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

Abstract

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 (λmax = 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. 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 (λmax = 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) 1The abbreviations used are: GFPgreen fluorescent proteinFLfirefly luciferaseCHOChinese hamster ovaryMLucMetridia luciferaseIPTGisopropyl-β-d-thiogalactopyranosideNPY2human neuropeptide Y receptor 2TricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. 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. 1Millar A.J. Short S.R. Chua N.H. Kay S.A. Plant Cell. 1992; 4: 1075-1087Google Scholar, 2Ando S. De Amicis F. Rago V. Carpino A. Maggiolini M. Panno M. Lanzino M. Mol. Cell. Endocrinol. 2002; 193: 121-128Google Scholar, 3Hough R.B. Avivi A. Davis J. Joel A. Nevo E. Piatigorsky J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8145-8150Google Scholar), bacterial luciferase (for review, see Refs. 4Kondo T. Strayer C.A. Kulkarni R.D. Taylor W. Ishiura M. Golden S.S. Johnson C.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5672-5676Google Scholar, 5Liu Y. Abaibou H. Fletcher H.M. Plasmid. 2000; 44: 250-261Google Scholar, 6Yoon K. Pellaroni L. Ramamoorthy K. Gaido K. Safe S. Mol. Cell. Endocrinol. 2002; 162: 211-220Google Scholar), the Ca2+-regulated photoprotein aequorin (for review, see Refs. 7Johnson C.H. Knight M.R. Kondo T. Masson P. Sedbrook J. Haley A. Trewavas A. Science. 1995; 269: 1863-1865Google Scholar, 8Langer I. Perret J. Vertongen P. Waelbroeck M. Robberecht P. Cell Calcium. 2001; 30: 229-234Google Scholar, 9Radford J.C. Davies S.A. Dow J.A. J. Biol. Chem. 2002; 277: 38810-38817Google Scholar), Renilla luciferase (for review, see Refs. 10Broussard D.R. Mertz J.A. Lozano M. Dudley J.P. J. Virol. 2002; 76: 2087-2099Google Scholar and 11Bhaumik S. Gambhir S.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 377-382Google Scholar), and GFP (for review, see Refs. 12Chalfie M. Tu Y. Euskirchen G. Ward W.W. Prasher D.C. Science. 1994; 263: 802-805Google Scholar, 13Ronen M. Rosenberg R. Shraiman B.I. Alon U. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10555-10560Google Scholar, 14Kostrouchova M. Housa D. Kostrouch Z. Saudek V. Rall J.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9254-9259Google Scholar). 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 (15Gandelman O. Allue I. Bowers K. Cobbold P. J. Biolumin. Chemilumin. 1994; 9: 363-371Google Scholar, 16Lembert N. Idahl L.A. Biochem. J. 1995; 305: 929-933Google Scholar). Despite the fact that the GFP possesses excellent properties as a reporter, it has some shortcomings (toxicity for living cells (for review, see Ref. 17Duisit G. Conrath H. Saleun S. Folliot S. Provost N. Cosset F. Sandrin V. Moullier P. Rolling F. Mol. Ther. 2002; 6: 446-454Google Scholar), slow maturation of chromophore (for review, see Ref. 18Bevis B.J. Glick B.S. Nat. Biotechnol. 2002; 20: 83-87Google Scholar), and the long half-life of the protein in mammalian cells (for review, see Ref. 19Bi J.X. Wirth M. Beer C. Kim E.J. Gu M.B. Zeng A.P. J. Biotechnol. 2002; 93: 231-242Google Scholar) that limit its application. Although the Ca2+-regulated photoproteins provide high sensitivity for monitoring gene expression (20Yoshida R. Sanchez-Bueno A. Tsuji F.I. Biochem. Biophys. Res. Commun. 1998; 242: 659-662Google Scholar) they have one disadvantage. To measure the photoprotein activity the synthesized apophotoprotein needs to be charged with coelenterazine substrate over periods of several h, and this delays answering the question about gene activity. To circumvent these limitations and difficulties, fusion proteins of luciferases and of GFP were created possessing sequences that induce secretion of the protein into the culture medium (21Liu J. Wang Y. Szalay A.A. Escher A. Luminescence. 2000; 15: 45-49Google Scholar, 22Liu S. Bugos R.C. Dharmasiri N. Su W.W. J. Biotechnol. 2001; 87: 1-16Google Scholar). The main advantage of using a secreted reporter protein is that it is possible to measure gene expression extracellularly without destroying the cells or tissues. This is particularly useful for time course studies of transcription activity or receptor/channel response kinetics. green fluorescent protein firefly luciferase Chinese hamster ovary Metridia luciferase isopropyl-β-d-thiogalactopyranoside human neuropeptide Y receptor 2 N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. There are bioluminescent organisms with naturally occurring secreted luciferases, and the cDNAs encoding some of them have been cloned. These include the marine ostracod Vargula hilgendorfii (23Thompson E.M. Nagata S. Tsuji F.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6567-6571Google Scholar), the deep sea shrimp Oplophorus gracilorostris (24Inouye S. Watanabe K. Nakamura H. Shimomura O. FEBS Lett. 2000; 481: 19-25Google Scholar), and the marine copepod Gaussia princeps (GenBank™/EBI accession number AY015993). The Vargula and Oplophorus luciferases have been expressed in mammalian cells (23Thompson E.M. Nagata S. Tsuji F.