The calcium-binding photoprotein clytin II: Expression of the preferred human codon-optimized clytin II gene in Chinese hamster ovary-K1 cells and its use in the G-protein-coupled receptor assays.

Clytin II (CLII) is a Ca2+-binding photoprotein and has been identified as an isotype of clytin I (CLI). CLII consists of apoCLII (an apoprotein) and 2-peroxide of coelenterazine (an adduct of molecular oxygen to coelenterazine), which is identical to the widely used Ca2+-binding photoprotein, aequorin (AQ). However, CLII triggered by Ca2+ exhibits a 4.5-fold higher maximum luminescence intensity (Imax) compared to both AQ and CLI, and it is approximately 5 times less sensitive to Ca2+ than AQ. To confirm the suitability of the preferred human codon-optimized CLII (pCLII) gene for cell-based G-protein-coupled receptor (GPCR) assays, a transformant stably expressing apoprotein of pCLII using the pCLII gene in the mitochondria of CHO-K1 cells was established and in situ regenerated pCLII in the cells were applied to the high-throughput screening system. An ATP-stimulated GPCR assay for endogenous P2Y purinergic receptors was confirmed using the established stable transformant.

Bioluminescent immunoassay for serine/threonine protein kinase activity using an aequorin-labeled monoclonal antibody and a synthetic peptide as a substrate.

A bioluminescent immunoassay system was developed to determine serine/threonine protein kinase activity using an aequorin-labeled monoclonal antibody and a synthetic peptide as the substrate. A monoclonal antibody against the synthetic phosphorylated serine peptide (K9P peptide) of histone H3 (19 amino acid residues), referred to as the H3S10P antibody, was chemically conjugated to maleimide-activated aequorin to prepare aequorin-labeled H3S10P (AQ-S-H3S10P). For the serine/threonine kinase assay, a non-phosphorylated serine peptide (K9C peptide) coated on a microplate was incubated with serine/threonine protein kinase in the presence of ATP and Mg2+. The resulting phosphorylated K9C peptides (K9P peptide) were identified using AQ-S-H3S10P. Thus, after the removal of unbound AQ-S-H3S10P though washing, the serine/threonine kinase activity was determined by the luminescence activity of aequorin from AQ-S-H3S10P bound to the K9P peptide. This assay system, in combination with the K9C peptide and AQ-S-H3S10P, could be used to screen inhibitors of various serine/threonine protein kinases in general.

Enzymatic sulfation of coelenterazine by human cytosolic aryl sulfotransferase SULT1A1: identification of coelenterazine C2-benzyl monosulfate by LC/ESI-TOF-MS.

Coelenterazine (CTZ) is known as a light-emitting source for the bioluminescence reaction in marine organisms. CTZ has two phenolic hydroxy groups at the C2-benzyl and C6-phenyl positions, and a keto-enol type hydroxy group at the C3-position in the core structure of imidazopyrazinone (= 3,7-dihydroimidazopyrazin-3-one). These hydroxy groups in CTZ could be sulfated by sulfotransferase(s), and the sulfates of Watasenia luciferin (CTZ disulfate at the C2- and C6-positions) and Renilla pre-luciferin (CTZ 3-enol sulfate) have been identified in marine organisms. To characterize the sulfation process of CTZ, human cytosolic aryl sulfotransferase SULT1A1 (SUTase) was used as a model enzyme. The sulfated products catalyzed by SUTase with 3'-phosphoadenosine 5'-phosphosulfate (PAPS) were analyzed by LC/ESI-TOF-MS. The product was the monosulfate of CTZ and identified as the C2-benzyl sulfate of CTZ (CTZ C2-benzyl monosulfate), but CTZ disulfate, CTZ 3-enol sulfate, and CTZ C6-phenyl monosulfate were not detected. The non-enzymatic oxidation products of dehydrocoelenterazine (dCTZ, dehydrogenated derivative of CTZ), coelenteramide (CTMD), and coelenteramine (CTM) from CTZ were also identified as their monosulfates.

Visualization of osteocalcin and bone morphogenetic protein 2 (BMP2) secretion from osteoblastic cells by bioluminescence imaging.

