Bioluminescence - The Vibrant Glow of Nature and its Chemical Mechanisms.

Bioluminescence, the mesmerizing natural phenomenon where living organisms produce light through chemical reactions, has long captivated scientists and laypersons alike, offering a rich tapestry of insights into biological function, ecology, evolution as well as the underlying chemistry. This comprehensive introductory review systematically explores the phenomenon of bioluminescence, addressing its historical context, geographic dispersion, and ecological significance with a focus on their chemical mechanisms. Our examination begins with terrestrial bioluminescence, discussing organisms from different habitats. We analyze thefireflies of Central Europe's meadows and the fungi in the Atlantic rainforest of Brazil. Additionally, we inspect bioluminescent species in New Zealand, specifically river-dwelling snails and mosquito larvae found in Waitomo Caves. Our exploration concludes in the Siberian Steppes, highlighting the area's luminescent insects and annelids. Transitioning to the marine realm, the second part of this review examines marine bioluminescent organisms. We explore this phenomenon in deep-sea jellyfish and their role in the ecosystem. We then move to Toyama Bay, Japan, where seasonal bioluminescence of dinoflagellates and ostracods present a unique case study. We also delve into the bacterial world, discussing how bioluminescent bacteria contribute to symbiotic relationships. For each organism, we contextualize its bioluminescence, providing details about its discovery, ecological function, and geographical distribution. A special focus lies on the examination of the underlying chemical mechanisms that enables these biological light displays. Concluding this review, we present a series of practical bioluminescence and chemiluminescence experiments, providing a resource for educational demonstrations and student research projects. Our goal with this review is to provide a summary of bioluminescence across the diverse ecological contexts, contributing to the broader understanding of this unique biological phenomenon and its chemical mechanisms serving researchers new to the field, educators and students alike.

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.

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.

Potential use of Cypridina luciferin for quantifying alpha 1-acid glycoprotein in human serum.

Alpha 1-acid glycoprotein (AGP) is an acute phase protein in mammals, including humans. The amount of AGP in human serum varies in response to certain diseases; thus, many efforts have been made to develop methods for quantifying human AGP. We recently discovered that luminescence occurs merely by mixing Cypridina luciferin with human AGP under human serum-free neutral or basic buffer conditions. In this study, we tested an application of Cypridina luciferin for quantifying AGP contained in human serum. Our luminescence spectrum measurements of Cypridina luciferin with human serum samples showed that the maximum emission wavelength with human serum (480 nm) differed from that with human AGP (464 nm) due to the abundant presence of endogenous human serum albumin (HSA). Furthermore, the luminescence intensities of Cypridina luciferin with human AGP in HSA-depleted human serum were consistent with those in a human serum-free basic buffer, but those in human serum were not. These results indicated that depletion of HSA in human serum was required to use Cypridina luciferin for quantifying AGP in human serum. Additionally, we found that the luminescence intensity of Cypridina luciferin with bovine AGP was approximately tenfold lower than that with human AGP.

Comparative Investigation of the Chemiluminescent Properties of a Dibrominated Coelenterazine Analog.

Chemi- and bioluminescence are remarkable light-emitting phenomena, in which thermal energy is converted into excitation energy due to a (bio)chemical reaction. Among a wide variety of chemi-/bioluminescent systems, one of the most well-known and studied systems is that of marine imidazopyrazinones, such as Coelenterazine and Cypridina luciferin. Due to the increasing usefulness of their chemi-/bioluminescent reactions in terms of imaging and sensing applications, among others, significant effort has been made over the years by researchers to develop new derivatives with enhanced properties. Herein, we report the synthesis and chemiluminescent characterization of a novel dibrominated Coelenterazine analog. This novel compound consistently showed superior luminescence, in terms of total light output and emission lifetime, to natural imidazopyrazinones and commercially available analogs in aprotic media, while being capable of yellow light emission. Finally, this new compound showed enhanced chemiluminescence in an aqueous solution when triggered by superoxide anion, showing potential to be used as a basis for optimized probes for reactive oxygen species. In conclusion, bromination of the imidazopyrazinone scaffold appears to be a suitable strategy for obtaining Coelenterazines with enhanced properties.

