Copepod Luciferases: Properties and Application as Bioluminescent Reporters
Secreted luciferases of marine copepods are relatively small proteins ranging from 18.4 to 24.2 kDa that catalyze the oxidation of their substrate, coelenterazine, to produce a bright blue bioluminescence.Highly homologous Gaussia and Metridia luciferases are the best known representatives of copepod luciferases. These luciferases share no sequence identity with other coelenterazine-dependent luciferases cloned to date, such as of Renilla-, Oplophorus-, and Cypridina-type. Today, the first cloned copepod luciferases, GLuc from Gaussia princeps and MLuc from Metridia longa are successfully applied as extremely sensitive bioluminescent reporters for biological and medical research including in vivo imaging. But the search for bioluminescent reporters with more advanced properties is continued.
With cDNA functional screening applied, we have shown that luciferase of Metridia longa is represented by multiple isoforms that were categorized into four types based on sequence features. Presumably, each type represents a separate non-allelic group of luciferase genes. These groups of MLuc isoforms differ in length (18.4 – 23.9 kDa) and share 53%, 68%, and 80% protein sequence identity with the group of luciferases having the longest sequences. The sequence alignment of the M. longa luciferase isoforms has shown a variable N-terminus of approximately 1/3 in length of the longest sequence followed by the conserved C-terminal region consisting of two non-identical repeats of ~70 amino acids. Each repeat contains highly conserved short motif of 32 amino acids with five highly conserved Cys residues suggesting the presence of up to 5 disulfide bonds per the protein molecule. These repeats of ~70 amino acids of M. longa luciferase expressed separately didn’t reveal any bioluminescent activity. Only entire conserved C-terminal domain displays bioluminescence activity. The variable N-terminus is not important for bioluminescence because MLuc variant with truncation of this region retains the activity.
Some bioluminescent properties of different types of MLuc isoforms of different types and GLuc were investigated using recombinant proteins produced in baculovirus/insect cell expression system. All investigated luciferase isoforms display a very high bioluminescent activity and extreme thermostability retaining up to 70% activity after 30-min incubation at 100°C. The most significant differences were found in kinetics, salt concentration requirement, and specific bioluminescence activity. In addition, the temperature optimum of bioluminescent reaction ranged from extremely low 4°C to 20°C. It is highly probable that such different properties of the luciferase isoforms provide adaptability of copepod M. longa to rapid change of environmental conditions during diurnal vertical migrations. It also suggests that these isoforms of different luminescence properties may have different applications depending on experimental tasks.