The secretions of osteocalcin and bone morphogenetic protein 2 (BMP2) from living osteoblastic cells were visualized for the first time using a method of video-rate bioluminescence imaging. The fusion proteins with Gaussia luciferase (GLase) for mouse osteocalcin and BMP2 (OC-GLase and BMP2-GLase, respectively) expressed in osteoblastic MC3T3-E1 cells were correctly processed and secreted. In the video images of exocytotic secretion, the luminescence spots of OC-GLase and BMP2-GLase disappeared rapidly and gradually, respectively, indicating different manners of these proteins in diffusion. Notably, a deletion mutant of BMP2 (Δ3BMP2-GLase) lacking three basic amino acid residues in the N-terminal region for binding to heparan sulfate showed rapidly disappearing luminescence spots. In our imaging conditions, the half-life of luminescence for the spots of Δ3BMP2-GLase (1.61 ± 0.20 s) was similar to that of OC-GLase (1.22 ± 0.14 s) but not to that of BMP2-GLase (4.31 ± 0.41 s). These results suggest that, in contrast to osteocalcin, the diffusion of BMP2 from cells occurred slowly after exocytosis. Thus, our bioluminescence imaging method is useful to study the diffusion properties of secreted proteins in exocytosis.

Reverse mutants of the catalytic 19 kDa mutant protein (nanoKAZ/nanoLuc) from Oplophorus luciferase with coelenterazine as preferred substrate.

Native Oplophorus luciferase (OpLase) and its catalytic 19 kDa protein (wild KAZ) show highest luminescence activity with coelenterazine (CTZ) among CTZ analogs. Mutated wild KAZ with 16 amino acid substitutions (nanoKAZ/nanoLuc) utilizes bis-coelenterazine (bis-CTZ) as the preferred substrate and exhibits over 10-fold higher maximum intensity than CTZ. To understand the substrate selectivity of nanoKAZ between CTZ and bis-CTZ, we prepared the reverse mutants of nanoKAZ by amino acid replacements with the original amino acid residue of wild KAZ. The reverse mutant with L18Q and V27L substitutions (QL-nanoKAZ) exhibited 2.6-fold higher maximum intensity with CTZ than that of nanoKAZ with bis-CTZ. The catalytic properties of QL-nanoKAZ including substrate specificity, luminescence spectrum, luminescence kinetics, luminescence products of CTZ, and luminescence inhibition by deaza-CTZ analogs were characterized and were compared with other CTZ-utilizing luciferases such as Gaussia and Renilla luciferases. Thus, QL-nanoKAZ with CTZ could be used as a potential reporter protein for various luminescence assay systems. Furthermore, the crystal structure of QL-nanoKAZ was determined at 1.70 Å resolution. The reverse mutation at the L18Q and V27L positions of α2-helix in nanoKAZ led to changes in the local structures of the α4-helix and the ß6- and ß7-sheets, and might enhance its binding affinity and oxidation efficiency with CTZ to emit light.

Enzymatic conversion of dehydrocoelenterazine to coelenterazine using FMN-bound flavin reductase of NAD(P)H:FMN oxidoreductase.

Coelenterazine (CTZ) is known as luciferin (a substrate) for the luminescence reaction with luciferase (an enzyme) in marine organisms and is unstable in aqueous solutions. The dehydrogenated form of CTZ (dehydrocoelenterazine, dCTZ) is stable and thought to be a storage form of CTZ and a recycling intermediate from the condensation reaction of coelenteramine and 4-hydroxyphenylpyruvic acid to CTZ. In this study, the enzymatic conversion of dCTZ to CTZ was successfully achieved using NAD(P)H:FMN oxidoreductase from the bioluminescent bacterium Vibrio fischeri ATCC 7744 (FRase) in the presence of NADH (the FRase-NADH reaction). CTZ reduced from dCTZ in the FRase-NADH reaction was identified by HPLC and LC/ESI-TOF-MS analyses. Thus, dCTZ can be enzymatically converted to CTZ in vitro. Furthermore, the concentration of dCTZ could be determined by the luminescence activity using the CTZ-utilizing luciferases (Gaussia luciferase or Renilla luciferase) coupled with the FRase-NADH reaction.

