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Multi-colored homologs of the green fluorescent protein from hydromedusa
Obelia sp.
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Published on 26 May 2011 on http://pubs.rsc.org | doi:10.1039/C1PP05068K
Galina V. Aglyamova,†a Marguerite E. Hunt,†b Chintan K. Modib and Mikhail V. Matz*a
Received 11th February 2011, Accepted 21st April 2011
DOI: 10.1039/c1pp05068k
The presence of green fluorescent protein (GFP) within the bioluminescent system of Obelia (Cnidaria,
Hydrozoa, Campanulariidae) was inferred shortly after the discovery of GFP in Aequorea. Despite the
enormous success of Aequorea GFP as a genetically encoded fluorescent label, Obelia GFP thus far has
been defeating attempts to clone it from the hydroid life cycle stage. Here, we report cloning of three
GFP-like fluorescent proteins (FPs) from Obelia medusa, representing cyan, green, and yellow spectral
types. Such color diversity has never been detected outside class Anthozoa, suggesting a more general
function for multi-colored fluorescence in cnidarians than has been previously hypothesized. An
unusual property of the new FPs is the formation of large soluble complexes of well-defined sizes and
molecular weights, corresponding to up to 128 individual polypeptides. This aligns well with the earlier
observation that luminescence in Obelia, unlike in Aequorea, is localized within subcellular granules,
which prompts further inquiry into the self-assembly properties of the new FPs and their interactions
with the photoprotein. The discovery of Obelia FPs fills the four-decade-old gap in the knowledge of
cnidarian bioluminescence and provides experimental material to further investigate the details of its
molecular mechanism.
Introduction
Fluorescent proteins (FPs) homologous to the green fluorescent
protein (GFP) from hydromedusa Aequorea victoria made a
great impact on biological research by providing the means to
genetically encode fluorescent labels, molecular tags, and intracellular environment sensors.1 Much of the progress in FP-based
technologies came as a result of the discovery of novel natural
FPs with unusual properties; therefore, there is still a considerable
interest in the characterization of natural FP diversity. To date,
the most well-characterized group of organisms in terms of
fluorescent proteins are reef Anthozoa, including sea anemones
(order Actiniaria2,3 ), tube anemones (order Ceriantharia4 ), mushroom anemones and button polyps (orders Corallimorpharia and
Zoanthidea5 ), soft corals (order Alcyonaria6 ), and especially hard
corals (order Scleractinia7–12 ). This focus on Anthozoa is not
surprising, provided the spectacular manifestation of their FPs
in the form of bright and often multi-colored fluorescence.13–17 At
the same time, the other class of the phylum Cnidaria – Hydrozoa,
from which the very first FP (GFP) was cloned18 – received
considerably less attention. Thus far, it has yielded 11 proteins,
most of which are green with only two exceptions: a yellow FP
from hydromedusa Phialidium sp.19 , and a non-fluorescent purple
a
Integrative Biology Section, University of Texas at Austin, 1 University
station C0930, Austin, Texas, 78712, United States
b
Institute for Cell and Molecular Biology, University of Texas at Austin, 1
University station C0930, Austin, Texas, 78712, United States
† These authors contributed equally to this work.
chromoprotein from an unidentified anthomedusa.19 No single
hydrozoan species previously studied has yielded multiple FPs
with different colors.
