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.

Chemical Mechanism of Fireworm Bioluminescence - A Theoretical Proposition.

Odontosyllis undecimdonta is a marine worm, commonly known as a fireworm, that exhibits bluish-green bioluminescence (BL). The luciferin (L) and oxyluciferin (OL) during fireworm BL have been experimentally identified in vitro. The L and OL are the respective starting point and ending point of a series of complicated chemical reactions in the BL. However, the chemical mechanism of the fireworm BL remains largely unknown. Before the experiments provided strong evidence for the mechanism, based on our previously successful studies on several bioluminescent systems, we theoretically proposed the chemical mechanism of the fireworm BL in this article. By means of the spin-flip and time-dependent density functional calculations, we clearly described the complete process from L to OL: under the catalysis of luciferase, L undergoes deprotonation and reacts with 3O2 to form a dioxetanone anion via the single-electron transfer mechanism; the dioxetanone anion decomposes into the OL at the first singlet excited state (S1) by the gradually reversible charge-transfer-induced luminescence mechanism; and the S1-OL emits light and deexcites to OL in the ground state.

Analysis of D-amino acid in Japanese post-fermented tea, Ishizuchi-kurocha.

The D-amino acid content of Ishizuchi-kurocha, a post-fermented tea produced in Ehime, Japan, was measured. Ishizuchi-kurocha mainly contains D-glutamic acid and D-alanine, but it also contains a small amount of D-aspartic acid. Two types of lactic acid bacteria, Lactiplantibacillus plantarum and Levilactobacillus brevis, are the main species involved in lactic acid fermentation during the tea fermentation process. Therefore, the D-amino acid-producing abilities of strains of these two species isolated from Ishizuchi-kurocha were examined. Specifically, the production of D-aspartic acid, D-alanine, and D-glutamic acid by L. brevis and L. plantarum strains was observed. The amount of D-aspartic acid produced by L. plantarum was low. D-glutamine was detected in culture supernatant but not in bacterial cells. D-arginine was detected in bacterial cells of the L. plantarum strains but not in the culture supernatant. Both the L. brevis and L. plantarum strains possessed at least three kinds of putative racemase genes: alanine racemase, glutamate racemase, and aspartate racemase. However, their expression and enzyme activity remain unknown. L. plantarum and L. brevis could play an important role in the production of D-amino acids in Ishizuchi-kurocha. In fact, Ishizuchi-kurocha is expected to possess the effective physiological activities of D-amino acids.

Molecular evolution of Cypridina noctiluca secretory luciferase for production of spectrum-shifted luminescence-emitting mutants and their application in nuclear receptor-reporter assays.

Luciferase is a popular enzyme used for biological analyses, such as reporter assays. In addition to a conventional reporter assay using a pair of firefly and Renilla luciferases, a simple multicolor reporter assay using multiple firefly or beetle luciferases emitting different color luminescence with a single substrate has been reported. Secretory luciferases have also been used for convenient sample preparation in reporter assays; however, reporter assay using secretory luciferase mutants that emit spectrum-shifted luminescence have not yet been reported. In this study, we generated blue- and red-shifted (-16 and 12 nm) luminescence-emitting Cypridina secretory luciferase (CLuc) mutants using multiple cycles of random and site-directed mutagenesis. Even for red-shifted CLuc mutant, which exhibited relatively low activity and stability, its enzymatic activity was sufficiently high for a luciferase assay (3.26 × 106 relative light unit/s), light emission was sufficiently prolonged (half-life is 3 min), and stability at 37°C was high. We independently determined the luminescence of these CLuc mutants using a luminometer with an optical filter. Finally, we replaced the commonly used reporters, firefly and Renilla luciferases used in a conventional nuclear receptor-reporter assay with these CLuc mutants and established a secretory luciferase-based single-substrate dual-color nuclear receptor-reporter assay.

Investigating the diversity of bioluminescent marine worm Polycirrus (Annelida), with description of three new species from the Western Pacific.

