Báo cáo khoa học: A new phospholipase A2 isolated from the sea anemone Urticina crassicornis – its primary structure and phylogenetic classification pptx

A new phospholipase A2 isolated from the sea anemone
Urticina crassicornis – its primary structure and
phylogenetic classification
ˇ
ˇ
ˇ
ˇ
ˇ
ˇ
Andrej Razpotnik1, Igor Krizaj2, Jernej Sribar2, Dusan Kordis2, Peter Macek1, Robert Frangez3,
William R. Kem4 and Tom Turk1
1
2
3
4

Biotechnical Faculty, Department of Biology, University of Ljubljana, Slovenia
ˇ
Department of Molecular and Biomedical Sciences, Jozef Stefan Institute, Ljubljana, Slovenia
Faculty of Veterinary Medicine, University of Ljubljana, Slovenia
Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, Gainesville, FL, USA

Keywords
enzymatic activity; phospholipase A2;
phylogenetic classification; sea anemone;
sequence; venom
Correspondence
T. Turk, Biotechnical Faculty, Department of
ˇ
Biology, University of Ljubljana, Vecna pot
111, SI-1000 Ljubljana, Slovenia

Fax: +386 1 257 33 90
Tel: +386 1 423 33 88
E-mail:
Database
The nucleotide sequence described in
this article has been submitted to
EMBL ⁄ GenBank ⁄ DDBJ under the accession
number EU003992
(Received 26 February 2010, revised 7 April
2010, accepted 8 April 2010)

Disulfide pairings and active site residues are highly conserved in secretory
phospholipases A2 (PLA2s). However, secretory PLA2s of marine invertebrates display some distinctive structural features. In this study, we report
the isolation and characterization of a PLA2 from the northern Pacific sea
anemone, Urticina crassicornis (UcPLA2), containing a C27N substitution
and a truncated C-terminal sequence. This novel cnidarian PLA2 shares
about 60% identity and almost 70% homology with two putative PLA2s
identified in the starlet sea anemone (Nematostella vectensis) genome
project. UcPLA2 lacks hemolytic and neurotoxic activities. A search of
available sequences revealed that Asn27-‘type’ PLA2s are present in a few
other marine animal species, including some vertebrates. The possibility
that the C27N replacement represents a structural adaptation for PLA2
digestion ⁄ activity in the marine environment was not supported by experiments testing the influence of ionic strength on UcPLA2 enzymatic activity.
Because of the highly divergent sequences among invertebrate group I
PLA2s, it is currently not possible to identify orthologous relationships. As
the Asn27-containing PLA2s are scattered among the other invertebrate
group I PLA2s, they do not constitute a new, monophyletic PLA2 clade.

doi:10.1111/j.1742-4658.2010.07674.x

Introduction
Phospholipases A2 (EC 3.1.1.4) (PLA2s) catalyze the
hydrolysis of the ester bond at the sn-2 position of
1,2-diacyl-sn-phosphoglycerides. These enzymes are
currently divided into 15 structural groups [1].
Recently, a new PLA2 from adipose tissue was characterized and tentatively placed into a new group
(group XVI) [2]. Some PLA2s are toxic and have been
found in the venoms of insects, mollusks, snakes, and

many marine invertebrates, such as cnidarians [3] or
sponges [4]. Marine invertebrate PLA2s are generally
poorly characterized, with some notable exceptions,
such as those from the starfish Acanthaster planci [5,6]
and Asterina pectinifera [7,8], and the cone snail
Conus magus (conodipine M) [9].
In contrast to the PLA2s from marine invertebrates,
snake venom PLA2s are very well known. These can

Abbreviations
AcPLA2, Adamsia carciniopados phospholipase A2; AtxC, ammodytoxin C; PDB, Protein Data Bank; PLA2, phospholipase A2; PyPC,
1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine; PyPG, 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoglycerol;
UcPLA2, Urticina crassicornis phospholipase A2.

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be cytotoxic, myotoxic, and ⁄ or neurotoxic; also, they
may interfere with blood coagulation and activate
inflammation processes [10,11]. In general, toxic PLA2s
have been classified into three structural groups [12].
Most of the Elapidae snake venoms contain group I
PLA2s, whereas Viperidae snake venoms harbor
group II PLA2s. Several PLA2s from both groups bind
with high specificity to presynaptic membranes. These
enzymes, also known as b-neurotoxins, block neuromuscular transmission by impairing the cycling of synaptic vesicles within motoneuron terminals [13].
Sea anemones are other, albeit much less well
known, sources of PLA2s. According to Nevalainen
et al. [3], the distribution of PLA2s among members
of the phylum Cnidaria is widespread, but their enzymatic activities vary significantly between different
species. There are only a few reports on isolated cnidarian PLA2s that have been characterized more
thoroughly. According to these reports, it seems that
cnidarian PLA2s form a structurally diverse group of
proteins, mainly showing cytolytic activity [14,15].
Aiptasia pallida nematocyst venom contains at least
three synergistically acting proteins, two of which are
PLA2s. One of these two enzymes is composed of
two isozymes with molecular masses of 45 and
43 kDa (forms a and b, respectively). The b-form was
purified to homogeneity. It is a single-chain glycoprotein with a pI of 8.8. The b-form contributes about
70% of the total phospholipase activity of the venom
[16,17]. A similar PLA2 probably exists in the venom
of the related sea anemone Aiptasia mutabilis, as
reported by Marino et al. [18]. Very recently, a PLA2
belonging to group III was isolated from the Brazilian sea anemone species Bunodosoma caissarum [19].