The hydrozoan genus Obelia (order Leptothecata, family
Campanulariidae) contains several well-known bioluminescent
species.20 This genus is the classical textbook example of the
hydrozoan biphasic life cycle, alternating between a sedentary
colonial hydroid and a solitary free-swimming medusa.21 In the
early 1970s, James Morin and co-workers thoroughly characterized the bioluminescence of the Obelia hydroids, including
spectroscopy,22 physiology,23 morphology,24 and biochemistry.22,25
These works have firmly established the existence of an accessory
green fluorescent protein within the Obelia bioluminescent system,
as well as in the jellyfish Aequorea.22,26 The GFP from Aequorea
victoria was cloned18 and soon became an indispensable research
tool after it was demonstrated that it could be used as a genetically
encoded fluorescent label.27 Obelia GFP to this day did not
enjoy such a success. Several attempts to clone Obelia GFP
from the hydroid life stage of the organism failed, including the
authors’ own earlier studies, as well as efforts of other research
groups (Konstantin A. Lukyanov, personal communication) To
our knowledge, no attempts have been made thus far to clone
GFP from an Obelia medusa, despite the earlier observation
that it also exhibits a characteristic green bioluminescence, and
moreover possesses additional yellowish fluorescence that is not
associated with bioluminescent regions.24 Here, we searched for
the elusive green fluorescent protein in the medusa stage, with
an additional hope of discovering GFP-like proteins of different
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Fig. 1 Morphology of Obelia medusa (A) and localization of fluorescence (B). Scale bar: 0.2 mm.
emission colors within a single species of Hydrozoa. We were also
encouraged by the fact that the putatively closely related GFP
from Aequorea is the only known natural FP that is effectively
monomeric (dissociation constant K d = 0.11 mM28 ), which makes
it a very versatile molecular tag.1
Phylogeny
Results
Amino acid sequences of the three Obelia FPs are very similar to
each other (Fig. 2A). In the phylogeny of all hydrozoan FPs known
to date, supplemented with non-hydrozoan outgroups, the three
new proteins cluster together within a clade of FPs from order
Lepthothecata (Fig. 2B), in accord with the taxonomic affiliation
of the genus Obelia.
Fluorescence of Obelia medusae
Spectral properties
The general morphology and fluorescence of Obelia sp. medusae
investigated here are shown in Fig. 1. The apparently orangefluorescent areas in Fig. 1B are most likely due to yellow FP, which
photographs orange because of the way the double-bandpass
FITC/TRITC filter partitions the excitation-emission ranges. The
orange-fluorescent areas include tentacular bulbs of the ring canal,
gonads, and stomach. Green fluorescence is localized predominantly in the oral arms and the tissue surrounding the gonads. In
other Obelia sp. individuals from the same plankton tow (which
may or may not be the same species), the orange fluorescence was
often much weaker than in the individual depicted on Fig. 1B, and
was replaced by green fluorescence. Note that the FITC/TRITC
filter used to obtain the fluorescent image presented in Fig.
1B does not discriminate between cyan and green FP color
types.
The spectral properties of the new proteins are summarized in Fig.
3 and Table 1. The emission curve of the cyan protein (Fig. 3A)
is not as blue-shifted as in many cyan FPs from Anthozoa,9,12 but
still is relatively wide and with a maximum below 500 nm, which is
characteristic of the cyan color class. Its excitation spectrum with
a single mode at 400 nm indicates that its chromophore exists in
a neutral ground state,29 rather than in anionic state like in most
natural FPs. The excitation and emission spectra of the green
protein (Fig. 3B), as well as its brightness characteristics (Table
1), are typical of natural FPs with a GFP-like chromophore in
anionic ground state.29 The yellow protein is notably similar in its
spectral characteristics (Fig. 3C, Table 1) to the long-wavelength
emitting mutant version of the GFP from A. victoria, YFP.30
FP-encoding transcripts in Obelia
All three new proteins were found to form massive soluble complexes. obeCFP and obeYFP failed to enter the gel, while obeGFP
was detected as a very low mobility band and a minor band of
higher mobility (Fig. 4A, B). In the more precise analysis using
size-exclusion chromatography, each protein eluted as a single
peak containing complexes of well-defined molecular masses,
according to the light scattering measurements (Fig. 4C). The
numbers of individual polypeptides per oligomer that most closely
matched the measured molecular masses were 128 for obeCFP,
67 for obeGFP, and 124 for obeYFP. These measurements were
in agreement with the qualitative pattern seen in SDS-PAGE
(Fig. 4A, B), with the only exception of a minor low-molecularweight fraction in obeGFP. This fraction was not detected by sizeexclusion chromatography, and therefore most likely represented
the result of partial denaturation of obeGFP oligomers in presence
of SDS.
