Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 286–297 Contents lists available at ScienceDirect Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol Mitrocomin from the jellyfish Mitrocoma cellularia with deleted C-terminal tyrosine reveals a higher bioluminescence activity compared to wild type photoprotein Ludmila P. Burakova a,1, Pavel V. Natashin a,b,1, Svetlana V. Markova a, Elena V. Eremeeva a, Natalia P. Malikova a, Chongyun Cheng b, Zhi-Jie Liu b,c,d,⁎, Eugene S. Vysotski a,⁎ a Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk 660036, Russia b National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China c Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming 650500, China d iHuman Institute, ShanghaiTech University, Shanghai 201210, China article info abstract Article history: The full-length cDNA genes encoding five new isoforms of Ca2+-regulated photoprotein mitrocomin from a small Received 8 April 2016 tissue sample of the outer bell margin containing photocytes of only one specimen of the luminous jellyfish Received in revised form 30 June 2016 Mitrocoma cellularia were cloned, sequenced, and characterized after their expression in Escherichia coli and sub- Accepted 30 June 2016 sequent purification. The analysis of cDNA nucleotide sequences encoding mitrocomin isoforms allowed sugges- Available online 2 July 2016 tion that two isoforms might be the products of two allelic genes differing in one amino acid residue (64R/Q) whereas other isotypes appear as a result of transcriptional mutations. In addition, the crystal structure of mitrocomin was determined at 1.30 Å resolution which expectedly revealed a high similarity with the structures of other hydromedusan photoproteins. Although mitrocomin isoforms reveal a high degree of identity of amino acid sequences, they vary in specific bioluminescence activities. At that, all isotypes displayed the identical biolu- minescence spectra (473–474 nm with no shoulder at 400 nm). Fluorescence spectra of Ca2+-discharged mitrocomins were almost identical to their light emission spectra similar to the case of Ca2+-discharged aequorin, but different from Ca2+-discharged obelins and clytin which fluorescence is red-shifted by 25– 30 nm from bioluminescence spectra. The main distinction of mitrocomin from other hydromedusan photoproteins is an additional Tyr at the C-terminus. Using site-directed mutagenesis, we showed that this Tyr is not important for bioluminescence because its deletion even increases specificactivityandefficiency of apo- mitrocomin conversion into active photoprotein, in contrast to C-terminal Pro of other photoproteins. Since genes in a population generally exist as different isoforms, it makes us anticipate the cloning of even more iso- forms of mitrocomin and other hydromedusan photoproteins with different bioluminescence properties. © 2016 Elsevier B.V. All rights reserved. 1. Introduction molecular oxygen or any other cofactor, the reaction strikingly differs from that of classical bioluminescent systems in which an enzyme Ca2+-regulated photoproteins are “precharged” bioluminescent (luciferase) catalyzes the oxidation of a smaller organic substrate mole- proteins that are triggered to emit light by addition of Ca2+ or certain cule (luciferin) with the creation of an excited state and light emission. other inorganic ions [1]. The reaction does not require the presence of This feature prompted Shimomura and Johnson to coin the term molecular oxygen or any other cofactor – the photoprotein and the trig- “photoprotein” to describe proteins that serve as sole organic molecular gering ion are the only components required for light emission. Since species in bioluminescent reaction systems [2]. the energy emitted as light is derived from the “charged” photoprotein, Though other kinds of photoproteins have been described, the ma- the molecule can react only once, i.e. it does not “turn over” as an en- jority of the presently known photoproteins are stimulated to lumines- zyme does. In this respect, as well as in the lack of a requirement for cence by calcium. Ca2+-regulated photoproteins are responsible for bioluminescence of a variety of marine organisms [1]. The best known and studied of these is aequorin, first isolated in 1962 by Shimomura ⁎ Corresponding authors. et al. from the jellyfish Aequorea victoria [3].AllCa2+-regulated E-mail addresses: [email protected] (Z.