Discussions of "jellyfish DNA" that can make "glowing" animals often refer totransgenic animals thatexpress the green fluorescent protein, not aequorin, although both originally derive from the same animal.
Work on aequorin began withE. Newton Harvey in 1921.[3] Though Harvey was unable to demonstrate a classicalluciferase-luciferin reaction, he showed that water could produce light from driedphotocytes and that light could be produced even in the absence of oxygen. Later,Osamu Shimomura began work into thebioluminescence ofAequorea in 1961. This involved tedious harvesting of tens of thousands of jellyfish from the docks inFriday Harbor, Washington.[1] It was determined that light could be produced from extracts with seawater, and more specifically, withcalcium.[2] It was also noted during the extraction the animal creates green light due to the presence of thegreen fluorescent protein, which changes the native blue light of aequorin to green.[4]
Aequorin is aholoprotein composed of two distinct units, theapoprotein that is calledapoaequorin, which has an approximate molecular weight of 21kDa, and theprosthetic groupcoelenterazine, the luciferin.[6] This is to say, apoaequorin is theenzyme produced in thephotocytes of the animal, and coelenterazine is the substrate whose oxidation the enzyme catalyzes. When coelenterazine is bound, it is called aequorin. Notably, the protein contains threeEF hand motifs that function as binding sites for Ca2+ ions.[7] The protein is a member of the superfamily of the calcium-binding proteins, of which there are some 66 subfamilies.[8]
Thecrystal structure revealed that aequorin binds coelenterazine and oxygen in the form of aperoxide, coelenterazine-2-hydroperoxide.[9] The binding site for the first two calcium atoms show a 20 times greater affinity for calcium than the third site.[10] However, earlier claims that only two EF-hands bind calcium[11] were questioned when later structures indicated that all three sites can indeed bind calcium.[12] Thus,titration studies show that all three calcium-binding sites are active but only two ions are needed to trigger the enzymatic reaction.[13]
Other studies have shown the presence of an internalcysteine bond that maintains the structure of aequorin.[14] This has also explained the need for athiol reagent likebeta mercaptoethanol in the regeneration of the protein since such reagents weaken thesulfhydryl bonds betweencysteine residues, expediting the regeneration of the aequorin.
Chemical characterization of aequorin indicates the protein is somewhat resilient to harsh treatments. Aequorin is heat resistant.[15] Held at 95 °C for 2 minutes the protein lost only 25% activity. Denaturants such as 6-M urea or 4-M guanidine hydrochloride did not destroy the protein.
Aequorin is presumably encoded in thegenome ofAequorea. At least four copies of the gene were recovered ascDNA from the animal.[16][17] Because the genome has not been sequenced, it is unclear if the cDNA variants can account for all of the isoforms of the protein.[18]
Early studies of thebioluminescence ofAequorea byE. Newton Harvey had noted that the bioluminescence appears as a ring around the bell, and occurs even in the absence of air.[19] This was remarkable because most bioluminescence reactions requireoxygen, and led to the idea that the animals somehow store oxygen.[20] It was later discovered that the apoprotein can stably bind coelenterazine-2-hydroperoxide, and oxygen is required for the regeneration to this active form of aequorin.[21] However, in the presence ofcalcium ions, the protein undergoes a conformational change and converts its prosthetic group, coelenterazine-2-hydroperoxide, into excitedcoelenteramide andCO2.[22] As the excited coelenteramide relaxes to the ground state, blue light (wavelength of 465 nm) is emitted. Before coelenteramide is exchanged out, the entire protein is still fluorescent blue.[23][24] because of the connection betweenbioluminescence andfluorescence, this property was ultimately important in the discovery of the luciferincoelenterazine.[25]
Since the emitted light can be easily detected with aluminometer, aequorin has become a useful tool inmolecular biology for the measurement of intracellular Ca2+ levels.[26] The early successful purification of aequorin led to the first experiments involving the injection of the protein into the tissues of living animals to visualize the physiological release of calcium in the muscle fibers of a barnacle.[27] Since then, the protein has been widely used in manymodel biological systems, includingzebrafish,[28]rats,mice, andcultured cells.[29][30][31][32]
Cultured cells expressing the aequoringene can effectively synthesize apoaequorin; however,recombinant expression yields only theapoprotein. Therefore it is necessary to addcoelenterazine into the culture medium of the cells to obtain a functional protein and thus use its bluelight emission to measure Ca2+ concentration. Coelenterazine is a hydrophobic molecule, and therefore is easily taken up across plant and fungalcell walls, as well as theplasma membrane of higher eukaryotes, making aequorin suitable as aCa2+ reporter in plants, fungi, and mammalian cells.[33][34]
Aequorin has a number of advantages over other Ca2+ indicators. Because the protein is large, it has a low leakage rate from cells compared tolipophilic dyes such asDiI. It lacks phenomena of intracellular compartmentalization or sequestration as is often seen forVoltage-sensitive dyes, and does not disrupt cell functions or embryo development. Moreover, the light emitted by the oxidation of coelenterazine does not depend on any optical excitation, so problems with auto-fluorescence are eliminated.[35] The primary limitation of aequorin is that the prosthetic group coelenterazine is irreversibly consumed to produce light, and requires continuous addition of coelenterazine into the media. Such issues led to developments of other genetically encoded calcium sensors including thecalmodulin-based sensorcameleon,[36] developed byRoger Tsien and thetroponin-based sensor,TN-XXL, developed by Oliver Griesbeck.[37]