From the original cloned cDNA library, 14 fluorescent colonies
(12 cyan and 2 yellow) were identified and sequenced. A second
round of cloning from the same cDNA library was performed
using a pair of primers bearing expression-facilitating sequences
at their 5¢-ends, which were designed to anneal to the 5¢- and
3¢-most portions of the identified ORFs. The primers were not
expected to discriminate between proteins of different color due
to nearly identical sequence within the corresponding portions of
their ORFs. Cloning of the product of this amplification yielded
predominantly fluorescent cyan and yellow colonies, but also
contained a small number of colonies that were fluorescing green.
12 colonies of each color were sequenced and matched to the
data obtained initially to confirm the sequences of the UTRs and
primer-annealing sites in cyan and yellow FPs.
Photochem. Photobiol. Sci.
Oligomerization
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Fig. 2 Sequences of Obelia GFP-like proteins. (A) Alignment of amino acid sequences of the three proteins against GFP from Aequorea victoria. In
obeGFP and obeYFP, the residues identical to obeCFP are denoted as dots. Question marks in obeGFP indicate unknown residues: these regions in
the expressed obeGFP were derived from cloning primers, which encoded the same sequence for all three proteins; the original obeGFP cDNA has not
been identified. The black line above the alignment denotes the chromophore-forming triad. The numeration above the alignment corresponds to residue
numbers in GFP. (B) Phylogenetic tree of all known GFP-like proteins from class Hydrozoa, with two anthozoan and one bilaterian outgroups. The
origin of the ctenophoran protein hbeeGFP requires verification, and may actually be hydrozoan.1 Bipartitions with posterior probability less than 0.95
were collapsed; all the bipartitions shown have posterior probability exceeding 0.98. Scale bar: 0.2 amino acid replacements per site.
Table 1 Fluorescence characteristics of the new proteins
Protein
Excitation max, nm
Emission max, nm
ME, M-1 cm-1
QY
Relative brightness
obeCFP
obeGFP
obeYFP
EGFP (reference)
400
502
514
489
499
515
528
509
36 000
78 000
77 000
55 000
0.62
0.62
0.57
0.6
0.68
1.47
1.33
1
Discussion
Sequences and spectral properties of Obelia FPs
The success of FP cloning from the medusa, while the previous
attempts to clone these proteins from the polyp life cycle stage
failed, may be attributable to a range of possible causes that affect
the success rate of the bacterial expression method. Possibly, FP
transcripts in the polyp have untranslatable sequences of the 5¢UTRs, or very long 3¢-UTRs that would render the cloning of
their full-length open reading frames unlikely, due to alternative
splicing or expression of different FP paralogs. It is also possible
that polyps simply express their FPs at lower levels.
The Obelia FPs described here fluoresce in three colors: cyan,
green, and yellow (Fig. 3), which is the first report of multi-colored
FPs from a hydrozoan representative. Cyan and yellow proteins
(obeCFP and obeYFP) were considerably more abundant than
the green (obeGFP) judging by the frequency of corresponding
clones during both stages of the cloning process (see Methods).
The sequences of all three Obelia FPs are very similar and
cluster together within the phylogeny of hydrozoan FPs (Fig. 2B),
indicating that the evolution of different Obelia colors happened
relatively recently and independently of occurrences of similar
colors elsewhere in the phylogeny. This pattern resembles the
repeated FP color diversifications within a group of reef-building
corals, Scleractinia.12
In contrast to Scleractinia, however, Obelia did not evolve a true
red-emitting protein with an extended chromophore structure.31,32
The yellow Obelia protein (obeYFP) is the second natural protein
with just slightly red-shifted excitation-emission characteristics,
the first one being phiYFP from a related hydromedusa Phialidium.19 With the emission maximum at 528 nm, obeYFP is
much less red-shifted than phiYFP (emission maximum 537 nm),
resembling more the mutant version of the GFP from A. victoria,
YFP,30 both in excitation-emission (Fig. 3C) and brightness
characteristics (Table 1). Similarly to phiYFP and YFP, the red
shift in obeYFP was most likely achieved by the incorporation
of tyrosine residue at position 203 (according to A. victoria GFP
numeration, Fig. 2A).
