Calcium-activated photoprotein
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
[
edit
]
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
[
edit
]
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 Ca
2+
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
[
edit
]
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
[
edit
]
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
CO
2
.
[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
[
edit
]
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 Ca
2+
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 Ca
2+
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
Ca
2+
reporter
in plants, fungi, and mammalian cells.
[33]
[34]
Aequorin has a number of advantages over other Ca
2+
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
[
edit
]
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 was set for October 2020.
[47]
[48]
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.
[49]
Dr.
Harriet Hall
, writing for
Science-Based Medicine
, noted that the Quincy-sponsored study (known as "Madison Memory Study") was negative, but that the company utilized
p-hacking
to find favorable results. She wrote that their cited safety studies were all rat studies and their claim that apoaequorin crosses the
blood?brain barrier
was based solely on a dog study.
[50]
The
American Pharmacists Association
warns that Apoaequorin "is unlikely to be absorbed to a significant degree; instead it degrades into amino acids".
[51]
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.
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