Fabrication of a New Lineage of Artificial Luciferases from Natural

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Fabrication of a New Lineage of Artificial Luciferases from Natural Luciferase Pools Sung Bae Kim,*,† Ryo Nishihara,‡ Daniel Citterio,‡ and Koji Suzuki‡ †

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Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan ‡ Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan S Supporting Information *

ABSTRACT: The fabrication of artificial luciferases (ALucs) with unique optical properties has a fundamental impact on bioassays and molecular imaging. In this study, we developed a new lineage of ALucs with unique substrate preferences by extracting consensus amino acids from the alignment of 25 copepod luciferase sequences available in natural luciferase pools. The primary sequence was first created with a sequence logo generator resulting in a total of 11 sibling sequences. Phylogenetic analysis shows that the newly fabricated ALucs form an independent branch, genetically isolated from the natural luciferases, and from a prior series of ALucs produced by our laboratory using a smaller basis set. The new lineage of ALucs were strongly luminescent in living mammalian cells with specific substrate selectivity to native coelenterazine. A single-residue-level comparison of the C-terminal sequences of new ALucs reveals that some amino acids in the C-terminal ends are greatly influential on the optical intensities but limited in the color variance. The success of this approach guides on how to engineer and functionalize marine luciferases for bioluminescence imaging and assays. KEYWORDS: artificial luciferase, bioluminescence, frequency analysis, coelenterazine, copepod luciferase

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INTRODUCTION Natural beetle and marine luciferases have been established from a large variety of insects, marine organisms, and prokaryotes.1,2 Conventionally, the establishment of new luciferases from light-emitting organisms in nature has been considered as the only method to expand the reporter pool. These are of course the result of Darwinian evolution3 under selection pressures that are not well suited to the needs of biomolecular imaging.4 Natural luciferases are therefore generally poor in optical intensities and stability, shortcomings that have been addressed by directed evolution in the laboratory.5 However, such random mutagenesis approaches are often slow and tedious, and crystallographic information on natural beetle and marine luciferases, which is essential for sitedirected mutagenesis, is rare.6 As an alternative approach, we recently showed a new method of creating artificial luciferases (ALucs) by extracting the consensus amino acids from the sequences of natural copepod luciferases available in the National Center for Biotechnology Information (NCBI) database.7 This strategy is based on the premise that the frequently occurring amino acids at a given position have a larger thermostabilizing effect compared with less frequent amino acids. This approach was originally developed for finding effective mutation sites and thus is called “consensus sequence-driven mutagenesis strategy” (CSMS).8,9 We applied the basic concept to the fabrication of © 2017 American Chemical Society

whole sequences of consensus amino acids, and here we extend the approach to a larger collection of 25 copepod luciferase homologues.10

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RESULTS AND DISCUSSION As shown in Figure 1, two primary sequences, ALuc41 and ALuc42, were created by extracting frequently occurring amino acids from the alignment of 25 copepod luciferase sequences (Figure 1C and D) with the help of the Web software WebLogo, version 2.8.2 (http://weblogo.berkeley.edu/logo. cgi).11 The newly fabricated ALucs were solely derived from the 25 new copepod sequences in the public database; the sequences of our previously developed ALucs7 were not included. As the primary sequence ALuc41 consists of variable N-terminal and two conserved domains, it was aligned in three rows. The aligned sequence was further modified: (i) to increase cases of the consensus amino acids between the N- and C-terminal domains because we empirically know that the higher homology allows the stronger bioluminescence intensities7,12 and (ii) to introduce multiply mutated amino acids at the C-terminal end of ALuc41 for generating the sibling sequences (ALuc43−ALuc51). The new artificial sequences Received: May 16, 2017 Revised: July 11, 2017 Published: July 25, 2017 594

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Figure 1. Fabrication of a primary sequence of new artificial luciferases from an alignment of natural copepod luciferases in public databases (NCBI BLAST and SIB BLAST). (A) The frequently occurring amino acids were extracted from the alignment with the help of the Web software WebLogo, version 2.8.2 (http://weblogo.berkeley.edu/logo.cgi).11 (B) An unrooted phylogenetic tree according to CLUSTALW, version 2.1. The blue and red circles mark the relative positions of existing and new artificial luciferases, respectively. Inset a shows the identity ranking of existing marine luciferases compared with ALuc41. The identity ranking was determined with the program BLASTP 2.5.1+ in the NCBI BLAST. (C) The relative optical intensities of ALuc41−45 compared with conventional luciferases (n = 5). The optical image in pseudocolor was developed with nCTZ.

