MOLECULAR EVOLUTION OF THE Ca<sup>2+</sup>‐BINDING PHOTOPROTEINS OF THE HYDROZOA

Tsuji, Frederick I.; Ohmiya, Yoshihiro; Fagan, Thomas F.; Toh, Hiroyuki; Inouye, Satoshi

doi:10.1111/j.1751-1097.1995.tb08713.x

Cited by 69 publications

(28 citation statements)

References 55 publications

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“…To date primary sequences have been determined for several luciferases (Renilla (32), Oplophorus (24), Pleuromamma, and Gaussia luciferases) and for several Ca 2ϩ -regulated photoproteins (33,34), all of which catalyze the luminescent oxidation of the same substrate, coelenterazine (35). Despite the fact that all of them use the same substrate, it is very intriguing and possibly significant to find no sequence similarity among these bioluminescent proteins.…”

Section: Resultsmentioning

confidence: 99%

Cloning and Expression of cDNA for a Luciferase from the Marine Copepod Metridia longa

Markova

Gölz

Frank

et al. 2004

Journal of Biological Chemistry

141

133

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Metridia longa is a marine copepod from which a blue bioluminescence originates as a secretion from epidermal glands in response to various stimuli. We demonstrate that Metridia luciferase is specific for coelenterazine to produce blue light ( max ‫؍‬ 480 nm). Using an expression cDNA library and functional screening, we cloned and sequenced the cDNA encoding the Metridia luciferase. The cDNA is an 897-bp fragment with a 656-bp open reading frame, which encodes a 219-amino acid polypeptide with a molecular weight of 23,885. The polypeptide contains an N-terminal signal peptide of 17 amino acid residues for secretion. On expression of the Metridia luciferase gene in mammalian Chinese hamster ovary cells the luciferase is detected in the culture medium confirming the existence of a naturally occurring signal peptide for secretion in the cloned luciferase. The novel secreted luciferase was tested in a practical assay application in which the activity of A2a and NPY2 G-protein-coupled receptors was detected. These results clearly suggest that the secreted Metridia luciferase is well suited as a reporter for monitoring gene expression and, in particular, for the development of novel ultrahigh throughput screening technologies.Continuous monitoring of dynamic changes in gene expression from living cells in response to various stimuli provides important information about cell physiology. For this purpose bioluminescent and fluorescent reporters have been introduced as tools for sensitive and convenient monitoring of gene expression. A cDNA encoding a bioluminescent or fluorescent reporter such as a luciferase or green fluorescent protein (GFP) 1 is fused to the promoter region of the target gene, and the construct is transfected to mammalian cells. The gene expression is monitored simply by measuring light emitted through an enzymatic reaction or fluorescence. To date several bioluminescent proteins have been widely and successfully used for optical monitoring of gene expression in living cells: firefly luciferase (FL) (for review, see Refs. 1-3), bacterial luciferase (for review, see Refs. 4 -6), the Ca 2ϩ -regulated photoprotein aequorin (for review, see Refs. 7-9), Renilla luciferase (for review, see Refs. 10 and 11), and GFP (for review, see . Although time course studies of gene expression have been determined by real time imaging with these reporter genes, intracellular assays present difficulties for continuous measurements in some cases. For instance, it is not easy to keep an intracellular concentration of luciferin at a constant level in the case of application of firefly and Renilla luciferases. In the case of FL, additional methods may be required to facilitate the passage of the substrate across the cell membrane. In addition, the intensity and stability of the bioluminescent response of FL in living cells are reported to be affected by concentrations of ATP, luciferin, and luciferin-luciferase complex (15, 16). Despite the fact that the GFP possesses excellent properties as a reporter, it has some short...

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Section: Resultsmentioning

confidence: 99%

Cloning and Expression of cDNA for a Luciferase from the Marine Copepod Metridia longa

Markova

Gölz

Frank

et al. 2004

Journal of Biological Chemistry

141

133

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“…Photoproteins contain a pseudo-EFhand motif, which does not bind Ca 2ϩ (6,13). Its position relative to the three EF hands in the primary sequence, which defines a pattern similar to the two EF-hand pairs of calmodulin, has suggested that photoproteins have evolved from a calmodulin ancestor gene toward bioluminescence (24). As underlined above, some of the present findings may apply to calmodulin and other Ca 2ϩ sensors that rely on several EF hands exhibiting different affinities to transduce Ca 2ϩ stimuli into biological signals.…”

