Bioluminescence of (R)-Cypridina Luciferin with Cypridina Luciferase.

Cypridina luciferin (CypL) is a marine natural product that functions as the luminous substrate for the enzyme Cypridina luciferase (CypLase). CypL has two enantiomers, (R)- and (S)-CypL, due to its one chiral center at the sec-butyl moiety. Previous studies reported that (S)-CypL or racemic CypL with CypLase produced light, but the luminescence of (R)-CypL with CypLase has not been investigated. Here, we examined the luminescence of (R)-CypL, which had undergone chiral separation from the enantiomeric mixture, with a recombinant CypLase. Our luminescence measurements demonstrated that (R)-CypL with CypLase produced light, indicating that (R)-CypL must be considered as the luminous substrate for CypLase, as in the case of (S)-CypL, rather than a competitive inhibitor for CypLase. Additionally, we found that the maximum luminescence intensity from the reaction of (R)-CypL with CypLase was approximately 10 fold lower than that of (S)-CypL with CypLase, but our kinetic analysis of CypLase showed that the Km value of CypLase for (R)-CypL was approximately 3 fold lower than that for (S)-CypL. Furthermore, the chiral high-performance liquid chromatography (HPLC) analysis of the reaction mixture of racemic CypL with CypLase showed that (R)-CypL was consumed more slowly than (S)-CypL. These results indicate that the turnover rate of CypLase for (R)-CypL was lower than that for (S)-CypL, which caused the less efficient luminescence of (R)-CypL with CypLase.

Gene Cloning and Functional Analysis of the Luciferase from Luminous Syllids of the Genus Odontosyllis.

The marine fireworm Odontosyllis spp. produce the bluish-green bioluminescence (BL). Despite years of research, molecular mechanisms of this unique luciferin-luciferase reaction have not been elucidated. Recently, the genes encoding luciferases of O. undecimdonta and O. enopla have been identified. Here, we describe gene cloning techniques for the luciferase of Odontosyllis spp. from a small number of specimens using highly sensitive mass spectrometry analysis in combination with RNA-sequencing. The luciferase activities of the cloned cDNAs are confirmed by BL assay in vitro using recombinant protein expressed in mammalian cells.

Host-Dependent Producibility of Recombinant Cypridina noctiluca Luciferase With Glycosylation Defects.

Cypridina noctiluca luciferase (CLuc) is a secreted luminescent protein that reacts with its substrate (Cypridina luciferin) to emit light. CLuc is known to be a thermostable protein and has been used for various research applications, including in vivo imaging and high-throughput reporter assays. Previously, we produced a large amount of recombinant CLuc for crystallographic analysis. However, this recombinant protein did not crystallize, probably due to heterogeneous N-glycan modifications. In this study, we produced recombinant CLuc without glycan modifications by introducing mutations at the N-glycan modification residues using mammalian Expi293F cells, silkworms, and tobacco Bright Yellow-2 cells. Interestingly, recombinant CLuc production depended heavily on the expression hosts. Among these selected hosts, we found that Expi293F cells efficiently produced the recombinant mutant CLuc without significant effects on its luciferase activity. We confirmed the lack of N-glycan modifications for this mutant protein by mass spectrometry analysis but found slight O-glycan modifications that we estimated were about 2% of the ion chromatogram peak area for the detected peptide fragments. Moreover, by using CLuc deletion mutants during the investigation of O-glycan modifications, we identified amino acid residues important to the luciferase activity of CLuc. Our results provide invaluable information related to CLuc function and pave the way for its crystallographic analysis.

An international validation study of the IL-2 Luc assay for evaluating the potential immunotoxic effects of chemicals on T cells and a proposal for reference data for immunotoxic chemicals.

To evaluate the immunotoxic effects of xenobiotics, we have established the Multi-ImmunoTox assay, in which three stable reporter cell lines are used to evaluate the effects of chemicals on the IL-2, IFN-γ, IL-1ß and IL-8 promoters. Here, we report the official validation study of the IL-2 luciferase assay (IL-2 Luc assay). In the Phase I study that evaluated five coded chemicals in three sets of experiments, the average within-laboratory reproducibility was 86.7%. In the Phase II study, 20 coded chemicals were evaluated at multiple laboratories. In the combined results of the Phase I and II studies, the between-laboratory reproducibility was 80.0%. These results suggested that the IL-2 Luc assay was reproducible both between and within laboratories. To determine the predictivity, we collected immunotoxicological information and constructed the reference data by classifying the chemical into immunotoxic compounds targeting T cells or others according to previously reported criteria. When compared with the reference data, the average predictivity of the Phase I and II studies was 75.0%, while that of additional 60 chemicals examined by the lead laboratory was 82.5%. Although the IL-2 Luc assay alone is not sufficient to predict immunotoxicity, it will be a useful tool when combined with other immune tests.

Luciferase gene of a Caribbean fireworm (Syllidae) from Puerto Rico.

The fireworms Odontosyllis spp. are globally distributed and well-known for their characteristic and fascinating mating behavior, with secreted mucus emitting bluish-green light. However, knowledge about the molecules involved in the light emission are still scarce. The fireworms are believed to emit light with a luciferin-luciferase reaction, but biochemical evidence of the luciferase is established for only one species living in Japan and no information is available for its luciferin structure. In this study, we identified a luciferase gene from a related Puerto Rican fireworm. We identified eight luciferase-like genes in this Puerto Rican fireworm, finding amino acid identities between Japanese and Puerto Rican luciferase-like genes to be less than 60%. We confirmed cross reactivity of extracts of the Japanese fireworm luciferin with a recombinant Puerto Rican luciferase (PR1). The emission spectrum of recombinant PR1 was similar to the crude extract of the native luciferase, suggesting that PR1 is a functional luciferase of this Puerto Rican fireworm. Our results indicate that the molecular mechanism of luminescence is widely conserved among fireworms.

