Cloning, Sequencing, Expression and Structural Investigation of Mnemiopsin from Mnemiopsis leidyi: An Attempt Toward Understanding Ca2+-Regulated Photoproteins

Abstract

A comparison of the two most famous groups of calcium-regulated photoproteins, cnidarians and ctenophores, showed unexpectedly high degree of structural similarity regardless of their low sequence identity. It was suggested these photoproteins can play an important role in understanding the structural basis of bioluminescence activity. Based on this postulate, in this study the cDNA of mnemiopsin from luminous ctenophore Mnemiopsis leidyi was cloned, expressed, purified and sequenced. The purified cDNA, with 621 base pairs, coded a 206 residues protein. Sequence of mnemiopsin showed 93.5 and 51% similarity to other ctenophore proteins and cnidarians, respectively. The cDNA encoding apo-mnemiopsin of M. leidyi was expressed in Escherichia coli. The purified apo-protein showed a single band on SDS-PAGE (molecular weight ~27 kDa). A semi-synthetic mnemiopsin was prepared using coelenterazine and EDTA and its luminescence activity was measured in the presence of CaCl(2). The results showed an optimum pH of 9.0 and lower calcium sensitivity compared to aequorin. Comparison of amino acid residues in substrate binding site indicated that binding pocket of ctenophores contains less aromatic residues than cnidarians. This can lead to a decline in the number of stacking interactions between substrate and protein which can affect the stability of coelenterazine in binding cavity. Structural comparison of photoproteins with low sequence identity and high 3D structural similarity, can present a new insight into the mechanism of light emission in photoproteins.

Expression and characterization of the calcium-activated photoprotein from the ctenophore Bathocyroe fosteri: Insights into light-sensitive photoproteins

“…We determined the optimal environment for coelenterazine binding and calcium-activated luminescence in BfosPP to be pH 8.5, with salt concentration similar to ocean molarity (500mM). These conditions are similar to the natural environment of Bathocyroe and to those published for berovin [4] and mnemiopsin-1,2 [2,3]. They differ markedly from those of cnidarian photoproteins, whose activity is highest at pH ~7.0 and without salt [7].…”

Genomic organization, evolution, and expression of photoprotein and opsin genes in Mnemiopsis leidyi: a new view of ctenophore photocytes

“…At the N-terminus, there are two regions where the ctenophore photoproteins have insertions of six to nine amino acids relative to the hydromedusae photoproteins. There are several differences in important functional residues, especially in residues that make up the coelenterazine binding cavity, between the two groups of photoproteins, which have been previously discussed [20]. These amino acid substitutions may be partially responsible for the functional differences seen between the two groups, such as the property of photoinactivation present in ctenophore photoproteins but not in hydromedusae photoproteins.…”

“…Berovin and bolinopsin from the ctenophores Beroe abyssicola [16,17] and Bolinopsis infundibulum [18,19] were subsequently cloned and sequenced. Recently, sequences for two photoproteins from Mnemiopsis leidyi , named mnemiopsin 1 and mnemiopsin 2 , have been reported [20,21]. …”

“…Previous studies have explored photoprotein phylogenetic relationships within jellyfish (hydromedusan) species [30], comparing both their sequences and structures [31]. Recently, Aghamaali et al [20] performed sequence comparisons of ctenophore and hydromedusae photoproteins. However, no investigations have gone beyond the hydromedusan and ctenophoran representatives, potentially obscuring the evolutionary history of this gene family by omitting sequences from other phyla.…”

Fluorescent Protein–photoprotein Fusions and Their Applications in Calcium Imaging

“…These photoproteins, which appear in organisms of the phylum Ctnenophora , have been characterized and found to be strongly inactivated by light. Berovin from Beroe abyssicola and mnemiopsin from Mnemiopsis leidyi were recently cloned. Although berovin sequence identity with aequorin is as low as 28%, three canonical EF‐hand Ca 2+ ‐binding sites were discovered, and it was successfully expressed in mammalian cells in culture.…”