Apoaequorin is an ingredient in Prevagen, which is marketed by Quincy Bioscience as a memory supplement. In 2017, the USFederal Trade Commission (FTC) charged the maker withfalsely advertising that the product improves memory, provides cognitive benefits, and is "clinically shown" to work.[38][39] According to the FTC, "the marketers of Prevagen preyed on the fears of older consumers experiencingage-related memory loss". Quincy said that it would fight the charges.[40][41][42]
Prior to the suit, a clinical trial run by researchers employed by Quincy Bioscience "found no overall benefit compared to a placebo for its primary endpoints involving memory and cognition", while the company's advertising misleadingly cited a few contestedsubgroup analyses that showed slight improvements.[43][44]
The suit (Spath, et al. v. Quincy Bioscience Holding Company, Inc., et al., Case No. 18-cv-12416, D. NJ.) was dismissed in the District court, but an appeal seeking to overturn the dismissal was filed. The suit was consolidated with another against Quincy Pharmaceuticals,Vanderwerff v. Quincy Bioscience (Case No. 17-cv-784, D. NJ), which was the lead case.[45]
On February 21, 2019, theUnited States Court of Appeals for the Second Circuit ruled that the FTC and the state of New York could proceed with their lawsuit against Quincy Bioscience for its claims that Prevagen can improve memory. The order came less than two weeks after the parties argued the case before a three-judge panel of the circuit, where company lawyers admitted they did not "dispute that if you look across the entire 211 people who completed the study there was no statistically significant difference". The court vigorously dismissed allegations by the company lawyers that the FTC pursued its action for political reasons.[46][47]
As of September 21, 2020[update], Quincy Bioscience agreed to settle the claims that it misrepresented its Prevagen products as supporting brain health and helping with memory loss. Under the terms of the settlement, eligible purchasers applying by October 26, 2020, for purchases made from 2007 through July 31, 2020, could recover refunds of up to $70.[50]
Dr.Harriet Hall, writing forScience-Based Medicine, noted that the Quincy-sponsored study (known as "Madison Memory Study") was negative, but that the company utilizedp-hacking to find favorable results. She wrote that their cited safety studies were all rat studies and their claim that apoaequorin crosses theblood–brain barrier was based solely on a dog study.[51][52] TheAmerican Pharmacists Association warns that Apoaequorin "is unlikely to be absorbed to a significant degree; instead it degrades into amino acids".[53]
On April 3, 2025, Quincy Bioscience, who was a co-plaintiff with Amazon, won a lawsuit against several sellers of counterfeit Prevagen on the Amazon website. A Washington federal judge awarded a combined total of $1,895,375.40 in default judgments.[54][55]
Prevagen is purported to be a target for theft in retail stores by organized retail crime groups. Several factors contribute to it being a popular target, including its price, demand, and marketing claims around its effectiveness. On April 24, 2020, The U.S. Circuit Court of Appeals for theSeventh Circuit upheld a ruling in favor of Quincy finding an online seller of Prevagen liable for selling Prevagen products in damaged condition and products the seller knew or should have known to have been stolen, ordering the seller to pay $480,968.13 in damages.[56] On August 24, 2023, the Florida Attorney General charged two members of a criminal ring[57] with stealing $10,000 worth of Prevagen and other items. On May 21, 2024, a multistate theft ring leader[58] was sentenced to federal prison for directing a crime ring that stole an estimated $9 million worth of merchandise, including Prevagen,Abreva,Zantac, and other products, over three years.
^abShimomura O, Johnson FH, Saiga Y (1962). "Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea".J Cell Comp Physiol.59 (3):223–39.doi:10.1002/jcp.1030590302.PMID13911999.
^Charbonneau H, Walsh KA, McCann RO, Prendergast FG, Cormier MJ, Vanaman TC (1985). "Amino acid sequence of the calcium-dependent photoprotein aequorin".Biochemistry.24 (24):6762–6771.doi:10.1021/bi00345a006.PMID2866797.
^Zhou Y, Yang W, Kirberger M, Lee HW, Ayalasomayajula G, Yang JJ (2006). "Prediction of EF-hand calcium-binding proteins and analysis of bacterial EF-hand proteins".Proteins.65 (3):643–655.doi:10.1002/prot.21139.PMID16981205.S2CID8904181.
^Masuda H, Takenaka Y, Shikamoto Y, Kagawa M, Mizuno H, Tsuji FI (2003). "Chromatography of isoforms of recombinant apoaequorin and method for the preparation of aequorin".Protein Expr. Purif.31 (2):181–187.doi:10.1016/s1046-5928(03)00186-4.PMID14550635.
^Shimomura O, Johnson FH, Morise H (1974). "Mechanism of the luminescent intramolecular reaction of aequorin".Biochemistry.13 (16):3278–3286.doi:10.1021/bi00713a016.PMID4152180.
^Rembold CM, Kendall JM, Campbell AK (January 1997). "Measurement of changes in sarcoplasmic reticulum [Ca2+] in rat tail artery with targeted apoaequorin delivered by an adenoviral vector".Cell Calcium.21 (1):69–79.doi:10.1016/s0143-4160(97)90098-1.PMID9056079.
^Yamano K, Mori K, Nakano R, Kusunoki M, Inoue M, Satoh M (2007). "Identification of the functional expression of adenosine A3 receptor in pancreas using transgenic mice expressing jellyfish apoaequorin".Transgenic Res.16 (4):429–435.doi:10.1007/s11248-007-9084-0.PMID17387626.S2CID19339429.
^Moran, Daniel L.; Underwood, Mark Y.; Gabourie, Taylor A.; Lerner, Kenneth C. (2016). "Effects of a Supplement Containing Apoaequorin on Verbal Learning in Older Adults in the Community".Adv Mind Body Med.30 (1):4–11.PMID26878676.
^FTC vs. Quincy Bioscience Holding Company, United States Court of Appeals for the Second Circuit, Case 17-3745, Document 257, February 21, 2019. Retrieved March 26, 2019.
