Biology:Aequorin

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Short description: Calcium-activated photoprotein
Aequorin 1
Aequorin 1EJ3.png
Aequorin ribbon diagram from PDB 1ej3 with prosthetic group coelenterazine in blue
Identifiers
OrganismAequorea victoria (Jellyfish)
SymbolN/A
UniProtP07164
Other data
EC number1.13.12.5

Aequorin is a calcium-activated photoprotein isolated from the hydrozoan Aequorea victoria.[1] Its bioluminescence was studied decades before the protein was isolated from the animal by Osamu Shimomura in 1962.[2] In the animal, the protein occurs together with the green fluorescent protein to produce green light by resonant energy transfer, while aequorin by itself generates blue light.

Discussions of "jellyfish DNA" that can make "glowing" animals often refer to transgenic animals that express the green fluorescent protein, not aequorin, although both originally derive from the same animal.

Apoaequorin, the protein portion of aequorin, is an ingredient in the dietary supplement Prevagen. The US Federal Trade Commission (FTC) has charged the maker with false advertising for its memory improvement claims.

Discovery

Work on aequorin began with E. Newton Harvey in 1921.[3] Though Harvey was unable to demonstrate a classical luciferase-luciferin reaction, he showed that water could produce light from dried photocytes and that light could be produced even in the absence of oxygen. Later, Osamu Shimomura began work into the bioluminescence of Aequorea in 1961. This involved tedious harvesting of tens of thousands of jellyfish from the docks in Friday Harbor, Washington.[1] It was determined that light could be produced from extracts with seawater, and more specifically, with calcium.[2] It was also noted during the extraction the animal creates green light due to the presence of the green fluorescent protein, which changes the native blue light of aequorin to green.[4]

While the main focus of his work was on the bioluminescence,[5] Shimomura and two others, Martin Chalfie and Roger Tsien, were awarded the Nobel Prize in 2008 for their work on green fluorescent proteins.

Structure

Aequorin is a holoprotein composed of two distinct units, the apoprotein that is called apoaequorin, which has an approximate molecular weight of 21 kDa, and the prosthetic group coelenterazine, the luciferin.[6] This is to say, apoaequorin is the enzyme produced in the photocytes 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 three EF 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]

The crystal structure revealed that aequorin binds coelenterazine and oxygen in the form of a peroxide, 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 internal cysteine bond that maintains the structure of aequorin.[14] This has also explained the need for a thiol reagent like beta mercaptoethanol in the regeneration of the protein since such reagents weaken the sulfhydryl bonds between cysteine 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.

Genetics

Aequorin is presumably encoded in the genome of Aequorea. At least four copies of the gene were recovered as cDNA 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]

Mechanism of action

Early studies of the bioluminescence of Aequorea by E. 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 require oxygen, 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 of calcium ions, the protein undergoes a conformational change and converts its prosthetic group, coelenterazine-2-hydroperoxide, into excited coelenteramide and CO2.[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 between bioluminescence and fluorescence, this property was ultimately important in the discovery of the luciferin coelenterazine.[25]

Applications

Since the emitted light can be easily detected with a luminometer, aequorin has become a useful tool in molecular 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 many model biological systems, including zebrafish,[28] rats, mice, and cultured cells.[29][30][31][32]

Cultured cells expressing the aequorin gene can effectively synthesize apoaequorin; however, recombinant expression yields only the apoprotein. Therefore it is necessary to add coelenterazine into the culture medium of the cells to obtain a functional protein and thus use its blue light emission to measure Ca2+ concentration. Coelenterazine is a hydrophobic molecule, and therefore is easily taken up across plant and fungal cell walls, as well as the plasma membrane of higher eukaryotes, making aequorin suitable as a Ca2+ 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 to lipophilic dyes such as DiI. It lacks phenomena of intracellular compartmentalization or sequestration as is often seen for Voltage-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 the calmodulin-based sensor cameleon,[36] developed by Roger Tsien and the troponin-based sensor, TN-XXL, developed by Oliver Griesbeck.[37]

Marketing and legal challenges

Apoaequorin is an ingredient in "Prevagen", which is marketed by Quincy Bioscience as a memory supplement. In 2017, the US Federal Trade Commission (FTC) charged the maker with falsely advertising that the product improves memory, provides cognitive benefits, and is "clinically shown" to work.[38] According to the FTC, "the marketers of Prevagen preyed on the fears of older consumers experiencing age-related memory loss". Quincy said that it would fight the charges.[39][40][41]

