Yoshihiro Ohmiya

MOLECULAR EVOLUTION OF THE Ca2+-BINDING PHOTOPROTEINS OF THE HYDROZOA

Abstract— Alignment of the primary structures of the hydrozoan photoproteins, aequorin, mitrocomin, clytin and obelin showed very strong amino acid sequence identities. The Ca2+‐binding sites of the proteins were found to be highly conserved. The Ca2+‐binding sites were also homologous to the Ca2+‐binding sites of other Ca2+‐binding proteins. However, aequorin, mitrocomin, clytin and obelin differed from other Ca2+‐binding proteins in that they contained a relatively large number of cysteine, tryptophan, histidine, proline and tyrosine residues, suggesting that these residues may have evolved as part of the light‐emitting mechanism. Construction of a phylogenetic tree showed that aequorin, mitrocomin, clytin and obelin form a closely related group of proteins.

Cloning, expression and sequence analysis of cDNA for the luciferases from the Japanese fireflies, Pyrocoelia miyako and Hotaria parvula

Abstract— Cloning and sequence analysis of cDNA for the luciferases of Pyrocoelia miyako and Hotaria parvula were carried out (GenBank accession numbers L39928 and L39929, respectively). The amino acid sequence, deduced from the nucleotide sequence, showed P. miyako luciferase to consist of 548 amino acid residues with a molecular weight of 60955, while the luciferase of H. parvula consisted of 548 amino acid residues with a molecular weight of 60 364. Pyrocoelia miyako luciferase showed 82.1 % homology with the luciferase of Photinus pyralis and less than 70% homology with other firefly luciferases, whereas H. parvula luciferase showed 98%, 82.5% and 81.2% homology with the luciferases of Luciola mingrelica, Luciola lateralis and Luciola cruciata, respectively. Two regions in the enzymes were found to be highly conserved. The amino acid sequences were used to construct a phylogenetic tree, which showed that the fireflies could be divided into two groups.

The structural origin of the color differences in the bioluminescence of firefly luciferase

Six chimeric mutants between Hotaria parvula (lambda max = 568 nm) and Pyrocoelia miyako (lambda max = 550 nm) luciferases were reconstructed to determine the structural origin of the color differences in firefly luciferase. Based on light-emitting color, five chimeric luciferases could be divided into two groups: the three green-emitting mutants, classified as P. miyako luciferase, and the two yellow-emitting mutants, classified as H. parvula luciferase. Their common fragments between Val-209 and Ala-318 within each group contain the active site for the color differences.Six chimeric mutants between Hotaria parvula (λmax=568 nm) and Pyrocoelia miyako (λmax=550 nm) luciferases were reconstructed to determine the structural origin of the color differences in firefly luciferase. Based on light‐emitting color, five chimeric luciferases could be divided into two groups: the three green‐emitting mutants, classified as P. miyako luciferase, and the two yellow‐emitting mutants, classified as H. parvula luciferase. Their common fragments between Val‐209 and Ala‐318 within each group contain the active site for the color differences.

Mass spectrometric evidence for a disulfide bond in aequorin regeneration

Tryptic digests of purified recombinant apoaequorin were analyzed, before and after reduction with DTT, by fast atom bombardment mass spectrometry. The results showed that apoaequorin contains a disulfide bond between Cys145 and Cys152 and that the reduction of this bond is involved in the regeneration of aequorin.

Application to processing system using intra-molecular BRET

Luciferases are used as the reporter gene for promoter activity, whereas a green fluorescent protein (GFP) is used as marker for cellular function and localization. Recently, bioluminescence resonance energy transfer (BRET) between luciferase and YFP is used for analysis of inter-molecular reaction such as ligand-receptor in the living cells. The neuropeptides nocistatin (NST) and nociceptin/orphanin FQ (Noc/OFQ) are derived from the same precursor protein, while NST exhibits antagonism against Noc/OFQ-actions. In this study, we attempt an intra-molecular BRET system for monitoring dynamic biological process of the production of NST and Noc/OFQ in the living cells. At first, we constructed a fusion protein (Rluc-GFP) covalently linking luciferase (Renilla luciferase; Rluc) to Aequorea GFP as an intra-molecular BRET partner. Furthermore, we inserted constructs of mouse NST and Noc/OFQ (Rluc-m-GFP) or bovine NST and Noc/OFQ (Rluc-b-GFP) containing a proteolytic cleavage motif (Lys-Arg) within Rluc-GFP. When these constructions were transfected into Cos7 cells, all fusion proteins had luciferase activity and specific fluorescence. Luminescence spectra of Rluc-GFP, Rluc-m-GFP and Rluc-b-GFP fusion proteins with DeepBlueC as a substrate showed two peaks centered at 400 nm and 510 nm, whereas Rluc showed one peak centered at 400 nm. These results indicate that the proteolytic cleavage motif inserted fusion proteins between luciferase and GFP are available for intra-molecular BRET systems at first step.