Multiple Cypridina Luciferase Genes in the Genome of Individual Ostracods, Vargula hilgendorfii (Cypridina hilgendorfii).

The genomic structure of the Cypridina luciferase gene in Vargula hilgendorfii (formerly Cypridina hilgendorfii) was determined with three λ phage clones (λ34, λ45, and λ61). The luciferase genes in clones λ34 and λ61 consisted of 13 exons and 12 introns, and clone λ45 only contained exons 1-5. The splicing sites of the luciferase genes in λ34 and λ61 were conserved completely with the consensus sequence. The translated luciferases had 555 amino acid residues, which were over 98.6% identical to those of cDNA clones as previously reported. In contrast, each intron in clones λ34, λ45, and λ61 varied significantly in length. To explain the variation of intron length among the three V. hilgendorfii luciferase genes, genomic DNA was isolated from a single V. hilgendorfii specimen, and the regions from exon 1-3 of the luciferase gene were amplified by polymerase chain reaction (PCR). PCR products with various lengths were detected and were confirmed as the luciferase gene fragments by Southern blot analysis. Furthermore, DNA sequence analysis indicated that at least seven luciferase gene groups might be present in the genome of a single specimen. Thus, multiple Cypridina luciferase genes exist in the genome of a single V. hilgendorfii specimen.

Formation of Coelenteramine from 2-Peroxycoelenterazine in the Ca2+ -Binding Photoprotein Aequorin.

Aequorin consists of apoprotein (apoAequorin) and (S)-2-peroxycoelenterazine (CTZ-OOH) and is considered to be a transient-state complex of an enzyme (apoAequorin) and a substrate (coelenterazine and molecular oxygen) in the enzymatic reaction. The degradation process of CTZ-OOH in aequorin was characterized under various conditions of protein denaturation. By acid treatment, the major product from CTZ-OOH was coelenteramine (CTM), but not coelenteramide (CTMD), and no significant luminescence was observed. The counterparts of CTM from CTZ-OOH were identified as 4-hydroxyphenylpyruvic acid (4HPPA) and 4-hydroxyphenylacetic acid (4HPAA) by liquid chromatography/electrospray ionization-time-of-flight mass spectrometry (LC/ESI-TOF-MS). In the luminescence reaction of aequorin with Ca2+ , similar amounts of 4HPPA and 4HPAA were detected, indicating that CTM is formed by two pathways from CTZ-OOH through dioxetanone anion and not by hydrolysis from CTMD.

Chiral deaza-coelenterazine analogs for probing a substrate-binding site in the Ca2+-binding photoprotein aequorin.

The Ca2+-binding photoprotein aequorin is a complex of apoAequorin (apoprotein) and (S)-2-peroxycoelenterazine. Aequorin can be regenerated by the incubation of apoAequorin with coelenterazine and molecular oxygen (O2). In this study, to investigate the molecular recognition of apoAequorin for coelenterazine using chemical probes, the chiral deaza-analogs of (S)- and (R)-deaza-CTZ (daCTZ) for coelenterazine and of (S)-2- and (R)-2-hydroxymethyl-deaza-CTZ (HM-daCTZ) for 2-peroxycoelenterazine were efficiently prepared by the improvement method. The chiral deaza-analogs of (S)-daCTZ and (S)-HM-daCTZ selectively inhibited the regeneration step to aequorin by binding the catalytic site of coelenterazine in the apoAequorin molecule. The crystal structures of the apoAequorin complexes with (S)-daCTZ and (S)-HM-daCTZ were determined, suggesting that the hydroxy moiety at the C6-hydroxyphenyl group and the carbonyl moiety of the imidazopyrazinone ring in coelenterazine are essential to bind the apoAequorin molecule through hydrogen bonding. Therefore, the chiral deaza-analogs of coelenterazine can be used as a probe to study the interaction between coelenterazine and the related proteins including photoprotein, luciferase, and coelenterazine-binding protein.