Figure 2. Significance of the C-terminal end of ALuc41−51, promoted by the super 2-dimensional molecular structure of ALuc30. (A) A super 2dimensional molecular structure of ALuc30. Inset a shows the C-terminal end of ALuc30; inset b aligns the C-terminal sequences of ALuc41−51. (B) The bioluminescence image of ALuc41−51 compared with conventional marine luciferases. The alphabets in white under the optical image mean amino acids mutated in the sequence. Inset a shows the absolute optical intensities normalized by integration time (s) and light-emitting area (mm2). The percentages in the bar graphs denote the remaining optical intensities after 5 min (n = 3).

were found to be considerably different from those of any existing marine luciferases: For example, the closest sequence was that of Pleuromamma xiphias, the maximal identity of which was approximately 77% of ALuc41 according to the program BLASTP 2.5.1+ in the NCBI BLAST (Figure 1B, inset a). The phylogenetic tree showed that the newly fabricated artificial luciferase 40 (ALuc40) series formed an independent branch,

which was isolated even from precedent ALuc series (Figure 1B). ALuc40s Survive in Mammalian Cells and Emit Strong Bioluminescence. Each new ALuc sequence tagged with an ER retention signal (KDEL) was expressed in COS-7 cells using the same mammalian expression plasmid. The relative optical intensities were evaluated in the presence of Promega’s assay buffer dissolving 0.1 mM native coelenterazine (nCTZ) 595

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Figure 3. Substrate-driven properties of the bioluminescence spectra of ALucs. (A) The peak heights of the bioluminescence spectra of various luciferases according to nCTZ (blue bars) and CTZh (red bars). The numbers on the bars indicate the fold intensities of each luciferase with nCTZ compared with CTZh. The red asterisk “*” denotes the maximal fold intensity difference with nCTZ and CTZh. The yellow shaded area shows the spectral heights of previously reported ALucs. Inset a presents a comparison of the chemical structures of nCTZ and CTZh. (B) Comparison of the bioluminescence spectra of ALuc16, -42, and -47 according to nCTZ (dotted line) and CTZh (solid line). The pink shaded area indicates wavelengths longer than 600 nm.

Figure 4. (A) Live cell bioluminescence imaging with conventional and newly established ALucs. The optical image was taken with a CCD camera equipped with the LAS-4000 system (Fujifilm) after simultaneous injection of furimazine, nCTZ, or 6piOH-CTZ with the use of a multichannel micropipette. Inset a shows the optical profile of the microslide containing COS-7 cells after the addition of nCTZ. (B) The absolute optical intensities of the live cells on the microslides after the addition of furimazine, nCTZ, or 6piOH-CTZ.

directly in cell lysates without correction for protein expression levels (Figure 1C). Thus, this assay identifies the best candidates for practical use under these standard bioassay conditions, not necessarily the most active among the isolated luciferase variants. The results showed the new sequences to mediate the production of stronger optical signals than the conventional marine luciferases used for comparison, GLuc and RLuc8.6-535, which were chosen because they are the most frequently used marine luciferases among researchers. For example, the optical signal from ALuc45 was 6.2-fold (±1.2 s.d.) more intense than from GLuc. Differences among the Artificial Luciferases Were Revealing. As shown in Figure 1C, ALuc43 and ALuc45 gave approximately 7- and 15-fold stronger bioluminescence than ALuc41, respectively. Only one amino acid distinguishes ALuc41 (187T) from ALuc 45 (187N), and only one amino acid differentiates ALuc43 (183T) and ALuc44 (185Q) from

ALuc41 (183K, 185V). Assuming that luciferase expression is comparable in these tests, which seems likely given the modest differences in sequence, these dramatic differences in light intensity suggests that positions 183, 185, and 187 at the Cterminal end of ALuc41 are key sites for enzymatic activity. C-Terminal End of ALuc41 Dominates the Optical Intensities. The supersecondary structure of ALuc30 based on X-ray crystal structure of the coelenterazine-binding protein (CBP; PDB accession numbers 2hps and 2hq8)12 shows the Cterminal end to be close to the C6 position of the bound lightgenerating substrate nCTZ (Figure 2A). This, along with the sequence comparisons discussed above, suggested that further modifications of C-terminal domain residues could be informative (Figure 2). Additional derivatives of ALuc45 were created by single mutations among residues 183−192 (Figure 2Ab). Among these candidates, the three brightest appeared in the intensity 596