Section: Discussionmentioning

confidence: 99%

Calcium dependence of aequorin bioluminescence dissected by random mutagenesis

Tricoire¹,

Tsuzuki²,

Courjean³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Aequorin bioluminescence is emitted as a rapidly decaying flash upon calcium binding. Random mutagenesis and functional screening were used to isolate aequorin mutants showing slow decay rate of luminescence. Calcium sensitivity curves were shifted in all mutants, and an intrinsic link between calcium sensitivity and decay rate was suggested by the position of all mutations in or near EF-hand calcium-binding sites. From these results, a low calcium affinity was assigned to the N-terminal EF hand and a high affinity to the C-terminal EF-hand pair. In WT aequorin, the increase of the decay rate with calcium occurred at constant total photon yield and thus determined a corresponding increase of light intensity. Increase of the decay rate was underlain by variations of a fast and a slow component and required the contribution of all three EF hands. Conversely, analyses of double EF-hand mutants suggested that single EF hands are sufficient to trigger luminescence at a slow rate. Finally, a model postulating that proportions of a fast and a slow light-emitting state depend on calcium concentration adequately described the calcium dependence of aequorin bioluminescence. Our results suggest that variations of luminescence kinetics, which depend on three EF hands endowed with different calcium affinities, critically determine the amplitude of aequorin responses to biological calcium signals.EF hand ͉ kinetics ͉ luminescence ͉ photoprotein ͉ transduction T he photoprotein aequorin is a stable luciferase intermediate formed from the reaction of the protein apoaequorin (luciferase) and the substrate coelenterazine (luciferin), which emits light upon Ca 2ϩ binding (1-5). Aequorin contains three EF-hand Ca 2ϩ -binding sites (6-8) located close to its N (EF1) or C terminus (EF2,3 pair). Aequorin mutagenesis and crystal structure suggest that these three EF hands indeed bind Ca 2ϩ , but their individual contribution to luminescence is still a matter of debate (9-13).The steep increase of luminescence intensity with [Ca 2ϩ ] makes aequorin a useful reporter of intracellular calcium signals (14). The formation of aequorin is a slow process (2), whereas the luminescence reaction is very fast and proceeds to completion in the continuous presence of Ca 2ϩ . The aequorin response thus occurs as a flash that decays exponentially and whose onset rate does not depend on [Ca 2ϩ ] (15). It has been observed early that the decay rate of this response increases with [Ca 2ϩ ], whereas the total light emitted (light integral) remains relatively constant (15). This suggests that the increase of luminescence intensity with [Ca 2ϩ ] is determined by variations of the decay rate but not of the light integral. In other words, the shorter the duration of the flash (i.e., the faster the decay), the larger the amplitude of the response (i.e., light intensity). However, the relationships among the intensity, the decay rate, and the integral of bioluminescence and their links to EF-hand occupancy have not been clearly established.To analyze the co...

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“…Several Ca 2ϩ -binding photoproteins have been described, e.g., aequorin, obelin, mitrocomin, and clytin, and some of them have been used to measure [Ca 2ϩ ] (351,407). Because these photoproteins emit visible bioluminescence by an intramolecular reaction in the presence of Ca 2ϩ , they offer simplicity in terms of required instrumentation and are not affected by photobleaching due to excitation illumination.…”

Section: ؉ -Binding Photoproteinsmentioning

confidence: 99%

Measurement of Intracellular Calcium

Takahashi

Camacho

Lechleiter

et al. 1999

Physiological Reviews

674

511

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To a certain extent, all cellular, physiological, and pathological phenomena that occur in cells are accompanied by ionic changes. The development of techniques allowing the measurement of such ion activities has contributed substantially to our understanding of normal and abnormal cellular function. Digital video microscopy, confocal laser scanning microscopy, and more recently multiphoton microscopy have allowed the precise spatial analysis of intracellular ion activity at the subcellular level in addition to measurement of its concentration. It is well known that Ca2+ regulates numerous physiological cellular phenomena as a second messenger as well as triggering pathological events such as cell injury and death. A number of methods have been developed to measure intracellular Ca2+. In this review, we summarize the advantages and pitfalls of a variety of Ca2+ indicators used in both optical and nonoptical techniques employed for measuring intracellular Ca2+ concentration.

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

Cited by 69 publications

References 55 publications

Cloning and Expression of cDNA for a Luciferase from the Marine Copepod Metridia longa

Cloning and Expression of cDNA for a Luciferase from the Marine Copepod Metridia longa

Calcium dependence of aequorin bioluminescence dissected by random mutagenesis

Measurement of Intracellular Calcium

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

Cited by 69 publications

References 55 publications

Cloning and Expression of cDNA for a Luciferase from the Marine Copepod Metridia longa

Cloning and Expression of cDNA for a Luciferase from the Marine Copepod Metridia longa

Calcium dependence of aequorin bioluminescence dissected by random mutagenesis

Measurement of Intracellular Calcium

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