[Novel Technology for Studying Insulin Secretion: Imaging and Quantitative Analysis by a Bioluminescence Method].

We developed a method of video-rate bioluminescence imaging to visualize proteins secreted from living cells. A protein of interest was fused to Gaussia luciferase (GLase), and the luminescence signals of secreted GLase with coelenterazine (luciferin) were visualized at a video-rate of 30-500 ms/frame by using a water-cooled EM-CCD camera. We established a subclonal rat INS-1E cell line, named iGL cells, stably expressing the fusion protein of insulin and GLase (Insulin-GLase). By stimulation with high glucose, 3D-cultured iGL cells showed synchronized oscillatory secretion of insulin for over 1 h, as similarly observed in an isolated rat pancreatic islet. In 2D-cultured iGL cells, the luminescence images indicated that synchronized insulin secretion was localized in intercellular spaces between cells. Further, the relative amount of insulin secretion from iGL cells was easily determined with a luminometer, and we demonstrated that cell-cell interaction of beta cells is fundamental to increase glucose-stimulated insulin secretion by synchronization. Thus, iGL cells would be valuable for studying oscillatory insulin secretion and evaluating anti-diabetic drugs. Our bioluminescence imaging method with GLase could be generally used for investigating protein secretion in 2D and 3D cell culture systems.

Efficient conversion to Cypridina luciferin from Cypridina luciferyl sulfate, coupled with enzymatic sulfation of acetic acid.

In Cypridina (Vargula) hilgendorfii, Cypridina luciferin is converted from Cypridina luciferyl sulfate by a sulfotransferase with adenosine 3', 5'-diphosphate (PAP), and is used for the luminescence reaction of Cypridina luciferase. We found that the luminescence activity of crude extracts of C. hilgendorfii was significantly stimulated by the addition of acetic acid. This stimulation may be explained by an efficient supply of PAP from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) catalyzed by a sulfotransferase. Thus, acetic acid acts as a sulfate acceptor from PAPS, followed by forming acetyl sulfate and PAP. The structure of acetyl sulfate was identified using mass spectrometry and it spontaneously decomposed to acetic acid and free sulfate ion in aqueous solutions. This enzymatic conversion from Cypridina luciferyl sulfate to Cypridina luciferin could be coupled with acetic acid and PAPS by a sulfotransferase.

Identification of a novel oxidation product from yellow fluorophore in the complex of apoPholasin and dehydrocoelenterazine.

The complex of the recombinant fusion protein of apoPholasin and glutathione S-transferase (GST-apoPholasin) with non-fluorescent dehydrocoelenterazine (dCTZ) (GST-apoPholasin/dCTZ complex) shows yellow fluorescence at 539 nm by excitation at 430 nm. The GST-apoPholasin/dCTZ complex with a fluorophore (dCTZ*) has considerably weak luminescence activity, converting slowly to a blue fluorescence protein with the emission peak at 430 nm. The main oxidation products from dCTZ* for blue fluorescence were identified as coelenteramine (CTM) and an unreported pyrazine derivative, 3-benzyl-5-(4-hydroxyphenyl)pyrazin-2(1H)-one (CTO) that was confirmed by chemical synthesis.

A novel yellow fluorescent protein of recombinant apoPholasin with dehydrocoelenterazine.

Pholasin is classified as a photoprotein and comprises apoPholasin (an apoprotein of pholasin) and an unknown prosthetic group as the light-emitting source. The luminescence reaction of pholasin is triggered by reactive oxygen species. Recombinant apoPholasin was recently expressed as a fusion protein of glutathione S-transferase (GST-apoPholasin) and purified from E. coli cells. By incubating non-fluorescent dehydrocoelenterazine (dCTZ, dehydrogenated form of CTZ) with GST-apoPholasin, the complex of GST-apoPholasin and dCTZ (GST-apoPholasin/dCTZ complex) was formed immediately and showed bright yellow fluorescence (λmax = 539 nm, excited at 430 nm). Unexpectedly, the fluorescent chromophore of the GST-apoPholasin/dCTZ complex was identified as non-fluorescent dCTZ. The luminescence intensity of the GST-apoPholasin/dCTZ complex was increased in a catalase-H2O2 system, but not in sodium hypochlorite.