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 contested subgroup analyses that showed slight improvements.[42][43]

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.[44]

On February 21, 2019, the United 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.[45][46]

On March 23, 2020, a federal magistrate judge in the United States District Court for the Southern District of Florida entered a report and recommendations certifying a nationwide class action for the class of consumers who purchased Prevagen over the previous four years.[47] The trial in the case is set for October 2020.[47][48]Template:Update-needed

Dr. Harriet Hall, writing for Science-Based Medicine, notes that the Quincy-sponsored study (known as "Madison Memory Study") was negative, but that the company utilized p-hacking to find favorable results. She notes that their cited safety studies are all rat studies and their claim that apoaequorin crosses the blood–brain barrier is based solely on a dog study.[49] The American Pharmacists Association warns that Apoaequorin "is unlikely to be absorbed to a significant degree; instead it degrades into amino acids".[50]

References

  1. ↑ 1.0 1.1 "A short story of aequorin.". Biol. Bull. (Biological Bulletin) 189 (1): 1–5. 1995. doi:10.2307/1542194. PMID 7654844. 
  2. ↑ 2.0 2.1 "Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea". J Cell Comp Physiol 59 (3): 223–39. 1962. doi:10.1002/jcp.1030590302. PMID 13911999. 
  3. ↑ "Studies on Bioluminescence. XIII. Luminescence in the Cœlenterate". Biological Bulletin 41 (5): 280–287. 1921. doi:10.2307/1536528. https://www.journals.uchicago.edu/doi/pdf/10.2307/1536528. 
  4. ↑ "Energy transfer in a bioluminescent system". J. Cell. Physiol. 77 (3): 313–318. 1971. doi:10.1002/jcp.1040770305. PMID 4397528. 
  5. ↑ "The discovery of aequorin and green fluorescent protein". J Microsc 217 (Pt 1): 1–15. 2005. doi:10.1111/j.0022-2720.2005.01441.x. PMID 15655058. 
  6. ↑ "Peroxidized coelenterazine, the active group in the photoprotein aequorin". PNAS USA 75 (3): 2611–2615. 1978. doi:10.1073/pnas.75.6.2611. PMID 275832. Bibcode: 1978PNAS...75.2611S. 
  7. ↑ "Amino acid sequence of the calcium-dependent photoprotein aequorin". Biochemistry 24 (24): 6762–6771. 1985. doi:10.1021/bi00345a006. PMID 2866797. 
  8. ↑ "Prediction of EF-hand calcium-binding proteins and analysis of bacterial EF-hand proteins". Proteins 65 (3): 643–655. 2006. doi:10.1002/prot.21139. PMID 16981205. 
  9. ↑ "The crystal structure of the photoprotein aequorin at 2.3 Å resolution". Nature 405 (6784): 372–376. 2000. doi:10.1038/35012659. PMID 10830969. Bibcode: 2000Natur.405..372H. 
  10. ↑ "Luminescence of aequorin is triggered by the binding of two calcium ions". Biochem. Biophys. Res. Commun. 211 (2): 359–363. 1995. doi:10.1006/bbrc.1995.1821. PMID 7794244. 
  11. ↑ "Luminescence of aequorin is triggered by the binding of two calcium ions". Biochemical and Biophysical Research Communications 211 (2): 359–363. 1995. doi:10.1006/bbrc.1995.1821. PMID 7794244. 
  12. ↑ "All three Ca2+-binding loops of photoproteins bind calcium ions: the crystal structures of calcium-loaded apo-aequorin and apo-obelin". Protein Sci. 14 (3): 663–675. 2005. doi:10.1110/ps.041142905. PMID 15689515. 
  13. ↑ "Titration of recombinant aequorin with calcium chloride". Biochem. Biophys. Res. Commun. 221 (1): 77–81. 1996. doi:10.1006/bbrc.1996.0548. PMID 8660347. 
  14. ↑ "Mass spectrometric evidence for a disulfide bond in aequorin regeneration". FEBS Lett. 332 (3): 226–228. 1993. doi:10.1016/0014-5793(93)80637-a. PMID 8405461. 