Excitation maximum at 400 nm in the cyan protein obeCFP,
ostensibly indicating a neutral ground state of the chromophore,29
is uncommon in natural FPs, and among cyan FPs has been
seen in only two coral proteins, psamCFP and mmilCFP.12 In
these two proteins, the sequence determinant of the unusual
excitation profile was shown to be glutamic acid in position 167
(GFP numeration).12 This position is occupied by glutamine in
all three Obelia proteins, and therefore cannot be responsible
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Fig. 3 Excitation (dashed lines) and emission (solid lines) spectra of the
new proteins: (A) obeCFP, (B) obeGFP, (C) obeYFP. The position of the
peaks (wavelength in nanometres) are indicated above each panel.
for differences in emission color or excitation spectrum. Another
residue that may be affecting obeCFP fluorescence characteristics
is Asp 148.33 The three Obelia proteins feature different side chains
at this position (Fig. 2), which makes it a particularly promising
candidate. The sequence determinants of color diversity on Obelia
FPs thus represent an interesting problem for future site-directed
mutagenesis studies.
Oligomerization
Contrary to our expectations inspired by the monomeric nature
of GFP from A. victoria,28,34 Obelia FPs were found to assemble
into complexes of up to 128 individual polypeptides (Fig. 4). The
majority of natural FPs tend to form very large aggregates when
expressed in bacteria,35 which is most likely attributable to nonspecific interactions between FP polypeptides. The aggregation
properties have been characterized in detail for the yellow FP
zFP538,36 cloned from a button polyp (class Anthozoa, order
Zoanthidae).5 The aggregates formed by zFP538 are extremely
large, variable in size (1000–10 000 individual polypeptides), and
dissociate upon dilution during gel-filtration chromatography. In
contrast to zFP538, Obelia FPs are of relatively small and welldefined sizes, and remain stable during chromatography (Fig. 4C).
Photochem. Photobiol. Sci.
Fig. 4 Oligomerization analysis. (A) Fluorescence of the SDS-PAGE gel
with either native (unheated, “u”) or denatured (heated, “h”) samples of
Obelia proteins. EGFP serves as the approximate monomeric size standard;
the positions of bands in the marker lane “M” are irrelevant for this image.
All the fluorescence in native obeCFP and obeYFP remains in the wells,
and the main fluorescent bulk of obeGFP barely enters the gel. (B) Same gel
as in (A), stained with Coomassie Blue to reveal bands of denatured protein
along with native bands. Numbers by the “M” lane indicate molecular
weight in kDa. (C) Size-exclusion HPLC elution profiles of Obelia proteins.
Molecular masses for each elution product (in megadaltons, MDa)
are indicated, according to light-scattering measurements. The numbers
in the parenthesis give the numbers of individual polypeptides in the
complex, assuming molecular weights calculated from the sequences of
the corresponding proteins.
An ability to form such well-defined and stable complexes is a novel
feature among FPs and may have important implications for their
molecular function. In the work that established the existence of a
green fluorescent protein within the Obelia bioluminescent system,
Morin and Hastings22 reported that bioluminescent material from
Obelia, unlike the one from Aequorea,26 was purified in the form of
sub-cellular granules, containing the photoprotein and the green
fluorescent protein. The structure of the granule prevented the
externally added Ca2+ ions from accessing the photoprotein and
triggering the luminescence. The granules could be dissociated by
the addition of freshwater, which resulted in green luminescence if
Ca2+ was also present. The formation of large soluble complexes
by individually expressed Obelia FPs may indicate their direct
involvement in the assembly of such photoprotein-enveloping
granules, and prompts further investigations into their selfassembly properties.