Expression, purification, and characterization of recombinant apoPholasin.

Pholasin is a reactive oxygen-sensitive photoprotein that consists of an apoprotein (apoPholasin) and an unknown chromophore. The preferred human codon-optimized apoPholasin gene was transiently expressed in mammalian cells and apoPholasin was detected using an anti-recombinant apoPholasin antibody. For the first time, we found that apoPholasin secreted into the culture medium could catalyze the oxidation of coelenterazine (CTZ, a luciferin) to produce continuous luminescence. The fusion protein of apoPholasin and glutathione S-transferase (GST-apoPholasin) was successfully expressed as a soluble form in bacterial cells using the cold induction system. The purified GST-apoPholasin also had luminescence activity with CTZ, showing the bioluminescence emission peak at 461 nm, and the resultant product showed purple blue fluorescence under 365 nm light. Unexpectedly, the main oxidation product of CTZ was identified as coelenteramine (CTM), not coelenteramide (CTMD).

Enzymatic Conversion of Cypridina Luciferyl Sulfate to Cypridina Luciferin with Coenzyme A as a Sulfate Acceptor in Cypridina (Vargula) hilgendorfii.

In the luminous ostracod Cypridina (presently Vargula) hilgendorfii, Cypridina luciferyl sulfate (3-enol sulfate of Cypridina luciferin) is converted to Cypridina luciferin by a sulfotransferase with 3'-phosphoadenosine-5'-phosphate (PAP) as a sulfate acceptor. The resultant Cypridina luciferin is used for the luciferase-luciferin reaction of Cypridina to emit blue light. The luminescence stimulation with major organic cofactors was examined using the crude extracts of Cypridina specimens, and we found that the addition of coenzyme A (CoA) to the crude extracts significantly stimulated luminescence intensity. Further, the light-emitting source in the crude extracts stimulated with CoA was identified as Cypridina luciferyl sulfate, and we demonstrated that CoA could act as a sulfate acceptor from Cypridina luciferyl sulfate. In addition, the sulfate group of Cypridina luciferyl sulfate was also transferred to adenosine 5'-monophosphate (5'-AMP) and adenosine 3'-monophosphate (3'-AMP) by a sulfotransferase. The sulfated products corresponding to CoA, 5'-AMP and 3'-AMP were identified using mass spectrometry. This is the first report that CoA can act as a sulfate acceptor in a sulfotransferase reaction.

Revalidation of recombinant aequorin as a light emission standard: Estimation of specific activity of Gaussia luciferase.

To validate the use of recombinant aequorin (reAequorin) as a light emission standard, the protein concentrations of highly purified reAequorin were determined by amino acid composition analysis, and the presence of active reAequorin was confirmed by the ratio of absorbance peak at 460 nm to that at 280 nm. The high correlation of the luminescence intensity with the protein concentration showed that reAequorin could be used for a light emission standard to study the luminescence properties of luciferases and to evaluate the detection sensitivity of luminometers. The specific activity of Gaussia luciferase with Imax was 7.5-fold higher than that of reAequorin and was calculated to be 3.8 × 1016 quanta/mg protein.

Video-Rate Bioluminescence Imaging of Degranulation of Mast Cells Attached to the Extracellular Matrix.

