An investigation of carotenoids and a carotenoid-binding protein in sea urchin gonad colour production
The sea urchin Evechinus chloroticus, known as kina, is endemic to New Zealand and is harvested for its edible gonads. Kina are culturally important to Maori and are also commercially harvested to supply roe to domestic and potentially international markets, where it may obtain up to NZ$500/kg. However, currently the export potential of Kina roe is limited by variations in colour between animals, which ranges from a desirable light yellow to dark brown and black. Sea urchin gonad colour is considered to be primarily determined by carotenoid pigment molecules obtained from the diet, which are then thought to be modified in the viscera. Carotenoids are then absorbed from the viscera and transported to the gonads, a process that is thought to be aided by carotenoid-protein interactions. The extracted carotenoid and protein profiles of light, medium and dark gonads of E. chloroticus and of Heliocidaris erthrogamma, a species of sea urchin commercially harvested in Australia, were analysed by RP-HPLC and by 2-D PAGE. The major carotenoid in the gonads of both species was found to be 9'-cis-echinenone with smaller amounts of all-trans-echinenone, lutein and (iso)zeaxanthin. However, few significant differences were observed between either the carotenoid or protein profiles of the light, medium and dark gonads of both species. This suggested that gonad colour variation is a complex process which is not accounted for by protein and carotenoid content. Carotenoid-binding proteins were targeted as potential facilitators of the apparent selective accumulation of 9'-cis-echinenone within the sea urchin gonad. Fractionation of E. chloroticus gonad homogenate resulted in the partial purification of a yellow/orange coloured protein of approximately 15 kDa in size. Analysis by RP-HPLC identified the yellow/orange chromophore as carotenoid, predominantly 9'-cis-echinenone. Therefore the complex was named the echinenone-binding protein (EBP). Mass spectrometry analysis of EBP, in-conjunction with a Mascot database search against the Strongylocentrotus purpuratus (purple sea urchin) genome, resulted in a match to an uncharacterised hypothetical protein. However, homology searches of the S. purpuratus protein sequence indicated that the protein was similar to members of the fatty acid-binding protein (FABP) family. FABPs are a sub-group of the calycin protein superfamily, which is renowned for binding and transporting small hydrophobic ligands. A de novo assembly of the E. chloroticus transcriptome provided the means to obtain the EBP cDNA sequence, enabling the protein to be produced recombinantly in an E. coli expression system. However, significant protein solubility issues were encountered during expression of recombinant EBP and subsequent purification. This was hypothesised to be due to the formation of intermolecular disulfide bond formation through the single cysteine amino acid at position 61 in EBP. The cysteine was mutated to serine to produce EBP-C61S. The tandem expression and purification of EBP and EBP-C61S indicated enhanced solubility and stability of EBP-C61S and the dimerisation effect was virtually eliminated. As a result, an approximately 7-fold increase in the quantity of purified protein was obtained for EBP-C61S compared to EBP. Ab initio secondary structure predictions for EBP, suggested a 10 beta-stranded structure with two short N-terminal alpha-helices. This was consistent with data obtained by circular dichroism which predicted a secondary structure consisting of 55% beta-sheet, 40% turns/unordered and a 5% helices for both EBP and EBP-C61S. The secondary structure predictions are consistent with the highly conserved structure observed amongst FABPs, which were able to be observed in the construction of a 3-D structure homology model. The model indicated that the FABP ligand-binding site would be able to accommodate carotenoids and provided an insight into the specificity of EBP for 9'-cis-echinenone. The model also suggested a transporter role for EBP, with the implication of involvement in the selective accumulation of 9'-cis-echinenone. Therefore EBP may have an important role in sea urchin gonad coloration.
Advisor: Carne, Alan
Degree Name: Doctor of Philosophy
Degree Discipline: Biochemistry
Publisher: University of Otago
Keywords: 9'-cis-echinenone; echinenone-binding; protein; sea; urchin; carotenoid
Research Type: Thesis