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6567-6571Google Scholar, 24Inouye S. Watanabe K. Nakamura H. Shimomura O. FEBS Lett. 2000; 481: 19-25Google Scholar, 25Thompson E.M. Nagata S. Tsuji F.I. Gene (Amst.). 1990; 96: 257-262Google Scholar, 26Inouye S. Ohmiya Y. Toya Y. Tsuji F.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9584-9587Google Scholar, 27Thompson E.M. Adenot P. Tsuji F.I. Renard J.-P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1317-1321Google Scholar, 28Tanahashi Y. Ohmiya Y. Honma S. Katsuno Y. Ohta H. Nakamura H. Honma K. Anal. Biochem. 2001; 289: 260-266Google Scholar), and the Vargula luciferase has shown promising results. For instance, in Chinese hamster ovary (CHO) cells transfected with a plasmid bearing the cDNA for Vargula luciferase it was demonstrated that the secretory process could be monitored in real time from individual cells (26Inouye S. Ohmiya Y. Toya Y. Tsuji F.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9584-9587Google Scholar). The attractiveness of secreted luciferases as reporters is a strong stimulus for the investigation and exploitation of new bioluminescent systems. Metridia longa is a small (∼1.5 mm) luminous marine copepod. The bioluminescence originates as a secretion from epidermal glands located in the head part and abdomen in response to mechanical, electrical, or chemical stimuli (29Evstigneev P.V. Bitukov E.P. Greze, B.N. Bioluminescence of Marine Copepods. Naukova Dumka, Kiev, Ukraine1990Google Scholar). Bioluminescence in M. longa may well serve as a defense mechanism against predators; the release of a luminous bolus from the animal is accompanied by rapid swimming that displaces the copepod away from its “glowing phantom.” Although the bioluminescence of M. longa was described many years ago (30Clarke G.L. Conover R.J. David C.N. Nicol J.A. J. Mar. Biolog. Assoc. U. K. 1962; 42: 541-564Google Scholar) no studies concerning the properties of the bioluminescent system were carried out until now. The present study provides information on the substrate specificity of Metridia luciferase (MLuc) and reports cloning, nucleotide sequence analysis of the cDNA encoding Metridia luciferase, expression of MLuc cDNA in Escherichia coli and mammalian cells, and verification of the suitability of MLuc for use as a reporter for monitoring gene expression. Collection of the Copepod M. longa—We collected the animals in Chupa Bay in the region of the White Sea biological station Kartesh from aboard the research vessel “Professor Kuznetsov” using a shutting plankton net at a depth of 100–50 m where this species dominates. Plankton were sorted with a transfer pipette under a stereomicroscope, and the M. longa specimens at stages IV–VI of development (0.3–1.5 mm in size) were transferred into a tank containing continuously aerated seawater at 4 °C, which was the temperature of the natural habitat of the copepod. The accumulated animals were filtered and quickly frozen with liquid nitrogen. Determination of Metridia Luciferase Substrate—For studies of the substrate specificity of Metridia luciferase about 30 freshly collected copepods were homogenized in 2 ml of 50 mm Hepes pH 7.2 at 0 °C. The suspension was centrifuged, the pellet was discarded, and the supernatant immediately was used for measurements. The was by of of coelenterazine or Vargula luciferin in into ml of the supernatant of M. longa of the cDNA and was from frozen copepods by the P. N. Anal. Biochem. 162: Scholar). was on and of this was to The expression cDNA library was in a with the cDNA library to the with the The was with about The and bacterial culture was carried out to the for the cDNA library For screening the a cDNA library was at of the was by a with to the The was to a and on the for the to The were until a bacterial with clearly the bacterial with was the with a into medium with mm and The suspension was into a culture and gene expression was with for at °C. The cell pellet from ml of suspension was in ml of m mm 50 mm pH at 0 °C. The cell was for bioluminescence activity with coelenterazine The from a was into that were as the individual from the were and for bioluminescence activity For the analysis of the the were to the plasmid in the E. coli to the with the cDNA library The cDNA was with the was used as a The was used for sequence and of Metridia Luciferase in E. of the MLuc sequence a for bacterial expression the of the protein To it we in the nucleotide sequence with amino acid sequence using a The plasmid with the luciferase gene was used as the for natural secreted proteins signal peptide secretion. is to that this secreted proteins are used as reporters in mammalian cells. In that the properties of the reporter protein without the signal peptide to be the results are to be The cDNA encoding the part of the luciferase gene without signal peptide was with a high to the For this we used the MLuc gene of the of as The of 50 of with of The fragment was cloned in the reading into the expression in The new for were by of the The was introduced into the and the was introduced into