the species. The pathway analysis showed that all the species can synthesize astaxanthin from β-carotene, ingested from dietary sources, via 3-hydroxyechinenone, canthaxanthin, zeaxanthin, adonirubin or adonixanthin. Further, using de novo assembled transcriptome of blue A. fossae (subclass Copepoda), we identified highly expressed homologous β-carotene hydroxylase enzymes and putative carotenoid-binding proteins responsible for astaxanthin formation and the blue phenotype. In blue O. dioica (class Appendicularia), corresponding putative genes were identified from the reference genome. Collectively, our data provide molecular evidences for the bioconversion and accumulation of blue astaxanthin-protein complexes underpinning the observed ecological functional equivalence and adaptive convergence among neustonic mesozooplankton.

Carotenoid-binding proteins; accessories to carotenoid function.

Understanding of the widespread biological importance of carotenoids is increasing. Accompanying this is the developing recognition that the interaction of carotenoids with other molecules, such as proteins, is also essential. Here the significance of carotenoid-protein interactions with respect to biological function is reviewed for three well characterised carotenoprotein complexes; crustacyanin, the orange carotenoid protein and glutathione-S-transferase P1. In addition a preliminary report is made on the recent partial purification of an echinenone-binding protein extracted from a New Zealand sea urchin, Evechinus chloroticus.

Surface plasmon resonance (SPR) studies on the interactions of carotenoids and their binding proteins

The xanthophyll carotenoids lutein and zeaxanthin constitute the major carotenoids of the macular pigment in the human retina where they are thought to act in part to prevent light induced oxidative damage associated with age-related macular degeneration (AMD). The highly selective uptake of these pigments is mediated by specific carotenoid-binding proteins (GSTP1 and StARD3) recently identified in our laboratory. Carotenoids are hydrophobic in nature, so we first systematically optimized carotenoid preparations that are nano-dispersed in aqueous buffers, and then we used a new-generation surface plasmon resonance (SPR) protocol called FastStep™, which is significantly faster than conventional SPR assays. We have explored carotenoid-binding interactions of five proteins: human serum albumin (HSA), β-lactoglobulin (LG), steroidogenic acute regulatory domain proteins (StARD1, StARD3) and glutathione S- transferase Pi isoform (GSTP1). HSA and LG showed relatively weak interaction with carotenoids (KD>1μM). GSTP1 evidenced high affinity and specificity towards zeaxanthin and meso-zeaxanthin with KD values 0.14±0.02μM and 0.17±0.02μM, respectively. StARD3 expressed a relative high specificity towards lutein with a KD value of 0.59±0.03μM, whereas StARD1 exhibited a relatively low selectivity and affinity (KD>1μM) towards the various carotenoids tested.

Human ocular carotenoid-binding proteins.

Two dietary carotenoids, lutein and zeaxanthin, are specifically delivered to the human macula at the highest concentration anywhere in the body. Whenever a tissue exhibits highly selective uptake of a compound, it is likely that one or more specific binding proteins are involved in the process. Over the past decade, our laboratory has identified and characterized several carotenoid-binding proteins from human retina including a pi isoform of glutathione S-transferase (GSTP1) as a zeaxanthin-binding protein, a member of the steroidogenic acute regulatory domain (StARD) family as a lutein-binding protein, and tubulin as a less specific, but higher capacity site for carotenoid deposition. In this article, we review the purification and characterization of these carotenoid-binding proteins, and we relate these ocular carotenoid-binding proteins to the transport and uptake role of serum lipoproteins and scavenger receptor proteins in a proposed pathway for macular pigment carotenoid delivery to the human retina.

Euglena Light-Harvesting Complexes Are Encoded by Multifarious Polyprotein mRNAs that Evolve in Concert

Light-harvesting complexes (LHCs) are a superfamily of chlorophyll- and carotenoid-binding proteins that are responsible for the capture of light energy and its transfer to the photosynthetic reaction centers. Unlike those of most eukaryotes, the LHCs of Euglena gracilis are translated from large mRNAs, producing polyprotein precursors consisting of multiple concatenated LHC subunits that are separated by conserved decapeptide linkers. These precursors are posttranslationally targeted to the chloroplast and cleaved into individual proteins. We analyzed expressed sequence tags from Euglena to further characterize the structural features of the LHC polyprotein-coding genes and to examine the evolution of this multigene family. Of the 19 different LHC transcriptional units we detected, 17 encoded polyproteins composed of both tandem and nontandem repeats of LHC subunits; organizations that likely occurred through unequal crossing-over. Of the 2 nonpolyprotein-encoding LHC transcripts detected, 1 evolved from the truncation of a polyprotein-coding gene. Duplication of LHC polyprotein-coding genes was particularly important in the LHCI gene family where multiple paralogous sequences were detected. Intriguingly, several of the individual LHC-coding subunits both within and between transcriptional units appeared to be evolving in concert, suggesting that gene conversion has been a significant mechanism for LHC evolution in Euglena.