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ACS Combinatorial Science order ALuc49 ≈ ALuc50 > ALuc45 (Figure 2B). The optical stabilities of the new ALucs were examined by comparing the optical intensities at 5 min after nCTZ injection with those observed immediately after nCTZ injection (Figure 2B, inset a). ALuc51 and ALuc48 maintained 49% and 40% of their initial optical intensities after 5 min. GLuc showed the poorest optical stability of 16% in the same experimental conditions. Substrate Selectivity. The relative substrate preference of the new lineage of ALucs was determined with the use of various coelenterazine analogues (Figure 3, Supporting Figure 2). Most analogues were not accommodated by these luciferase variants, except nCTZ (Supporting Figure 2). The previously reported ALuc16 and ALuc30 were able to use both nCTZ and CTZh, whereas the new lineage of ALucs presented a high preference for nCTZ (Figure 3). The most biased optical intensities were observed with ALuc47; that is, the intensity of the nCTZ−ALuc47 pair was found to be 217-fold brighter than that of CTZh−ALuc47. This type of substrate preference may reflect changes in the binding or turnover properties of the new ALucs or may reflect established preferences of copepod enzymes, as the relative brightness of the natural enzymes with various CTZ analogues has not been established. The emission wavelengths of the new luciferases were unchanged, remaining in the 487−500 nm range (Figure 3B). In the case of ALuc42, the percentage of red light emitted (longer than 600 nm) was approximately 4% of the total. Substitution of T187 with N (ALuc45), Y (ALuc49), F (ALuc50), or W (ALuc51) gave rise to no change in emission wavelength, in spite of the additional opportunity provided by these mutations to create additional π−π stacking or hydrogen bonds with the substrate. Live Cell Bioluminescence Imaging with Conventional and Newly Established ALucs. The live cell images of conventional and newly established ALucs were determined in COS-7 cells grown in 6-channel microslides (Figure 4). NanoLuc and RLuc8.6-535 have been previously reported as the brightest among marine luciferase in live cells.9,13,14 Thus, their live cell images were compared with those of ALuc49 (Figure 4A). The results showed the expected substrate selectivity: furimazine and 6piOH-CTZ selectively illuminated NanoLuc and RLuc8.6-535, respectively, whereas nCTZ dominantly illuminated ALuc49 in live cells. Molecular Tension Probes. The useful features of the new lineage of ALucs were shown by applying the ALucs to a singlechain bioluminescent probe, referred to as a “bioluminescent capsule”, where a full-length ALuc is initially designed to be fixed at the plasma membrane (Supporting Figure 3). Two such probes, designated P47 and P49 to reflect their use of ALuc47 and ALuc49, respectively, were created. The addition of the apoptosis inducer staurosporine (STS) increased the optical intensities of P47 and P49 by 50% and 28%, respectively. This is attributed to the caspase-induced release of full-length ALucs from the capsules. The free ALucs are brighter than ALucs fixed in the plasma membrane.

nCTZ, which contains a para-hydroxyphenyl group at the C-2 position of the imidazolone ring, over CTZh, which is missing the hydroxyl group at that site (Figure 3). These variants do not accommodate iodine or fluorine at this position. These observations suggest that the small variations in amino acid composition in the new ALucs are localized at the luciferase binding site. In contrast, the active site of the old lineage of ALucs (ALuc16−34) is more promiscuous concerning functional groups at the C-2 imidazoline benzyl group; for example, ALuc30 activates CTZh, CTZf, and CTZi with high selectivity.12 The varying substrate preferences for marine luciferases open exciting possibilities for multiplexed assay systems, where the optical signals may be pinpointed by adding specific substrates. The success of this approach is doubtless due to the highly conserved nature of the copepod luciferases and, for such families, highlights the potential value of focusing on the degrees of conservation at the single-residue level.