-J. Liu), [email protected] (E.S. Vysotski). photoproteins isolated to date are one-subunit globular proteins, in 1 These authors contributed equally to this work. the inner cavity of which there is a non-covalently bound peroxy- http://dx.doi.org/10.1016/j.jphotobiol.2016.06.054 1011-1344/© 2016 Elsevier B.V. All rights reserved. L.P. Burakova et al. / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 286–297 287 substituted coelenterazine molecule, 2-hydroperoxycoelenterazine [4– and the choice of which one to use depends on the biological task 6]. The bioluminescence reaction is an oxidative decarboxylation of 2- to be addressed. hydroperoxycoelenterazine with the elimination of one mole of carbon The amino acid sequences of hydromedusan Ca2+-regulated dioxide and the generation of the protein-bound product (called photoproteins from different organisms reveal a high degree of identity coelenteramide) in an excited state [7,8]. The excited coelenteramide (76–64%) [42,43]. These photoproteins, except mitrocomin, contain Pro then relaxes to its ground state with the production of blue light of a residue at C-terminus. The C-terminal Pro is important for photoprotein broad spectrum with its maximum in a range 465–495 nm, depending bioluminescence because its deletion or replacement destroys the lumi- on the photoprotein type [9]. nescence capacity of aequorin [44,45]. However, it was found later that Although Ca2+-regulated photoproteins have been detected in the addition of a peptide consisting of eight amino acid residues to the many (N25) different coelenterates [10], cloning and sequence analysis C-terminus of a cysteine-free aequorin mutant [46] or Tyr residue to have been performed only for four hydromedusan photoproteins the obelin C-terminus [47] has no drastic effect on their biolumines- (aequorins from Aequorea victoria [11–13], Aequorea coerulescens [14], cence. In contrast to other hydromedusan photoproteins, a C-terminal and Aequorea macrodactyla [15], clytins from Clytia gregaria [16–18] residue in mitrocomin is tyrosine. The reason of that is unclear. and Clytia hemisphaerica [19], mitrocomin from Mitrocoma cellularia In the present study, we report the cloning of cDNA genes encoding [20], obelins from Obelia longissima [21,22] and Obelia geniculata [23]) several isotypes of the Ca2+-regulated photoprotein mitrocomin from and three photoproteins of ctenophores [24–28], which are extremely the jellyfish Mitrocoma cellularia, their expression in E. coli cells, purifi- sensitive to light, i.e. in contrast to hydromedusan photoproteins they cation and characterization of several mitrocomin isospecies, and the lose the ability to bioluminescence on exposure to light over the entire crystal structure of mitrocomin determined at 1.30 Å resolution. In addi- absorption spectrum [1]. Apophotoproteins expressed in Escherichia coli tion, we quantitatively characterize an effect of deletion of the C-termi- can be converted into active photoproteins by incubating them with nal tyrosine on mitrocomin bioluminescence. 2+ coelenterazine under Ca -free conditions in the presence of O2 and re- ducing agents [29]. While coelenterazine binding with apoprotein re- 2. Materials and Methods quires milliseconds [30], its following conversion into 2-hydroperoxy adduct occurs much slower [31]. 2.1. Collection of the Jellyfish M. cellularia and Preparation of the cDNA Ex- Ca2+-regulated photoproteins draw a strong interest due to their pression Library wide analytical potential. Their main application derives from the ability to emit light upon Ca2+ binding. Natural aequorin and obelin embedded The jellyfish M. cellularia was collected in August 2001 at Friday Har- in living cells were the first indicators of intracellular calcium and have bor Laboratories, University of Washington. The tissue sample been used to detect calcium ions in various types of living cells [32,33]. (~100 mg) was excised from the outer bell margin containing Although Ca2+-regulated photoproteins, mostly aequorin, are still the photocytes of only one jellyfish specimen and immediately used for indicators of choice for some applications, they have now been largely mRNA isolation. The poly(A) + mRNA was directly isolated with eclipsed by the fluorescent tetracarboxylate calcium indicators intro- Straight A's mRNA Isolation Kit (Novagen) and approximately 1 μg duced by Tsien [34]. These substances rapidly gained popularity because (~1/10 part) was employed to synthesize cDNA. The synthesis of cDNA of their ready availability, stability, and ease of introduction into cells and the cDNA library construction were carried out using the SMART [35]. The cloning of cDNAs encoding apophotoproteins opened new av- cDNA library construction kit (Clontech) according to the accompanying enues for utilizing photoproteins, by expressing the recombinant protocol. The first-strand cDNA was synthesized with PowerScript re- apophotoprotein