Implications for biological function
The Obelia example makes it tempting to speculate that multicolored fluorescence serves a wider general purpose in cnidarians
than suggested by some of the previous hypotheses formulated for
coral FPs, which assume the link to symbiosis with dinoflagellate
algae (“zooxanthellae”), such as photoprotection37,38 or regulatory
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photosynthesis modulation.39 Still, although the complement of
FP colors in corals also typically includes cyan, green, and
red-shifted varieties,9,12 the similarity with Obelia FPs may be
superficial. Obelia FPs are not as diverse in their spectral
properties as coral FPs, and clearly participate in at least one
function that does not apply to corals: bioluminescence.22 Notably,
bioluminescence seems to be not the only function they serve:
in Obelia medusae, the only bioluminescent region is the ring
canal,24 whereas fluorescence is observed in other parts as well,
such as oral arms, stomach, and gonads (ref. 25, Fig. 1). One
alternative possibility is prey attraction,40 which seems rather
unlikely due to very unusual morphology and feeding habits of
Obelia medusa: instead of being a predator like other, larger,
hydromedusae, the tiny Obelia appears to be a microphagous
filtrator that is able to efficiently feed on bacteria.41 This notion
is supported by the fact that the only identifiable items in the
gut content of Obelia medusae were tests of tintinnids (minute
planktonic ciliates) instead of crustaceans or their larvae.42 The
aposematic interaction with visual daytime predators19 is yet
another possible function, requiring experimental verification.
It is also possible that the function of Obelia FPs is tied to
some other aspect of their biochemistry rather than fluorescence,
such as deactivation of reactive oxygen species.43 Finally, one
intriguing, although highly speculative, hypothesis is suggested
by the recent discovery that at least some FPs are capable
of photo-reduction of various compounds in the cell.44 If so,
Obelia FPs may mediate the regeneration of coelenterazine (Obelia
luciferin,45 ) following its oxidation in the bioluminescent reaction.
This exciting possibility can now be explored using recombinant
Obelia FPs in combination with Obelia photoprotein (obelin),
which was cloned almost two decades ago.46
Experimental
Specimens
The medusae were collected in a surface plankton tow at midday,
at Friday Harbor, Seattle, WA, USA, in July 2007, examined
for fluorescence using stereomicroscope MZ FL III (Leica,
Bannockburn, IL), and photographed using Canon G6 camera attached to the microscope with the FITC-TRITC double-bandpass
filter set (Chroma Technology # 51004v2). The medusae were
identified to the genus Obelia (Cnidaria, Hydrozoa, Leptothecata,
Campanulariidae); no further taxonomic keys exist to identify
species of this genus by the medusoid life cycle stage only.47 The
individuals exhibiting multiple fluorescent colors (Fig. 1B) were
selected for FP cloning.
Bacterial expression library
Total RNA from 4 medusae was extracted using RNAqueous
Micro kit (Ambion; Austin, TX). The cDNA was synthesized
and bacterial expression libraries were constructed as described
previously.48 Briefly, the cDNA was produced and amplified using
the SMARTer cDNA amplification kit (Clontech; Mountain View,
CA, USA), but using a modified primer to achieve preferential
amplification of longer cDNA fragments49 that would be more
likely to contain full-length coding regions. Then, this cDNA
was re-amplified with three primers of different length to ensure
coverage of three possible reading frames upon fusion with the
vector-encoded lacZ peptide. The product of amplification was
ligated into pGEM-T vector (Promega; Madison, WI, USA)
and transformed into Top10 E. coli strain (Stratagene; Cedar
Creek, TX, USA). The transformants were plated onto agar
plates containing the inducer of the lac promoter and screened
for fluorescent colonies using fluorescent stereomicroscope MZ
FL III (Leica, Bannockburn, IL, USA) after incubating overnight
at 37 ◦ C. The screening was repeated after an additional several
days of incubation at room temperature. A total of about 5 ¥ 105
transformed colonies were screened.