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.

Human Serum Enhances Biomimicry of Engineered Tissue Models of Bone and Cancer.

For decades, fetal bovine serum (FBS) has been used routinely for culturing many cell types, based on its empirically demonstrated effects on cell growth, and the lack of suitable non-xenogeneic alternatives. The FBS-based culture media do not represent the human physiological conditions, and can compromise biomimicry of preclinical models. To recapitulate in vitro the features of human bone and bone cancer, we investigated the effects of human serum and human platelet lysate on modeling osteogenesis, osteoclastogenesis, and bone cancer in two-dimensional (2D) and three-dimensional (3D) settings. For monitoring tumor growth within tissue-engineered bone in a non-destructive fashion, we generated cancer cell lines expressing and secreting luciferase. Culture media containing human serum enhanced osteogenesis and osteoclasts differentiation, and provided a more realistic in vitro mimic of human cancer cell proliferation. When human serum was used for building 3D engineered bone, the tissue recapitulated bone homeostasis and response to bisphosphonates observed in native bone. We found disparities in cell behavior and drug responses between the metastatic and primary cancer cells cultured in the bone niche, with the effectiveness of bisphosphonates observed only in metastatic models. Overall, these data support the utility of human serum for bioengineering of bone and bone cancers.

A small molecule UPR modulator for diabetes identified by high throughput screening.

Unfolded protein response (UPR) is a stress response that is specific to the endoplasmic reticulum (ER). UPR is activated upon accumulation of unfolded (or misfolded) proteins in the ER's lumen to restore protein folding capacity by increasing the synthesis of chaperones. In addition, UPR also enhances degradation of unfolded proteins and reduces global protein synthesis to alleviate additional accumulation of unfolded proteins in the ER. Herein, we describe a cell-based ultra-high throughput screening (uHTS) campaign that identifies a small molecule that can modulate UPR and ER stress in cellular and in vivo disease models. Using asialoglycoprotein receptor 1 (ASGR) fused with Cypridina luciferase (CLuc) as reporter assay for folding capacity, we have screened a million small molecule library and identified APC655 as a potent activator of protein folding, that appears to act by promoting chaperone expression. Furthermore, APC655 improved pancreatic ß cell viability and insulin secretion under ER stress conditions induced by thapsigargin or cytokines. APC655 was also effective in preserving ß cell function and decreasing lipid accumulation in the liver of the leptin-deficient (ob/ob) mouse model. These results demonstrate a successful uHTS campaign that identified a modulator of UPR, which can provide a novel candidate for potential therapeutic development for a host of metabolic diseases.

A small molecule UPR modulator for diabetes identified by high throughput screening

Unfolded protein response (UPR) is a stress response that is specific to the endoplasmic reticulum (ER). UPR is activated upon accumulation of unfolded (or misfolded) proteins in the ER's lumen to restore protein folding capacity by increasing the synthesis of chaperones. In addition, UPR also enhances degradation of unfolded proteins and reduces global protein synthesis to alleviate additional accumulation of unfolded proteins in the ER. Herein, we describe a cell-based ultra-high throughput screening (uHTS) campaign that identifies a small molecule that can modulate UPR and ER stress in cellular and

Luminescence of Cypridina Luciferin in the Presence of Human Plasma Alpha 1-Acid Glycoprotein.

The enzyme Cypridina luciferase (CLase) enables Cypridina luciferin to emit light efficiently through an oxidation reaction. The catalytic mechanism on the substrate of CLase has been studied, but the details remain to be clarified. Here, we examined the luminescence of Cypridina luciferin in the presence of several proteins with drug-binding ability. Luminescence measurements showed that the mixture of human plasma alpha 1-acid glycoprotein (hAGP) and Cypridina luciferin produced light. The total value of the luminescence intensity over 60 s was over 12.6-fold higher than those in the presence of ovalbumin, human serum albumin, or bovine serum albumin. In the presence of heat-treated hAGP, the luminescence intensity of Cypridina luciferin was lower than in the presence of intact hAGP. Chlorpromazine, which binds to hAGP, showed an inhibitory effect on the luminescence of Cypridina luciferin, both in the presence of hAGP and a recombinant CLase. Furthermore, BlastP analysis showed that hAGP had partial amino acid sequence similarity to known CLases in the region including amino acid residues involved in the drug-binding ability of hAGP. These findings indicate enzymological similarity between hAGP and CLase and provide insights into both the enzymological understanding of CLase and development of a luminescence detection method for hAGP.