  15. ↑ "Blue fluorescent protein from the calcium-sensitive photoprotein aequorin is a heat-resistant enzyme, catalyzing the oxidation of coelenterazine". FEBS Lett. 577 (1–2): 105–110. 2004. doi:10.1016/j.febslet.2004.09.078. PMID 15527769. 
  16. ↑ "Cloning and expression of the cDNA coding for aequorin, a bioluminescent calcium-binding protein". Biochem. Biophys. Res. Commun. 126 (3): 1259–68. 1985. doi:10.1016/0006-291X(85)90321-3. PMID 2579647. 
  17. ↑ "Cloning and sequence analysis of cDNA for the luminescent protein aequorin". Proc. Natl. Acad. Sci. U.S.A. 82 (10): 3154–58. 1985. doi:10.1073/pnas.82.10.3154. PMID 3858813. Bibcode: 1985PNAS...82.3154I. 
  18. ↑ "Chromatography of isoforms of recombinant apoaequorin and method for the preparation of aequorin". Protein Expr. Purif. 31 (2): 181–187. 2003. doi:10.1016/s1046-5928(03)00186-4. PMID 14550635. 
  19. ↑ "Oxygen and Luminescence, with a Description of Methods for Removing Oxygen from Cells and Fluids". Biological Bulletin 51 (2): 89–97. 1926. doi:10.2307/1536540. https://www.biodiversitylibrary.org/part/10295. 
  20. ↑ Bioluminescence. Academic Press. 1952. 
  21. ↑ "Regeneration of the photoprotein aequorin". Nature 256 (5514): 236–238. 1975. doi:10.1038/256236a0. PMID 239351. Bibcode: 1975Natur.256..236S. 
  22. ↑ "Mechanism of the luminescent intramolecular reaction of aequorin". Biochemistry 13 (16): 3278–3286. 1974. doi:10.1021/bi00713a016. PMID 4152180. 
  23. ↑ "Calcium binding, quantum yield, and emitting molecule in aequorin bioluminescence". Nature 227 (5265): 1356–1357. 1970. doi:10.1038/2271356a0. PMID 4393938. Bibcode: 1970Natur.227.1356S. 
  24. ↑ "Blue fluorescent protein from the calcium-sensitive photoprotein aequorin: catalytic properties for the oxidation of coelenterazine as an oxygenase". FEBS Lett. 580 (8): 1977–1982. 2006. doi:10.1016/j.febslet.2006.02.065. PMID 16545379. 
  25. ↑ "Chemical nature of bioluminescence systems in coelenterates". Proceedings of the National Academy of Sciences 72 (4): 1546–1549. 1975. doi:10.1073/pnas.72.4.1546. PMID 236561. Bibcode: 1975PNAS...72.1546S. 
  26. ↑ "Recombinant aequorin and recombinant semi-synthetic aequorins. Cellular Ca2+ ion indicators". Biochem. J. 270 (2): 309–312. 1990. doi:10.1042/bj2700309. PMID 2400391. 
  27. ↑ "Calcium transients in single muscle fibers". Biochem. Biophys. Res. Commun. 29 (2): 229–234. 1967. doi:10.1016/0006-291x(67)90592-x. PMID 4383681. 
  28. ↑ "Transient expression of apoaequorin in zebrafish embryos: extending the ability to image calcium transients during later stages of development". Int. J. Dev. Biol. 50 (6): 561–569. 2006. doi:10.1387/ijdb.062151cc. PMID 16741871. http://www.ijdb.ehu.es/web/descarga/paper/062151cc. 
  29. ↑ "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. January 1997. doi:10.1016/s0143-4160(97)90098-1. PMID 9056079. 
  30. ↑ "Identification of the functional expression of adenosine A3 receptor in pancreas using transgenic mice expressing jellyfish apoaequorin". Transgenic Res. 16 (4): 429–435. 2007. doi:10.1007/s11248-007-9084-0. PMID 17387626. 
  31. ↑ "Measurement of intracellular calcium using bioluminescent aequorin expressed in human cells". Anal. Biochem. 209 (2): 343–347. 1993. doi:10.1006/abio.1993.1132. PMID 8470808. 
  32. ↑ "Aequorin-based measurements of intracellular Ca2+ signatures in plant cells". Biol. Proced. Online 4: 105–118. 2002. doi:10.1251/bpo40. PMID 12734562. PMC 145563. http://www.biologicalprocedures.com/bpo/arts/1/40/m40.htm. 