Novel application of Macrolampis sp2 firefly luciferase for intracellular pH-biosensing in mammalian cells.

Bioluminescence is widely used in biosensors. Firefly luciferase-based bioluminescent sensors are among the most popular ones. Firefly luciferases are pH-sensitive, displaying a large red shift at acidic pH, a property that has been considered undesirable for most applications. Currently, biosensors that can detect intracellular pH are in demand, and some fluorescent biosensors are available. However, pH sensors using bioluminescence have not been used yet. Thus, we decided to harness a firefly luciferase to measure the intracellular pH in mammalian cells. For this purpose, we engineered the luciferase derived from Macrolampis sp2 firefly to localize it on the cytosol or nucleus, in order to observe pH variation in these compartments during biological activities. We first calibrated the emission ratios (R = Igreen/Ired) at different pH values. As expected, we observed a red shift of light emission under acidic conditions when the cells were subjected to different pH conditions in the presence of the K+/H+ ionophore, nigericin. Based on these results, we concluded that this firefly luciferase can be used as a diagnostic tool for measuring the intracellular pH variation in pathogenic cells or in cells during apoptosis. This is the first example of real time-monitoring of pH change using color tuning luciferase.

Novel gene encoding a unique luciferase from the fireworm Odontsyllis undecimdonta.

Luciferases identified or engineered so far emit violet, blue, blue-green, green, yellow, red or near infra-red light. The unique and beautiful bluish-green bioluminescence of fireworms Odontosyllis spp. has attracted particular interest, however, their molecular basis is totally unknown partly due to the difficulty of animal collection. Here we report a novel type of luciferase gene from the Japanese fireworm O. undecimdonta. The major SDS-PAGE band of the luminous mucus showed luciferase activity. A highly sensitive mass spectrometry analysis in combination with RNA sequencing technique revealed that this band was product of a single gene with no homology to any other sequences in public databases. The recombinant protein of this putative luciferase gene expressed in mammalian cells produced the same unique bluish-green emission peak as the fireworm crude extract, indicating that this novel gene is the genuine fireworm luciferase with an evolutionary different origin from other luciferases previously described. Our findings extend the repertoire of luciferin/luciferase system to previously unavailable wavelength range.

Effects of N-Glycosylation Deletions on Cypridina Luciferase Activity.

Cypridina luciferase (Cluc), a secreted luminescent protein identified from Cypridina noctiluca, has two N-glycosylation sites. In this study, we evaluated the effects of N-glycosylation on Cluc properties by creating site-directed mutagenic modifications at the consensus sequence for N-glycosylation (Asn-X-Ser/Thr). Eight variants consisting of four single- and double-residue mutants each were characterized. The producibility and relative specific activity were apparently reduced in mutant Cluc although the thermostability and secretion efficiency were not affected. These results suggested that N-glycosylation modifications and the proper amino acid sequence of the N-glycan binding sites of Cluc are required for the complete protein folding to form a stable catalytic center, for the proper conformation of substrate-protein interaction residues, or for both and that defects in the glycosylation modification are not related to secretion process and stability of the protein.

Identification and molecular characterization of the beta-ketoacyl-[acyl carrier protein] synthase component of the Arabidopsis mitochondrial fatty acid synthase.

Substrate specificity of condensing enzymes is a predominant factor determining the nature of fatty acyl chains synthesized by type II fatty acid synthase (FAS) enzyme complexes composed of discrete enzymes. The gene (mtKAS) encoding the condensing enzyme, beta-ketoacyl-[acyl carrier protein] (ACP) synthase (KAS), constituent of the mitochondrial FAS was cloned from Arabidopsis thaliana, and its product was purified and characterized. The mtKAS cDNA complemented the KAS II defect in the E. coli CY244 strain mutated in both fabB and fabF encoding KAS I and KAS II, respectively, demonstrating its ability to catalyze the condensation reaction in fatty acid synthesis. In vitro assays using extracts of CY244 containing all E. coli FAS components, except that KAS I and II were replaced by mtKAS, gave C(4)-C(18) fatty acids exhibiting a bimodal distribution with peaks at C(8) and C(14)-C(16). Previously observed bimodal distributions obtained using mitochondrial extracts appear attributable to the mtKAS enzyme in the extracts. Although the mtKAS sequence is most similar to that of bacterial KAS IIs, sensitivity of mtKAS to the antibiotic cerulenin resembles that of E. coli KAS I. In the first or priming condensation reaction of de novo fatty acid synthesis, purified His-tagged mtKAS efficiently utilized malonyl-ACP, but not acetyl-CoA as primer substrate. Intracellular targeting using green fluorescent protein, Western blot, and deletion analyses identified an N-terminal signal conveying mtKAS into mitochondria. Thus, mtKAS with its broad chain length specificity accomplishes all condensation steps in mitochondrial fatty acid synthesis, whereas in plastids three KAS enzymes are required.

The biosynthetic pathway for lipoic acid is present in plastids and mitochondria in Arabidopsis thaliana.

In eukaryotes, the biosynthetic pathway for lipoic acid is present in mitochondria. However, it has been hypothesized that, in plants, the biosynthetic pathway is present in plastids in addition to mitochondria. In this study, Arabidopsis thaliana LIP1p cDNA for a plastidial form of lipoic acid synthase has been identified. We show that it encodes a lipoic acid synthase by demonstrating its ability to complement an Escherichia coli mutant lacking lipoic acid synthase activity. We also show that LIP1p is targeted to chloroplasts. These findings suggest that the biosynthetic pathway for lipoic acid is present not only in mitochondria but also in plastids.