Phylogenetic analysis
The sequences of the new proteins were added to the alignment of all hydrozoan FPs, which also included two anthozoan
FPs and one arthropod FP as outgroups. The accession numbers of the aligned sequences were: GFP, P42212; acorNOFP,
AY151052; aldersGFP, ACC54354; amacGFP, AF435432;
clytiaGFP, 2HPW_A; anm1GFP1, AY485334; anm1GFP2,
AY485335; hbeeGFP, ACX47247; anm2CP, AY485336; rrenGFP,
AF372525; DsRed, AF168419; cpGFP, AB185173; abeGFP,
HQ699261; Ember, HQ699262. The phylogenetic analysis was
performed using MrBayes 3.1,50 with the following settings:
aamodelpr = mixed, ngen = 1000000, printfreq = 500, samplefreq =
200, nchains = 4, burnin = 2500.
Expression and purification of fluorescent proteins
The fluorescent clones from the expression library were sequenced
using Sanger method. The sequences of the clones were assembled
using SeqMan II software (Lasergene 7.2 package; Dnastar, Madison, WI, USA). After identification of the complete open reading
frames (ORFs), they were amplified from the original cDNA
using primers bearing 5¢-heels to facilitate bacterial expression and
purification of the protein, as described earlier.9 Briefly, the primer
corresponding to the start of the ORF had a 5¢-heel with stop
codons in three frames to terminate any overlapping translation,
followed by a Shine-Dalgarno sequence (the ribosome binding
site) and a six-base linker before the initiation codon, while the
primer corresponding to the end of the ORF encoded six histidine
residues inserted before the stop codon, to facilitate metal-affinity
purification of the expression product. The amplification products
were ligated into pGEM-T vector (Promega; Madison, WI, USA)
and transformed into Z-competent XJb autolysis E. coli cells
(Zymo Research, Orange, CA, USA). The fluorescent clones
from the resulting plates were streaked onto new Luria-Bertaniagar plates and grown for 1–4 days at room temperature to
accumulate bacterial biomass for protein purification. The product
of expression was purified from the bacteria using metal-affinity
chromatography on Ni-NTA agarose (Qiagen, Valencia, CA,
USA) according to the manufacturer’s protocol.
Spectroscopy
The excitation and emission spectra of all the bacterial expression
products were measured using LS-50B spectrofluorometer (Perkin
Elmer), and corrected for the photomultiplier sensitivity. The
brightness parameters (molar extinction coefficient, ME, and the
quantum yield of fluorescence, QY) were measured as described
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previously.12,19 Briefly, to obtain ME, the absorption of the
protein at the maximum was divided by the concentration of
the protein possessing fully mature chromophore, determined
from the chromophore’s absorption at 445 nm in the alkalidenatured protein preparation (ME = 44 000 M-1 cm-1 ). In the
QY measurement using a relative method, the integrated total
fluorescence of the new proteins was compared to the one of EGFP
(Biovision, Mountain View, CA; QY = 0.651 ) at the same range of
absorptions.
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Oligomerization analysis
9
10
11
The oligomeric state of the bacterial expression products was
initially evaluated by SDS-PAGE of unheated protein samples
in a 4–15% gradient gel with SDS-Tris-Glycine buffers (BioRad, Hercules, CA, USA). This method takes advantage of the
fact that natural FPs typically retain their native structure and
fluorescence in presence of SDS at low temperature.35,52 As globule
size standards the monomeric EGFP (BioVision, Mountain View,
CA, USA) and the tetrameric DsRed (Takara Bio - Clontech,
Mountain View, CA, USA) were used. The molecular weight of
the oligomers was more accurately measured by means of sizeexclusion HPLC with combined detection via multi-angle light
scattering and refractometry,53 using Wyatt MALS instrumentation (Wyatt Technology; Santa Barbara, CA, USA). The buffer
used during chromatography contained 10 mM HEPES, 100 mM
NaCl, pH 7.4.
Acknowledgements
12
13
14
15
16
17
18
We thank Dr Austen Riggs and Mrs Claire Riggs (UT) for assistance with oligomerization analysis and insightful discussions.
This work was supported by the National Institutes of Health
grants R01 GM078247 and R01 GM066243, and National Science
Foundation grant IOS-1052461 to M.V.M.
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