Bioluminescence, a phenomenon observed widely in organisms ranging from bacteria to metazoans, has a significant impact on the behaviour and ecology of organisms. Among bioluminescent organisms, Polycirrus, which has unique emission wavelengths, has received attention, and advanced studies such as RNA-Seq have been conducted, but they are limited to a few cases. In addition, accurate species identification is difficult due to lack of taxonomic organization. In this study, we conducted comprehensive taxonomic survey of Japanese Polycirrus based on multiple specimens from different locations and described as three new species: Polycirrus onibi sp. nov., P. ikeguchii sp. nov. and P. aoandon sp. nov. The three species can be distinguished from the known species based on the following characters: (i) arrangement of mid-ventral groove, (ii) arrangement of notochaetigerous segments, (iii) type of neurochaetae uncini, and (iv) arrangement of nephridial papillae. By linking the bioluminescence phenomenon with taxonomic knowledge, we established a foundation for future bioluminescent research development. We also provide a brief phylogenetic tree based on cytochrome c oxidase subunit I (COI) sequences to discuss the evolution of bioluminescence and the direction of future research.

High sensitivity and low-cost flavin luciferase (FLUXVc)-based reporter gene for mammalian cell expression.

Luciferase-based gene reporters generating bioluminescence signals are important tools for biomedical research. Amongst the luciferases, flavin-dependent enzymes use the most economical chemicals. However, their applications in mammalian cells are limited due to their low signals compared to other systems. Here, we constructed Flavin Luciferase from Vibrio campbellii (Vc) for Mammalian Cell Expression (FLUXVc) by engineering luciferase from V. campbellii (the most thermostable bacterial luciferase reported to date) and optimizing its expression and reporter assays in mammalian cells which can improve the bioluminescence light output by >400-fold as compared to the nonengineered version. We found that the FLUXVc reporter gene can be overexpressed in various cell lines and showed outstanding signal-to-background in HepG2 cells, significantly higher than that of firefly luciferase (Fluc). The combined use of FLUXVc/Fluc as target/control vectors gave the most stable signals, better than the standard set of Fluc(target)/Rluc(control). We also demonstrated that FLUXVc can be used for testing inhibitors of the NF-κB signaling pathway. Collectively, our results provide an optimized method for using the more economical flavin-dependent luciferase in mammalian cells.

Quantitative Analysis of Bioluminescence Optical Signal.

Bioluminescence is light emission based on the luciferin-luciferase enzymatic reaction in living organisms. Optical signals from bioluminescence (BL) reactions are available for bioanalysis and bioreporters for gene expression, in vitro, in vivo, and ex vivo bioimaging, immunoassay, and other applications. Although there are numerous bioanalysis methods based on BL signal measurements, the BL signal is measured as a relative value, and not as an absolute value. Recently, some approaches have been established to completely quantify the BL signal, resulting in, for instance, the redetermination of the quantum yield of the BL reaction and counting the photon number of the BL signal at the single-cell level. Reliable and reproducible understanding of biological events in the bioanalysis and bioreporter fields can be achieved by means of standardized absolute optical signal measurements, which is described in an International Organization for Standardization (ISO) document.

An international validation study of the interleukin-2 luciferase leukocyte toxicity test (IL-2 Luc LTT) to evaluate potential immunosuppressive chemicals and its performance after use with the interleukin-2 luciferase assay (IL-2 Luc assay).

We previously reported that the IL-2 Luc LTT can detect immunosuppressive effects of drugs that are attributed to their antimitotic activity. Here, we report an official validation study of the IL-2 Luc LTT. In the Phase I study that evaluated five coded chemicals, the within-laboratory reproducibility of three independent laboratories was 100.0%. In the combined results of the Phase I and II studies that evaluated 20 coded chemicals, the between-laboratory reproducibility was 92.0%. When compared with the reference data based on the previously-reported immunotoxicological information, the predictivity of the combined Phase I and II studies was 76.0% for Lab A and 72.0% for Labs B and C. In contrast, in the study in which the lead laboratory examined 37 non-pharmaceutical chemicals, the predictivity of the IL-2 Luc LTT and the IL-2 Luc assay was 48.6% and 64.9%, respectively, whereas that of the combined assays was 74.3%. It is clear that an integrated approach combining multiple assays is necessary for the development of in vitro immunosuppression testing. These data suggest that the IL-2 Luc LTT alone is not sufficient as a component of the integrated approach, but the combination of the IL-2 Luc assay and IL-2 Luc LTT is promising.

Genome analysis of Phrixothrix hirtus (Phengodidae) railroad worm shows the expansion of odorant-binding gene families and positive selection on morphogenesis and sex determination genes.