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.

Development of the Bioluminescent Immunoassay for the Detection of 5-Hydroxymethylcytosine in Dinoflagellate.

Cypridina luciferase is a bioluminescent enzyme that has been used as a reporter either in gene expression reporter assays or in immunoassays accompanied by an antibody. To develop a novel bioluminescent assay for the detection of 5-hydroxymethylcytosine, we first conjugated Cypridina luciferase to the antibody against 5-hydroxymethylcytosine. Next, we performed modifications of guanine bases in the genome DNA samples with 4-azidophenylglyoxal and the biotinylation via the azide-Staudinger ligation, which allowed streptavidin to capture and immobilize the genome DNA samples under mild conditions. The detection of 5-hydroxymethylcytosine in the genome DNA samples was performed with the conjugates between Cypridina luciferase and the reduced antibody, which was also confirmed by a surface plasmon resonance assay with the antibody alone. The results obtained from the bioluminescent assay were in good agreement with that of the surface plasmon resonance assay. We succeeded in the detection of 5hmC in the genome DNA samples from the dinoflagellate Pyrocystis Lunula by using this method.

Efficient production of glycosylated Cypridina luciferase using plant cells.

Cypridina noctiluca luciferase has been utilized for biochemical and molecular biological applications, including bioluminescent enzyme immunoassays, far-red luminescence imaging, and high-throughput reporter assays. Some of these applications require a large amount of purified luciferase. However, conventional protein expression systems are not capable of producing sufficient quantities of protein with a high quality and purity without laborious and costly purification processes. To improve the productivity and expand the breadth of possibilities for Cypridina luciferase applications, we employed a variety of secretion expression systems, including yeast, mammalian cells, and silk worms. In this study, we established a simple production procedure using plant cell cultures. The plant cell culture BY-2 efficiently secreted luciferase, which was easily purified using a simple one-step ion-exchange chromatography method. The production yield was 20-30 mg of luciferase per liter of culture medium, and its Km for the luciferin (0.45 µM) was similar to that of the native protein. Additionally, we characterized its glycosylation pattern and confirmed that the two potential N-glycosylation sites were modified with plant-type oligosaccharide chains. Interestingly, the oligosaccharide chains could be trimmed without any detectable decrease in recombinant protein activity. Therefore, the results of our study indicate that this method offers a more cost-effective production method for Cypridina luciferase than conventional methods.

Long-term ex vivo and in vivo monitoring of tumor progression by using dual luciferases.

We propose a new concept of tumor progression monitoring using dual luciferases in living animals to reduce stress for small animals and the cost of luciferin. The secreted Cypridina luciferase (CLuc) was used as an ex vivo indicator to continuously monitor tumor progression. On the other hand, the non-secreted firefly luciferase was used as an in vivo indicator to analyze the spatial distribution of the tumor at suitable time points indicated by CLuc. Thus, the new monitoring systems that use dual luciferases are available, allowing long-term bioluminescence imaging under minimal stress for the experimental animals.

Investigation on 1O2 generation ability of di-sulfonic phthalocyanine zinc isomers using an HPLC-CL system.

An HPLC system combining a chemiluminescence detector was applied to estimate the singlet oxygen ((1)O2) generation ability of di-sulfonic phthalocyanine zinc (ZnPcS2 ) isomers. As photosensitizers, ZnPcS2 produces (1)O2 in air-saturated solutions under photoirradiation. The latter reacts with methyl Cypridina luciferin analogue (MCLA) to initiate chemiluminescence. This photoinduced chemiluminescence (PCL) of MCLA provides an easy method for evaluating the isomers' (1)O2 generation ability during a simultaneous HPLC separation procedure. The cis-isomers and trans-isomers of ZnPcS2 show different (1)O2 generation abilities, which are in accordance with differences in their absorption spectra.