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EXPERIMENTAL PROCEDURES Design of ALucs by Extracting Frequently Occurring Amino Acids from the Alignment of Copepod Luciferases. Twenty-five sequences of marine planktonic copepod luciferases were collected from the literature,10,15 transformed into FASTA format (text-based single-letter amino acid codes), and aligned to determine amino acid frequency with the Web software WebLogo, version 2.8.2 (http://weblogo.berkeley. edu/logo.cgi).11 The initially suggested primary sequence was named “ALuc41”. ALuc41 contains two consecutive amino acids in the sequence of ALuc41 were further modified by embedding a His-tag and elongating the highly variable region in the N-terminal side, which was named “ALuc42.” A series of sibling sequences of ALucs was further fabricated from the prototypical sequence by substituting several amino acids in the C-terminal region with new ones (Supporting Figure 1). Lastly, the endoplasmic reticulum (ER) retention signal KDEL was added at the C-terminal end of each new sequence instead of the N-terminal secretion peptide common to copepod luciferases. The average length and molecular weight (MW) of the newly made artificial sequences were 195.7 (±5.7 s.d.) AAs and 21.2 (±0.7 s.d.) kD, respectively. The average theoretical isoelectric point (pI) was 5.7 (±0.5 s.d.). Synthesis of cDNA Constructs Encoding the New Lineage of Artificial Luciferases. The murine codonoptimized cDNA constructs encoding the artificially designed amino acid sequences (ALuc41−51) were custom-synthesized on order by Eurofins Genomics (Tokyo, Japan) (Supporting Figure 1A). The synthesized cDNAs were subcloned into pcDNA3.1(+) (Invitrogen) by using the specific restriction sites HindIII and XhoI for expression in mammalian cells. For the comparison studies, cDNA encoding GLuc or RLuc8.6-535 were subcloned intot the same pcDNA3.1(+) vector. In case of the cDNA encoding GLuc, the 3′-terminal were tagged with an oligomer encoding “KDEL” for the intracellular retention. The overall sequence fidelity was confirmed with the use of a sequencing service provided by Eurofins Genomics (Tokyo, Japan). Determination of the Sequential Identity Ranking and Phylogenetic Trees of the New Lineage of Artificial Luciferases. The rooted and unrooted phylogenetic trees of the artificially designed ALucs and existing marine luciferases were determined by using CLUSTALW, version 2.1 (Figure 1B; Supporting Figure 1B). The maximal identities and similarity rankings of the new ALucs were also compared

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CONCLUSIONS We have previously created a series of artificial luciferases by extracting the frequently occurring amino acids in a bundle of aligned copepod luciferase sequences.7 Using a larger data pool, we created here a new lineage of ALucs that is phylogenetically distinctive from any existing luciferases, including previously reported ALucs, as shown in Figure 1B. In addition to brighter output of light, the new ALucs are selective in their use of 597

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Live Cell Bioluminescence Imaging with Marine Luciferases. The live cell images and optical profiles of NanoLuc (Promega), RLuc8.6-535, and ALuc49 were determined with the use of the COS-7 cells grown in 6channel microslides (μ-Slide VI0.4; ibidi) (Figure 4). The COS-7 cells in 6-channel microslides were transiently transfected with the pcDNA 3.1(+) vector encoding NonoLuc, RLuc8.6-535, or ALuc49. Sixteen hours after the incubation, the cells in each channel were rinsed once with HBSS buffer (Gibco) and simultaneously bathed with 60 μL of furimazine, 6piOH-CTZ, or nCTZ dissolved in HBSS buffer (final concentration = 10−4 M) by using a multichannel micropipette (Gilson). The microslides were immediately set in the dark chamber of the LAS-4000 image analyzer (Fujifilm), and the corresponding optical intensities from the microslides were integrated for 20 s. The optical intensities and profiles from the living COS-7 cells in the microslide were normalized by measured time (sec) and area (mm2); the unit was RLU/sec/ mm2, and the specific image analysis software Multi Gauge, version 3.1 (Fujifilm), was used. Construction of Single-Chain Bioluminescent Probes with ALucs. The advantages of using the newly fabricated ALucs in bioassays were determined with the use of singlechain bioluminescent probes (bioluminescent capsules) carrying full-length ALuc47 or ALuc49 (Supporting Figure 3). The corresponding probes were named P47 and P49, respectively. The working mechanism of bioluminescent capsules has been previously shown by the authors.17 Briefly, the capsule is designed to carry a full-length ALuc, the C-terminal end of which is linked to a membrane localization signal (MLS) through a known substrate sequence of caspases (DEVD). The luciferase fixed in the capsule is dissected by active caspase 3 and is eventually diffused freely to the cytosol. The free luciferase has higher optical activities than the fixed one to the plasma membrane.