the The was with and by and into a and expression The plasmid was verification of the nucleotide the plasmid was introduced into E. coli for expression of luciferase. For protein the E. coli was with at in medium containing and with mm the culture an of of the was for For activity measurements on the expressed E. coli cells were by in ml of the m mm 50 mm pH and at 0 °C. The bioluminescence was by rapid of of coelenterazine in into a cell containing ml of cell in at The bioluminescence were with an were with the with the of MLuc and FL in and a and a were used for expression. The and were by the Metridia cDNA sequence from into the of and To produce the the FL (GenBank™/EBI accession number sequence was from the plasmid in the cells were used for and for The of cells with was with to the The plasmid was with for The transfected cells were in medium with 2 The were by of the medium for MLuc or cell for of Metridia cells were transfected with or and were in medium with The MLuc and FL were at time of with of The were from or The were transferred to of coelenterazine in or of FL assay concentrations in the firefly luciferin, of ATP, mm mm 30 and mm were The bioluminescence was by over a time of 30 with a To the intracellular MLuc and FL cells were and with The assay was by 50 of the cell in or with of coelenterazine in or with of the FL assay The bioluminescence was as described of A2a and NPY2 G-protein-coupled and were by the human receptor A2a cDNA into the and the human neuropeptide Y receptor NPY2 cDNA into the of The cDNAs encoding A2a (GenBank™/EBI accession number and NPY2 (GenBank™/EBI accession number were cloned from human using and were expressed in cells. is a cell containing the The cells plasmid were with for 4 The cells with were by neuropeptide Y for 4 in the of In these was by to of Metridia substrate specificity of Metridia luciferase was with a of The of Vargula luciferin to a of copepods not produce detected the of coelenterazine a of light with of and intensity on the of not A of coelenterazine to a new The bioluminescence of the is blue with at 480 The of the light emitted by the well with the bioluminescence from the copepods on of a cDNA and M. longa cDNA library about been several activity were The cDNA is an 897-bp fragment with a region from the The open reading contains which a 219-amino acid polypeptide with of 23,885. the is a region by a MLuc encodes for This number of residues that cloned luciferase may which may be for bioluminescent activity or for a protein luciferases that use coelenterazine as a substrate or the Ca2+-regulated photoproteins W.W. M. Proc. Natl. Acad. Sci. U. S. A. Scholar, F.I. Ohmiya Y. H. S. 1995; Scholar, Wang J. 2002; Scholar), Metridia luciferase has and A of the protein with the and the MLuc sequence as one the luciferase from G. The of of the Gaussia luciferase sequence to the MLuc sequence is and for nucleotide and amino acid the copepods G. princeps and M. longa to the this could for the the Gaussia and Metridia luciferase In the luciferase cloned from a of the of the (GenBank™/EBI accession number not the Metridia and Gaussia luciferases. This that is a of high of of luciferases the luminous the luminous species of which are in various and (29Evstigneev P.V. Bitukov E.P. Greze, B.N. Bioluminescence of Marine Copepods. Naukova Dumka, Kiev, Ukraine1990Google Scholar). To date sequences have been determined for several luciferases W.W. M. Proc. Natl. Acad. Sci. U. S. A. Scholar), Oplophorus (24Inouye S. Watanabe K. Nakamura H. Shimomura O. FEBS Lett. 2000; 481: 19-25Google Scholar), and Gaussia and for several Ca2+-regulated photoproteins F.I. Ohmiya Y. H. S. 1995; Scholar, Wang J. 2002; Scholar), of which the of the coelenterazine Mar. Biol. 1990; Scholar). Despite the fact that of them use the it is and to no sequence these bioluminescent are the of Metridia luciferase and Vargula luciferase (23Thompson E.M. Nagata S. Tsuji F.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6567-6571Google Scholar) that the of a A to the and of signal peptide in protein sequences the existence of an N-terminal signal peptide of 17 amino with the with to the cloned cDNA encodes the secreted luciferase, and this is in with the of secreted The Metridia luciferase sequence analysis the of of residues high This suggest that the MLuc gene could have by of one the sequence were in the MLuc The MLuc nucleotide sequence analysis that the sequence is an sequence that is to the from the protein and that is used in expression This of the with the of the For bacterial expression the of was from the sequence with amino acid sequence to the of additional To cDNA encoding Metridia luciferase, we the expression that we used for the cDNAs encoding Ca2+-regulated photoproteins from 1992; Scholar) and Wang J. 2002; Scholar). This has and The of expression is the it is in cDNAs for proteins of or activity In addition, the in assay has to be The advantage of expression the protein of of one polypeptide and be with high specificity and the expression be with high This of cDNA for proteins an in assay needs to be advantage is