Mass spectrometry analysis and transcriptome sequencing reveal glowing squid crystal proteins are in the same superfamily as firefly luciferase

Abstract

The Japanese firefly squid Hotaru-ika (Watasenia scintillans) produces intense blue light from photophores at the tips of two arms. These photophores are densely packed with protein microcrystals that catalyse the bioluminescent reaction using ATP and the substrate coelenterazine disulfate. The squid is the only organism known to produce light using protein crystals. We extracted microcrystals from arm tip photophores and identified the constituent proteins using mass spectrometry and transcriptome libraries prepared from arm tip tissue. The crystals contain three proteins, wsluc1–3, all members of the ANL superfamily of adenylating enzymes. They share 19 to 21% sequence identity with firefly luciferases, which produce light using ATP and the unrelated firefly luciferin substrate. We propose that wsluc1–3 form a complex that crystallises inside the squid photophores, and that in the crystal one or more of the proteins catalyses the production of light using coelenterazine disulfate and ATP. These results suggest that ANL superfamily enzymes have independently evolved in distant species to produce light using unrelated substrates.

New Zealand glowworm (Arachnocampa luminosa) bioluminescence is produced by a firefly-like luciferase but an entirely new luciferin

“…In fact, there is a growing group of bioluminescent systems that are evolutionarily distinct and are known to require ATP to produce light, but use entirely different luciferins. Three of these systems use enzymes related to acyl-CoA synthetases to carry out bioluminescence: the bioluminescent beetles/firefly 63 , the Japanese firefly squid ( Watasenia scintillans ) 64 – 66 , and now the Arachnocampa glowworm. The Siberian earthworm ( Fridericia heliota ) also uses ATP and an entirely different luciferin to produce light 67 , 68 ; we await further research to see if the F. heliota system also uses an enzyme related to acyl-CoA synthetases.…”

Symplectin evolved from multiple duplications in bioluminescent squid

“…Of these, one was highly similar to symplectin, including a symplectin-specific indel of two amino acids found in only one other protein (in P. hoylei ). Candidate luciferases were already identified from W. scintillans ( Gimenez et al, 2016 ) belonging to another protein family, so it is unclear what the role would be of the symplectin homologs identified here, or whether they have photoprotein activity with dhCtz.…”

“…For the proteins themselves, three inventions of luciferases have been found in family of adenylating enzymes: for fireflies, the New Zealand glow worm Arachnocampa luminosa ( Sharpe et al, 2015 ), and the squid W. scintillans ( Gimenez et al, 2016 ). This may suggest that members of this protein family has some propensity for becoming luciferases.…”

“…Nonetheless, some key protein components have been identified in two squid species. The luciferase of the firefly squid Watasenia scintillans was recently identified ( Gimenez et al, 2016 ), belonging to the same protein superfamily as firefly luciferases. The other protein comes from the purpleback flying squid Sthenoteuthis oualaniensis , where a 500-amino acid photoprotein has been cloned and characterized ( Isobe et al, 2008 ) and was named symplectin.…”

Advances in bioluminescence imaging: new probes from old recipes

“…In 2016, Sharpe and colleagues reported the isolation of bioluminescent, crystalline protein assemblies in the Japanese firefly squid. [8] The crystals comprise three different—but homologous—proteins that catalyze light emission with coelenterazine-disulfate and ATP (Figure 1B). …”