Non-photochemical quenching of fluorescence in cyanobacteria

The pathways of energy dissipation of excessive absorbed energy in cyanobacteria in comparison with that in higher plants are discussed. Two mechanisms of non-photochemical quenching in cyanobacteria are described. In one case this quenching occurs as light-induced decrease of the fluorescence yield of long-wavelength chlorophylls of the photosystem I trimers induced by inactive reaction centers: P700 cation-radical or P700 in triplet state. In the other case, non-photochemical quenching in cyanobacteria takes place with contribution of water-soluble protein OCP (containing 3 -hydroxyechinenone) that induces reversible quenching of allophycocyanin fluorescence in phycobilisomes. The possible evolutionary pathways of the involvement of carotenoid-binding proteins in non-photochemical quenching are discussed comparing the cyanobacterial OCP and plant PsbS protein.

Vertebrate and invertebrate carotenoid-binding proteins

In invertebrates and vertebrates, carotenoids are ubiquitous colorants, antioxidants, and provitamin A compounds that must be absorbed from dietary sources and transported to target tissues where they are taken up and stabilized to perform their physiological functions. These processes occur in a specific and regulated manner mediated by high-affinity carotenoid-binding proteins. In this mini-review, we examine the published literature on carotenoid-binding proteins in vertebrate and invertebrate systems, and we report our initial purification and characterization of a novel lutein-binding protein isolated from liver of Japanese quail ( Coturnix japonica).

The basis for colorless hemolymph and cocoons in the Y-gene recessive Bombyx mori mutants: a defect in the cellular uptake of carotenoids

Bombyx mori is an excellent model for the study of carotenoid-binding proteins (CBP). In previous papers, we identified and molecularly characterized a CBP from the Y-gene dominant mutants. In the present study, we attempted to correlate and establish lipid metabolism and distribution in these mutants. When [3H]-triolein was fed to the mutants, typical patterns of uptake of labeled fatty acids from midgut to hemolymph and subsequent delivery to fat body and silk glands were obtained in all mutants. Further analysis of lipid and carotenoid profiles revealed that the yellow coloration in the hemolymph associated with lipophorin is not attributed to a difference in lipophorin concentrations among the mutants, nor to its lipid composition, but rather to its carotenoid content. Lipophorin of the Y+I mutant exhibited the highest concentration of total carotenoids of 55.8μg/mg lipophorin compared to 3.1μg/mg in the +Y+I mutant, 1.2μg/mg in the YI mutant and 0.5μg/mg in the +YI mutant. Characteristic retention time in HPLC of the different classes of carotenoids of lipophorin identified the presence of lutein as the major chromophore (62–77%), followed by β-carotenes (22–38%). Although lutein and β-carotene content of mutants’ lipophorin differed significantly, the ratio of lutein to β-carotene of 3:1 was not different among mutants. Similarly, lipid compositions of mutant silk glands were not significantly different, but carotenoid contents were. The significantly high concentration of lutein in the Y+I mutant silk gland represented more than 160-fold increase compared to +Y+I mutant (p<0.001). In this report, we conclude that lipid metabolism in the mutants is not defected and that the molecular basis for colorless hemolymph and cocoons is a defect in the cellular uptake of lutein associated with the Y-gene recessive mutants.

Assembly of pigment-protein complexes in carotenoid-deficient membranes of plastids from wheat seedlings treated with norflurazon.

The pyridazinone-type herbicide norflurazon SAN 9789 inhibiting the biosynthesis of long-chain carotenoids results in significant decrease in PS II core complexes and content of light-harvesting complex (LHC) polypeptides. At the same time, early light-induced proteins (ELIP) with molecular masses of 20.5-16.5 and 13.5 kD disappear in norflurazon-treated seedlings grown under intermittent (pulsed) light, confirming the hypothesis that they are carotenoid-binding proteins. Full disappearance of Chl a forms at 668, 676, and 690 nm and a sharp decrease in Chl b form at 648 nm in treated seedlings grown under 30 or 100 lx light intensity shows close contact of these forms with carotenoids in the thylakoid membrane. The band shift from 740 to 720 nm in the low-temperature fluorescence spectrum (77 K) suggests a disturbance of energy transfer from LHC to the Chl a form at 710-712 nm.