that the cDNA encoding a functional protein is and this some on application of The cDNAs for the of luciferases and from may be for the expression in E. coli and of Metridia analysis of the E. coli cells the plasmid an additional protein with a molecular to that for Metridia luciferase cell from cells activity with coelenterazine in with the of protein in these cells. of the synthesized protein is accumulated in For of luciferase the cell was in mm pH and with at 0 °C. The was centrifuged, and the supernatant was The pellet was with and mm pH the were with at 4 °C. to measurements of the of MLuc the was no The pellet was in m for at 4 and The m of the The m was on a with mm pH The proteins were with a m in the this the of luciferase with protein The bioluminescent from the reaction with high MLuc has a at that is in with that for luciferase (λmax = 480 This clearly that Metridia luciferase be from the of MLuc and FL in cell MLuc and FL were to the secreted and were transfected with or in cell or cell cells transfected with these or in with cells were This clearly that Metridia luciferase is for mammalian cells. cell MLuc and FL were in cell and in the The cells FL activity with the firefly luciferin in the cell The cells MLuc luciferase activity with coelenterazine in the cell and in the The transfected cells secreted luciferase and it accumulated in the To the time course of the secretion the medium was the cells were with and medium was at the of the time course The MLuc luciferase activity be detected the medium is The cells transfected with without cDNA no luciferase activity with These results that the cloned cDNA encodes secreted luciferase. of of A2a and NPY2 G-protein-coupled with MLuc as a receptors a of proteins that to a of and chemical stimuli G.L. N. 1989; Scholar). the receptors have been successfully introduced for To that the activity of G-protein-coupled it may be to use ultrahigh throughput screening to chemical The secreted Metridia luciferase has for development of ultrahigh throughput screening the of this novel luciferase for monitoring expression of encoding the A2a and NPY2 receptors to the G-protein-coupled receptors The receptor A2a is a receptor that the intracellular concentration through of The receptor NPY2 is a receptor that the intracellular concentration through of The and were transfected into cells MLuc under the of a of cell the encoding receptors were with the for 4 h, and MLuc activity was in the culture medium The medium of the A2a cells by a high MLuc bioluminescent The medium of the A2a cells that were not with a level of bioluminescence to that for the medium from cells transfected with the The MLuc activity determined in the medium of the NPY2 cells with is for the medium of the cells by neuropeptide Y. The of the NPY2 receptor results in a of the MLuc transcription activity provides important information about cell The application of secreted bioluminescent reporters is a for this monitoring it of gene expression extracellularly without destroying the cells or tissues. occurring secreted luciferases are for this and this investigation of new bioluminescent systems. To date the cDNAs encoding several secreted luciferases have been cloned (23Thompson E.M. Nagata S. Tsuji F.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6567-6571Google Scholar, 24Inouye S. Watanabe K. Nakamura H. Shimomura O. FEBS Lett. 2000; 481: 19-25Google Scholar). Vargula luciferase has been expressed in mammalian cells and has shown promising properties as a reporter E.M. Nagata S. Tsuji F.I. Gene (Amst.). 1990; 96: 257-262Google Scholar, 26Inouye S. Ohmiya Y. Toya Y. Tsuji F.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9584-9587Google Scholar, 27Thompson E.M. Adenot P. Tsuji F.I. Renard J.-P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1317-1321Google Scholar, 28Tanahashi Y. Ohmiya Y. Honma S. Katsuno Y. Ohta H. Nakamura H. Honma K. Anal. Biochem. 2001; 289: 260-266Google Scholar). its the of Vargula luciferase as a secreted luciferase tested as a reporter is from the deep sea shrimp O. gracilorostris (24Inouye S. Watanabe K. Nakamura H. Shimomura O. FEBS Lett. 2000; 481: 19-25Google Scholar). This luciferase is a complex of and and the one the of The cDNA encoding the protein was expressed in cells, and the sequence a natural signal for secretion at the the culture medium activity. The luciferase from the marine copepod G. princeps possesses a naturally occurring signal for are no studies of it as a reporter in mammalian cells. The cloned secreted Metridia luciferase possesses promising properties as a reporter the bioluminescent reaction by this luciferase is coelenterazine and molecular and specific in the that coelenterazine not with mammalian proteins to produce the assay is sensitive light be detected with high sensitivity by The molecular of Metridia luciferase for Vargula luciferase, for is an these properties clearly suggest that Metridia luciferase may application for studies and in for the development of novel ultrahigh throughput screening technologies. We and for the of a cDNA We and for and on the