Research ArticleEVOLUTIONARY BIOLOGY

Kleptoprotein bioluminescence: Parapriacanthus fish obtain luciferase from ostracod prey

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Science Advances  08 Jan 2020:
Vol. 6, no. 2, eaax4942
DOI: 10.1126/sciadv.aax4942
  • Fig. 1 P. ransonneti and its bioluminescence.

    (A) Lateral view of the fish under white light. Body length, 8 cm. (B) Lateral and ventral views of the thoracic and anal luminous organs (TL and AL; blue lines). The cross sections for immunohistochemistry (Fig. 3) were made at lines a to e. (C) A ventral view of in vivo bioluminescence. Photo credit: Manabu Bessho-Uehara, MBARI.

  • Fig. 2 Properties and identification of the P. ransonneti luciferase.

    (A) Bioluminescence spectra of V. hilgendorfii (dotted line) from the dissected thoracic light organ of Parapriacanthus (solid black line) and of in vitro L-L reaction using Parapriacanthus luciferase and vargulin (solid blue line). (B) Distribution of luciferase activity. PC, pyloric caeca; IN, intestine; DM, dorsal muscle. (C) Western blot using anti-cypridinid luciferase antibody. The relative molecular weight of the band detected in TL and AL corresponds to that of V. hilgendorfii luciferase. (D) Peptide fragments of the purified Parapriacanthus luciferase detected by quadrupole orthogonal acceleration–time-of-flight tandem mass spectrometry (qTOF-MS/MS) mapped onto the amino acid sequence of C. noctiluca luciferase (red). Theoretical cleavage positions by trypsin and lysyl endopeptidase, lysine (K) and arginine (R), are shown in bold.

  • Fig. 3 Localization of luciferase in the thoracic and anal light organs.

    The levels of sections in (A) to (E) correspond to the positions a to e shown in Fig. 1B. Left: Fluorescent signals of anti-cypridinid luciferase antibody labeled by Alexa Fluor 488 (A, C, and E) or fluorescein (B and D). Scale bars, 100 μm. Middle: Merged images of fluorescent and phase contrast microscopic images. Right: Nissl-stained sections. Scale bars, 1 mm. The left and middle panels correspond to the red boxes in the right panel. Light organs are surrounded by reflectors (thick black area in the bright field). (A to C) The thoracic light organ is composed of tubular structures with the luciferase signal. The number of tubes decreases from anterior to posterior, but the diameter of each becomes larger (A to C). Luciferase is detected on the epidermal cell surface and in the cytosol. (D) The anal light organ has villi-like structures and is separated by a reflector from the intestine. The luciferase signal is primarily on the epidermal cell surface. (E) Granular substructures with the luciferase signals are spread in the cytosol of the epidermal cells in the villi. K, keel; M, muscle; TM, translucent muscle; OE, esophagus; OV, ovary; P, pylorus; PF, pelvic fin; R, reflector; ST, stomach.

  • Fig. 4 Vargula feeding experiment.

    (A) The values are given in percent relative to the mean of the original luciferase activities of extracts from freshly caught fish (1 week). Activity of specimens fed with nonluminous fish for 1 week, 3 months, and 1 year (gray) declined and subsequently increased when fed with V. hilgendorfii for 2 weeks (1 year + 2 weeks) and 1 month (1 year + 1 month) (blue). NC, no enzyme control. (B) Peptides detected by qTOF-MS/MS are highlighted in red on the amino acids of C. noctiluca luciferase (CnocLuc) and V. hilgendorfii luciferase (VhilLuc). It can be seen that in varying regions, peptide fragments matched VhilLuc, indicating a dietary source. The amino acid identity between CnocLuc and VhilLuc is 84%. The disagreements of amino acid residues between two luciferases were indicated by dots under the alignment. Theoretical cleavage positions by protease treatment are shown in bold. Putative signal peptide at the N terminus (33) is indicated by the blue underline. (C) Schematic view of the detected peptides. Full-length luciferase sequences are indicated by gray lines. The peptides that are common to both luciferases are shown by bold black lines. Peptides specific to either luciferase are shown as bold red lines.

  • Table 1 Purification of Parapriacanthus luciferase.

    Protein was calculated by the extract volume and concentration as determined by absorbance at 280 nm. rlu, relative light unit; HPLC, high-performance liquid chromatography.

    Purification
    method
    Activity
    (rlu)
    Protein
    (mg)
    Specific
    activity
    (rlu/mg)
    Purity
    (fold)
    Crude extract19714076261.20754751
    HiTrap Q132804652.35565126275
    Sephadex
    G-75
    120627591.82664614888
    HPLC Mono Q
    1 (fractions
    21-27)
    17184220.1710231190136
    HPLC Mono Q
    2 (fractions
    35-42)
    2090200.028709167115

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/2/eaax4942/DC1

    Supplementary Text

    Fig. S1. Light organ and luciferin storage organ in P. ransonneti.

    Fig. S2. Bioluminescence of P. ransonneti.

    Fig. S3. Luminescence spectra.

    Fig. S4. Purification of P. ransonneti luciferase.

    Fig. S5. Immunohistochemistry of thoracic and anal light organs.

    Fig. S6. In vitro luciferase expression.

    Fig. S7. Genomic PCR.

    Fig. S8. Immuno–pull-down assay.

    Fig. S9. Proteolytic resistance and stability of luciferases.

    Fig. S10. Phylogenetic relationship of P. ransonneti specimens used in this study.

    Table S1. Materials for RNA-seq.

    External database S1. DRA file of RNA-seq (DRA submission DRA008133) includes raw reads generated from thoracic light organ, anal light organ, pyloric caeca, intestine, and dorsal muscle of P. ransonneti and whole body of V. hilgendorfii.

    References (3947)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Text
    • Fig. S1. Light organ and luciferin storage organ in P. ransonneti.
    • Fig. S2. Bioluminescence of P. ransonneti.
    • Fig. S3. Luminescence spectra.
    • Fig. S4. Purification of P. ransonneti luciferase.
    • Fig. S5. Immunohistochemistry of thoracic and anal light organs.
    • Fig. S6. In vitro luciferase expression.
    • Fig. S7. Genomic PCR.
    • Fig. S8. Immuno–pull-down assay.
    • Fig. S9. Proteolytic resistance and stability of luciferases.
    • Fig. S10. Phylogenetic relationship of P. ransonneti specimens used in this study.
    • Table S1. Materials for RNA-seq.
    • External database S1. DRA file of RNA-seq (DRA submission DRA008133) includes raw reads generated from thoracic light organ, anal light organ, pyloric caeca, intestine, and dorsal muscle of P. ransonneti and whole body of V. hilgendorfii.
    • References (3947)

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