Degranulation refers to the secretion of inflammatory mediators, such as histamine, serotonin, and proteases, that are stored within the granules of mast cells and that trigger allergic reactions. The amount of these released mediators has been measured biochemically using cell mass. To investigate degranulation in living single cells, fluorescence microscopy has traditionally been used to observe the disappearance of granules and the appearance of these discharged granules within the plasma membrane by membrane fusion and the movement of granules inside the cells. Here, we developed a method of video-rate bioluminescence imaging to directly detect degranulation from a single mast cell by measuring luminescence activity derived from the enzymatic reaction between Gaussia luciferase (GLase) and its substrate coelenterazine. The neuropeptide Y (NPY), which was reported to colocalize with serotonin in the secretory granules, fused to GLase (NPY-GLase) was efficiently expressed in rat basophilic leukemia (RBL-2H3) cells, a mast-cell line, using a preferred human codon-optimized gene. Bioluminescence imaging analysis of RBL-2H3 cells expressing NPY-GLase and adhered on a glass-bottomed dish showed that the luminescence signals from the resting cells were negligible, while the luminescence signals of the secreted NPY-GLase were repeatedly detected after the addition of an antigen. In addition, this imaging method was applicable for observing degranulation in RBL-2H3 cells that adhered to the extracellular matrix (ECM). These results indicated that video-rate bioluminescence imaging using GLase will be a useful tool for detecting degranulation in single mast cells adhered to a variety of ECM proteins.

Slow luminescence kinetics of semi-synthetic aequorin: expression, purification and structure determination of cf3-aequorin.

cf3-Aequorin is one of the semi-synthetic aequorins that was produced by replacing 2-peroxycoelenterazine (CTZ-OOH) in native aequorin with a 2-peroxycoelenterazine analog, and it was prepared using the C2-modified trifluoromethyl analog of coelenterazine (cf3-CTZ) and the histidine-tagged apoaequorin expressed in Escherichia coli cells. The purified cf3-aequorin showed a slow luminescence pattern with half-decay time of maximum intensities of luminescence of 5.0 s. This is much longer than that of 0.9 s for native aequorin, and its luminescence capacity was estimated to be 72.8% of that of native aequorin. The crystal structure of cf3-aequorin was determined at 2.15 Å resolution. The light source of 2-peroxytrifluoromethylcoelenterazine (cf3-CTZ-OOH) was stabilized by the hydrogen-bonding interactions at the C2-peroxy moiety and the p-hydroxy moiety at the C6-phenyl group. In native aequorin, three water molecules contribute to stabilizing CTZ-OOH through hydrogen bonds. However, cf3-aequorin only contained one water molecule, and the trifluoromethyl moiety at the C2-benzyl group of cf3-CTZ-OOH interacted with the protein by van der Waals interactions. The slow luminescence kinetics of cf3-aequorin could be explained by slow conformational changes due to the bulkiness of the trifluoromethyl group, which might hinder the smooth cleavage of hydrogen bonds at the C2-peroxy moiety after the binding of Ca2+ to cf3-aequorin.

Single-step purification of recombinant Gaussia luciferase from serum-containing culture medium of mammalian cells.

A dihydrofolate reductase-deficient Chinese hamster ovary (CHO-K1/dhfr-) cell line stably expressing Gaussia luciferase with a histidine-tag sequence at the carboxyl terminus (GLase-His) was established. Recombinant GLase-His was purified from serum-containing culture medium by single-step Ni-chelate column chromatography in the presence of 2 M NaCl and 0.01% Tween 20. The protein yield of GLase-His with over 95% purity was 0.5 mg from 0.9 L of the cultured medium. The enzymatic properties of purified GLase-His were characterized. Interestingly, non-ionic detergent Tween 20 stabilized and stimulated GLase-His activity and its luminescence activity was stimulated 2-fold by the synergistic effect of 0.01% Tween 20 and 150 mM NaCl.

A fusion protein of the synthetic IgG-binding domain and aequorin: Expression and purification from E. coli cells and its application.

Aequorin is a Ca2+-binding photoprotein that is a complex of apoaequorin (apoAQ) and 2-peroxycoelenterazine. In this study, the fusion protein (ZZ-apoAQ) composed of the synthetic IgG-binding domain (ZZ domain) derived from Staphylococcus aureus protein A and apoAQ was expressed into the periplasmic space of Escherichia coli cells. ZZ-apoAQ was highly purified using Ni-chelate affinity chromatography followed by IgG affinity chromatography. ZZ-AQ was prepared from purified ZZ-apoAQ by incubation with coelenterazine and was characterized, including its luminescence properties. ZZ-AQ could be used as a reporter for detecting IgG and the measurable range of IgG coated on a 96-well plate was 1-1000 ng/mL.