with those of existing marine luciferases by using an alignment search tool provided by the NCBI BLAST (BLASTP 2.5.1+; http://www.ncbi.nlm.nih.gov/). Figure 1B, inset a, shows the maximal sequential identity rankings. Determination of the Absolute Optical Intensities of the New ALucs and Comparison with Conventional Luciferases. The relative optical properties of ALuc41−45 were examined in COS-7 cells derived from African green monkey kidney fibroblasts. COS-7 cells were grown in Dulbecco’s minimal essential medium (DMEM; high glucose) (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (P/S) (Gibco) (Figure 1C). The COS-7 cells, grown in a 96-well microplate (Nunc), were transiently transfected with the pcDNA3.1(+) plasmids encoding each ALuc (Supporting Figure 1) or the existing marine luciferases, that is, Gaussia princeps luciferase (Gluc; GenBank AAG54095.1)16 or Renilla reniformis luciferase 8.6− 535 (RLuc8.6-535),5 as internal references by using the lipofection reagent TransIT-LT1 (Mirus). Sixteen hours after the transfection, the cells in each well were lysed with 50 μL of lysis buffer (E291A; Promega). An aliquot of the lysates (10 μL) was transferred into a fresh 96-well optical bottom microplate (Nunc) by using an 8-channel micropipette. The relative optical intensities in the plate were also determined with the use of an image analyzer (LAS-4000; Fujifilm) immediately after simultaneous injection of 40 μL of the RLuc assay buffer carrying nCTZ by using a multichannel micropipette (Figure 1C). The absolute optical intensities shown in Figure 2B were determined by applying the same protocol as that in Figure 1C. The bioluminescence intensities were normalized by integration time (s) and light-emitting area (mm2). The normalized bioluminescence intensities were subsequently expressed in relative luminescence units per second−area (RLU/sec/mm2). Substrate Specificity of the New Lineage of ALucs. The substrate-specific bioluminescence of the new lineage of ALucs was examined with the use of various coelenterazine analogues (Figure 3; Supporting Figure 2). The COS-7 cells grown in a 96-well microplate were transiently transfected with a pcDNA3.1(+) vector encoding one of the following marine luciferases: GLuc, RLuc8.6-535, ALuc16, ALuc30, and ALuc41−48. Sixteen hours after the transfection, the cells were lysed, and an aliquot of the lysates (20 μL) was carefully transferred to a 200-μL-volume microtube. The lysate in the microtube was mixed with 60 μL of the substrate solution dissolving nCTZ or CTZh and then was immediately transferred into the dark chamber of a precision spectrophotometer (AB-1850; ATTO), which can simultaneously acquire all emitted photons ranging from 391 to 789 nm. Similarly, the substrate specificity of the new lineage of ALucs was examined with the use of a larger number of CTZ analogues (Supporting Figure 2). COS-7 cells expressing ALucs were prepared by applying the same protocol as that in Figure 3. The cells were lysed and then an aliquot of the lysates (10 μL) was carefully transferred to a 96-well optical bottom microplate, simultaneously mixed with 40 μL of each coelenterazine variant (nCTZ, CTZ400, CTZh, CTZn, CTZi, CTZf, CTZcp, CTZhcp, CTZ fcp, and CTZip) by using a multichannel micropipette, and placed in an image analyzer (LAS-4000; Fujifilm). The relative optical intensities were immediately integrated for 30 s.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscombsci.7b00081. A specified alignment of the sequences of the newly created ALucs, a rooted phylogenetic tree of the newly created ALucs and existing marine luciferases according to CLUSTALW, version 2.1, aligned sequences of 25 copepod luciferases, substrate-specific bioluminescence intensities of the new lineage of ALucs, illumination of caspase activities with ALuc47- and ALuc49-based bioluminescent probes, HPLC spectra showing the purity of the substrates nCTZ and CTZh, relative optical intensities of new ALucs in photon counts, and experimental procedures (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Sung Bae Kim: 0000-0002-7033-9056 Daniel Citterio: 0000-0001-7420-045X Author Contributions

S.B.K. and R.N. wrote the main manuscript and prepared all the figures. All authors reviewed the manuscript. 598

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the Japan Society for the Promotion of Science (JSPS) (Grants 26288088, 15KK0029, 16K14051, 24225001, and 17H01215).

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REFERENCES

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