エビガラスズメ幼虫の真皮細胞カロチノイド結合タンパク質(eCBP)の特性

エビガラスズメ幼虫の真皮細胞カロチノイド結合タンパク質(eCBP)の特性

Structure and Function of the Water-Soluble Carotenoid-Binding Proteins of Cyanobacteria

The orange carotenoid protein (OCP) and its derivative, the red carotenoid protein (RCP), appear to play important photoprotective roles in cyanobacteria. Structural and functional characterization is gradually elucidating the specific details of how carotenoid-protein interactions, including the role of six methionine residues oriented toward the pigment, contribute to the spectral and functional properties of these proteins.

β-carotene is the intermediate exported from the chloroplast during accumulation of secondary carotenoids in Haematococcus pluvialis

Recent findings on the localization in Haematococcus pluvialis of anearly and a late enzyme in astaxanthin synthesis have suggested thesequential involvement of two compartments, the chloroplast (site of earlysynthesis) and the cytoplasmic lipid vesicles. Here the intermediatetransported across the chloroplast envelope into the cytoplasm is elucidatedby means of the inhibitors diphenylamine (DPA) and2-(4-chlorophenylthio)-triethylamine (CPTA). Based on the amounts of theintermediates accumulated upon inhibitor treatment and their occurrencein the lipid vesicle fractions, it is concluded that the transport takes placeafter β-ionone ring cyclization. A non-vesicle mechanism of β-iononecarotenoid transport from the chloroplast towards the cytoplasmic lipidvesicles via carotenoid binding proteins is suggested.

The orange carotenoid protein of Synechocystis PCC 6803

A water soluble protein with the carotenoid 3′-hydroxyequinenone bound to it has been purified from the cyanobacterium Synechocystis PCC 6803. Based on partial amino acid sequencing of the protein, oligonucleotides were synthesized and used as primers for PCR to obtain a substantial fragment of the gene. This DNA was sequenced and the sequence data and the size of the protein indicate that the protein is encoded by gene slr 1963 in the Kazusa DNA sequence data bank containing the Synechocystis 6803 genome. This protein is very similar to 3′-hydroxyechinenone proteins found in several other cyanobacteria but it shows very little resemblance in its amino acid or gene sequence to other carotenoid binding proteins. The protein binds 1–2 molecules of 3′-hydroxyechinenone and is slowly cleaved by proteases in the cell extract to give a molecule of approximately half the original mass which retains the carotenoid and which shows a striking change in color.

Carotenoid-binding proteins of photosystem II.

The distribution of the photosynthetic pigments of the chlorophyll-binding proteins or photosystem-II membranes, isolated from dark-adapted maize leaves was determined. Most (80%) of a xanthophyll, violaxanthin, was found in the three minor chlorophyll-a/b proteins CP24, CP26 and CP29 whose function is unknown. Violaxanthin is the precursor of zeaxanthin, which is involved in dissipating excess excitation energy into heat [Demmig-Adams, B. (1991) Biochim. Biophys. Acta 1020, 1-24] under conditions of high transmembrane pH gradient [Gilmore, A. M. & Yamamoto, H. Y. (1992) Proc. Natl Acad. Sci. USA 89, 1899-1903]. We propose that a role for the minor photosystem-II chlorophyll-a/b proteins is the regulation of energy transfer to the reaction centre. It was also confirmed that the photosystem II reaction centre (D1-D2-cytochrome b559) contains beta-carotene as the only carotenoid. However, the two other chlorophyll-a-binding proteins of photosystem II, CP47 and CP43, bind not only beta-carotene, but also the xanthophyll lutein, previously thought to be restricted to chlorophyll-a/b proteins.

Co-regulation of a gene homologous to early light-induced genes in higher plants and beta-carotene biosynthesis in the alga Dunaliella bardawil

Dunaliella bardawil, a unicellular green alga that can be induced to accumulate massive amounts of beta-carotene, is particularly suitable for studies of carotenogenesis regulation and its links to developmental and adaptive processes in the chloroplast. A cDNA clone corresponding to a transcript accumulating coordinately with carotenogenesis induction was isolated by differential hybridization of a cDNA library made from intensely illuminated cells. This transcript was also abundant in algal mutants able to accumulate beta-carotene under relatively low light intensity. DNA sequence analysis indicates that cbr (for carotene biosynthesis-related) is closely related to early light-induced genes (elip) of higher plants. Similarity also exists between repeated oligopeptide motifs in Cbr and Cab proteins of photosystems I and II. Three upstream direct repeats in cbr include an octamer and hexamer related to mammalian sterol regulatory elements. The relationship between cbr transcript and beta-carotene accumulation, the structural similarity between Cbr and Cab proteins, and the presence of potential SRE-1 elements lead us to propose that Cbr represents novel carotenoid binding proteins, whose synthesis might be coordinated with carotenogenic enzymes via an evolutionary conserved regulatory mechanism.