Among bioluminescent beetles of the Elateroidea superfamily, Phengodidae is the third largest family, with 244 bioluminescent species distributed only in the Americas, but is still the least studied from the phylogenetic and evolutionary points of view. The railroad worm Phrixothrix hirtus is an essential biological model and symbolic species due to its bicolor bioluminescence, being the only organism that produces true red light among bioluminescent terrestrial species. Here, we performed partial genome assembly of P. hirtus, combining short and long reads generated with Illumina sequencing, providing the first source of genomic information and a framework for comparative analyses of the bioluminescent system in Elateroidea. This is the largest genome described in the Elateroidea superfamily, with an estimated size of ∼3.4 Gb, displaying 32 % GC content, and 67 % transposable elements. Comparative genomic analyses showed a positive selection of genes and gene family expansion events of growth and morphogenesis gene products, which could be associated with the atypical anatomical development and morphogenesis found in paedomorphic females and underdeveloped males. We also observed gene family expansion among distinct odorant-binding receptors, which could be associated with the pheromone communication system typical of these beetles, and retrotransposable elements. Common genes putatively regulating bioluminescence production and control, including two luciferase genes corresponding to lateral lanterns green-emitting and head lanterns red-emitting luciferases with 7 exons and 6 introns, and genes potentially involved in luciferin biosynthesis were found, indicating that there are no clear differences about the presence or absence of gene families associated with bioluminescence in Elateroidea.

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.

Luciferin Synthesis and Pesticide Detection by Luminescence Enzymatic Cascades.

D-Luciferin (D-LH2 ), a substrate of firefly luciferase (Fluc), is important for a wide range of bioluminescence applications. This work reports a new and green method using enzymatic reactions (HELP, HadA Enzyme for Luciferin Preparation) to convert 19 phenolic derivatives to 8 D-LH2 analogues with ≈51 % yield. The method can synthesize the novel 5'-methyl-D-LH2 and 4',5'-dimethyl-D-LH2 , which have never been synthesized or found in nature. 5'-Methyl-D-LH2 emits brighter and longer wavelength light than the D-LH2 . Using HELP, we further developed LUMOS (Luminescence Measurement of Organophosphate and Derivatives) technology for in situ detection of organophosphate pesticides (OPs) including parathion, methyl parathion, EPN, profenofos, and fenitrothion by coupling the reactions of OPs hydrolase and Fluc. The LUMOS technology can detect these OPs at parts per trillion (ppt) levels. The method can directly detect OPs in food and biological samples without requiring sample pretreatment.

Violet bioluminescent Polycirrus sp. (Annelida: Terebelliformia) discovered in the shallow coastal waters of the Noto Peninsula in Japan.

Terebellidae worms have large numbers of tentacles responsible for various biological functions. Some Terebellidae worms whose tentacles emit light are found around the world, including exceptional violet-light-emitting Polycirrus spp. found in Europe and North America. However, there is no video-recorded observation of the luminous behavior of such unique species in nature, and the genetic information related to their ecology are lacking. Here, for the first time, we video-recorded the violet-light-emitting behavior of an undescribed Japanese worm in its natural habitat. The worm was designated as Polycirrus sp. ISK based on morphological observations, and the luminescence spectrum showed a peak at 444 nm, which is an exceptionally short wavelength for bioluminescence in a shallow coastal water environment. An analysis of differentially expressing genes based on separate RNA-Seq analysis for the tentacles and the rest of body revealed the specific expression of genes that are probably involved in innate immunity in the tentacles exposed to predators. We also found a Renilla luciferase homologous gene, but coelenterazine was not detected in the worm extract by analyses using a liquid chromatography and a recombinant Renilla luciferase. These results will promote an understanding of the ecology and luminescence mechanisms of luminous Polycirrus spp.

Rapid reprogramming of tumour cells into cancer stem cells on double-network hydrogels.

Cancer recurrence can arise owing to rare circulating cancer stem cells (CSCs) that are resistant to chemotherapies and radiotherapies. Here, we show that a double-network hydrogel can rapidly reprogramme differentiated cancer cells into CSCs. Spheroids expressing elevated levels of the stemness genes Sox2, Oct3/4 and Nanog formed within 24 h of seeding the gel with cells from any of six human cancer cell lines or with brain cancer cells resected from patients with glioblastoma. Human brain cancer cells cultured on the double-network hydrogel and intracranially injected in immunodeficient mice led to higher tumorigenicity than brain cancer cells cultured on single-network gels. We also show that the double-network gel induced the phosphorylation of tyrosine kinases, that gel-induced CSCs from primary brain cancer cells were eradicated by an inhibitor of the platelet-derived growth factor receptor, and that calcium channel receptors and the protein osteopontin were essential for the regulation of gel-mediated induction of stemness in brain cancer cells.