  33. ↑ "Measurement of Ca2+ concentrations in living cells". Prog Biophys Mol Biol 40 (1–2): 1–114. 1982. doi:10.1016/0079-6107(82)90011-6. PMID 6758036. 
  34. ↑ "Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells". EMBO J 14 (22): 5467–5475. 1995. doi:10.1002/j.1460-2075.1995.tb00233.x. PMID 8521803. 
  35. ↑ "Recombinant apoaequorin acting as a pseudo-luciferase reports micromolar changes in the endoplasmic reticulum free Ca2+ of intact cells". Biochem J 318 (2): 383–387. 1996. doi:10.1042/bj3180383. PMID 8809023. 
  36. ↑ "Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin". Nature 388 (6645): 882–887. 1997. doi:10.1038/42264. PMID 9278050. Bibcode: 1997Natur.388..882M. 
  37. ↑ "Genetically encoded indicators of cellular calcium dynamics based on troponin C and green fluorescent protein". J Biol Chem 279 (14): 14280–14286. 2004. doi:10.1074/jbc.M312751200. PMID 14742421. 
  38. ↑ Hamilton, Martha M. (September 11, 2021). "Does the supplement Prevagen improve memory? A court case is asking that question.". The Washington Post. https://www.washingtonpost.com/health/prevagen-memory-loss-does-it-work/2021/09/10/53e5d3e8-f3a6-11eb-a49b-d96f2dac0942_story.html. Retrieved September 11, 2021. 
  39. ↑ Fox, Maggie (January 9, 2017). "Jellyfish Memory Supplement Prevagen Is a Hoax, FTC Says". NBC News. https://www.nbcnews.com/health/health-care/jellyfish-memory-supplement-prevagen-hoax-ftc-says-n704886. 
  40. ↑ Li, David K. (January 9, 2017). "Schneiderman slams Prevagen as a 'clear-cut fraud' in lawsuit". New York Post. https://nypost.com/2017/01/09/schneiderman-slams-prevagen-as-a-clear-cut-fraud-in-lawsuit/. 
  41. ↑ "Prevagen's Fishy Memory Claims Under Fire by Federal Regulators". Truth in Advertising. January 9, 2017. https://www.truthinadvertising.org/prevagens-fishy-memory-claims-fire-federal-regulators/. 
  42. ↑ University of California Berkeley School of Public Health, Health After 50, "Forget Jellyfish Protein", Winter, 2017–18, p. 6
  43. ↑ "Prevagen: How Can This Memory Supplement Flunk Its One Trial and Still Be Advertised as Effective?". Center for Science in the Public Interest. November 20, 2017. https://cspinet.org/news/prevagen-how-can-memory-supplement-flunk-its-one-trial-and-still-be-advertised-effective. 
  44. ↑ "Quincy Bioscience's Prevagen Supplement October 2018", Truth in Advertising, October 2018. Retrieved November 14, 2018.
  45. ↑ 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.
  46. ↑ "Prevagen Is Going to the Dogs", Truth in Advertising, February 22, 2019. Retrieved March 26, 2019.
  47. ↑ 47.0 47.1 Mora, Michael A. (March 24, 2020). "Federal Magistrate Judge Recommends Certifying Nationwide Prevagen Class Action in Florida". Daily Business Review (law.com). https://www.law.com/dailybusinessreview/2020/03/20/032020classactionprevagen/. 
  48. ↑ "Report and Recommendations on Plaintiff's Motion for Class Certification". United States District Court, Southern District of Florida, Miami Division. March 19, 2020. https://drive.google.com/file/d/1OOYG9hDSMic38NxVh7MFNXD27Q--Ncch/view?usp=embed_facebook. 
  49. ↑ Hall, Harriet (4 December 2018). "Reader's Digest Promotes Prevagen". https://sciencebasedmedicine.org/readers-digest-promotes-prevagen/. 
  50. ↑ Hume, Anne. "Apoaequorin for memory enhancement?". Pharmacist.com. https://www.pharmacist.com/apoaequorin-memory-enhancement. 

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