Synthetic poly(2-acrylamido-2-methylpropanesulfonic acid) gel induces chondrogenic differentiation of ATDC5 cells via a novel protein reservoir function.

We previously demonstrated that a synthetic negatively charged poly(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) gel induced chondrogenic differentiation of ATDC5 cells. In this study, we clarified the underlying molecular mechanism, in particular, focusing on the events that occurred at the interface between the gel and the cells. Gene expression profiling revealed that the expression of extracellular components was enhanced in the ATDC5 cells that were cultured on the PAMPS gel, suggesting that extracellular proteins secreted from the ATDC5 cells might be adsorbed in the PAMPS gel, thereby contributing to the induction of chondrogenic differentiation. Therefore, we created "Treated-PAMPS gel," which adsorbed various proteins secreted from the cultured ATDC5 cells during 7 days. Proteomic analysis identified 27 proteins, including extracellular matrix proteins such as Types I, III, and V collagens and thrombospondin (THBS) in the Treated-PAMPS gel. The Treated-PAMPS gel preferentially induced expression of chondrogenic markers, namely, aggrecan and Type II collagen, in the ATDC5 cells compared with the untreated PAMPS gel. Addition of recombinant THBS1 to the ATDC5 cells significantly enhanced the PAMPS-induced chondrogenic differentiation, whereas knockdown of THBS1 completely abolished this response. In conclusion, we demonstrated that the PAMPS gel has the potential to induce chondrogenic differentiation through novel reservoir functions, and the adsorbed THBS plays a significant role in the induction.

In Vivo Simultaneous Analysis of Gene Expression by Dual-Color Luciferases in Caenorhabditis elegans.

Both fluorescent and luminescent observation are widely used to examine real-time gene expression patterns in living organisms. Several fluuorescent and luminescent proteins with specific optical properties have been developed and applied for simultaneous, multi-color observation of more than two gene expression profiles. Compared to fluorescent proteins, however, the application of multi-color luminescent imaging in living organisms is still limited. In this study, we introduced two-color luciferases into the soil nematode C. elegans and performed simultaneous analysis of two gene expression profiles. Using a green-emitting luciferase Eluc (emerald luciferase) and red-emitting luciferase SLR (stable luciferase red), the expression patterns of two genes were simultaneously observed in single animals from embryonic to adult stages over its whole life span. In addition, dual gene activities were observed at the single embryo level, with the simultaneous observation of morphological changes. These are the first application of a two-color luciferase system into a whole animal and suggest that precise relationship of expression patterns of multiple genes of interest can be analyzed over the whole life of the animal, dependent on the changes in genetic and/or environmental conditions.

Quantitative immunohistochemistry using an antibody-fused bioluminescent protein.

We established a quantitative detection method for immunohistochemistry based on a reference standard light-emitting diode, protein microarray and antibody-fused bioluminescent protein. In this procedure, we calibrated the bioluminescence imaging system and prepared the calibration curve between antigen and antibody-fused bioluminescent protein using a protein microarray. Then we converted the detecting light signal to antigen count via absolute photon number in the bioluminescent images; there was a resulting threefold difference in the target antigen number between normal and cancerous tissues. Our technique can easily compare immunohistological images and evaluate tumor progression in quantitative pathological diagnosis.

Mechanically Assisted Bioluminescence with Natural Luciferase.

Mechanochemical analogues have recently been established for several enzymatic reactions, but they require periodic interruption of the reaction for sampling, dissolution, and (bio)chemical analysis to monitor their progress. By applying a mechanochemical procedure to induce bioluminescence analogous to that used by the marine ostracod Cypridina (Vargula) hilgendorfii, here we demonstrate that the light emitted by a bioluminescent reaction can be used to directly monitor the progress of a mechanoenzymatic reaction without sampling. Mechanical treatment of Cypridina luciferase with luciferin generates bright blue light which can be readily detected and analyzed spectroscopically. This mechanically assisted bioluminescence proceeds through a mechanism identical to that of bioluminescence in solution, but has higher activation energy due to being diffusion-controlled in the viscous matrix. The results suggest that luciferases could be used as light-emissive reporters of mechanoenzymatic reactions.