The N-terminal amino acid sequence of the B. caissarum PLA2 shows significant homology with PLA2s
present in bee and gila monster lizard (Heloderma
suspectum) venoms. PLA2 activity was also detected
in homogenized sea anemone tissues, including the
tentacles and acontia (structures involved in hunting
and defense) of the sea anemone Adamsia carciniopados
(AcPLA2) [20]. Nested RT-PCR with degenerate
primers and RACE was used to clone the PLA2 from
this animal. AcPLA2 contains a putative prepropeptide of 37 amino acids, ending with a basic doublet,
followed by a mature protein of 119 amino acids,
including 12 cysteines. AcPLA2 has more than 50%
similarity with other known secretory-type PLA2s.
The C-terminal extension, typical of group II and
group X PLA2s, is absent. The predicted molecular
mass and pI of the mature protein are 13.5 and
9.1 kDa, respectively. This PLA2 clearly differs from
the one from A. pallida.
2642

Here we report on the isolation, cloning and characterization of a novel PLA2 from the northern Pacific
sea anemone Urticina crassicornis (UcPLA2). This new
PLA2 (UniProt UcPLA2 A7LCJ2) is homologous with
AcPLA2, and, with regard to many structural features,
is also similar to Elapidae snake neurotoxic PLA2s
belonging to group I, suggesting a similar functional
role in snake and cnidarian venoms. However,
UcPLA2 has some unusual structural features, most
notably an asparagine at position 27 instead of cysteine, which is present in the majority of known group I
and group II PLA2s. This replacement is rare in invertebrate PLA2s, and has not been found yet in vertebrate toxic and nontoxic PLA2s of group I and
group II, with a single exception, a sea lamprey PLA2.

Also, in UcPLA2 there is a C-terminal truncation of
six amino acids, including a cysteine, so the usual pairing between Cys27 and Cys126 is not possible.
Recently, several similar proteins were also detected
during the starlet sea anemone (Nematostella vectensis)
genome project, implying that this type of PLA2 might
be more widespread among the Cnidaria [21,22].
Besides the Asn27 PLA2, numerous other group I
PLA2s have been discovered that cannot be easily
incorporated into the existing classification scheme,
resulting in a growing problem in the comprehensive
evolutionary classification of the secretory PLA2 superfamily [1]. In this study, we have provided some new
insights into the possible origin, distribution, diversity,
evolution and classification of the metazoa-specific
group I PLA2 family.

Results
Isolation of wild-type UcPLA2
The exudates resulting from milking of U. crassicornis
specimens contained some mucus, in which the sea
anemones were covered. It was therefore filtered
through a 0.45 lm membrane prior to concentration,
in order to avoid clogging of the ultrafiltration membrane. The concentrated sample was applied to a gel
filtration column in order to desalt the sample and
exchange sea water for an appropriate buffer
(Fig. 1A). Fractions with the highest absorbance and
rate of hemolysis were further separated on a cation
exchange column (Fig. 1B). The elution profile showed
three completely separated peaks, the final two of
which exhibited hemolytic activity. SDS ⁄ PAGE
showed the pattern of the last peak separated into two

bands (Fig. 1D). The N-terminal amino acid analysis
revealed that the protein of  14 kDa was homologous
to PLA2s (UcPLA2), whereas the protein of  20 kDa

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New PLA2 from Urticina crassicornis

A. Razpotnik et al.

A

B

C

D

Fig. 1. Isolation of native UcPLA2. Native UcPLA2 was isolated from the exudate in a three-step chromatography procedure. (A) Gel filtration
on a Superdex 75 HR 10 ⁄ 30 FPLC column in 10 mM ammonium acetate (pH 5.3) running buffer at a flow rate of 0.5 mLỈmin)1. Separation
was repeated several times with 0.5 mL aliquots. (B) Hemolytic fractions were pooled and separated on a Mono S HR 5 ⁄ 5 cation exchange
column at a flow rate of 1 mLỈmin)1 in the same ammonium acetate buffer as used for gel filtration. Bound proteins were eluted with a linear gradient of 0.0–1.0 M NaCl. (C) Peak 3 fractions were subjected to RP-HPLC, using a Vydac 218TP C18, 4.6 · 250 mm, 7 lm reverse
phase column. Proteins were eluted with an acetonitrile gradient of 0–70% (v ⁄ v) in 0.1% trifluoroacetic acid (v ⁄ v) at 1 mLỈmin)1. (D) A sample of the cation exchange chromatography peak 3 separated on a 12.5% SDS ⁄ PAGE gel.

was homologous to cytolytic proteins of the actinoporin family, which are probably responsible for the
hemolytic activity observed in fractions containing
UcPLA2. The 20 kDa protein was removed by
RP-HPLC to obtain sequentially pure UcPLA2, which
was not hemolytic (Fig. 1C).

The purification yield of UcPLA2 decreased with
each subsequent milking, in proportion to the length
of time for which sea anemones were held in captivity.
In preparations from the third and fourth milkings,
UcPLA2 was only barely detectable.

the four bases might occur. Performance of 3¢-RACE
with the degenerate primer on the RACE-ready cDNA
resulted in a predominant  600 bp product with some
minor impurities when the PCR reaction products
were separated by agarose electrophoresis (not shown).
The PCR product was cloned and sequenced. The
gene-specific reverse primer was constructed from
the cDNA sequence obtained by 3¢-RACE, and a
5¢-RACE reaction was carried out. Again, the
 500 bp PCR product was cloned and sequenced.
The complete cDNA sequence was obtained by
combining the 3¢-RACE and 5¢-RACE sequences.

N-terminal sequencing of wild-type UcPLA2
Wild-type UcPLA2 was sequenced from its N-terminus
to obtain the information necessary for the synthesis
of a suitable DNA primer. Clear signal was legible up
to amino acid 25. The main signal corresponded to the
following sequence: NLLQFSSMIKCATGRSAWKYDNYGN.
RACE
A degenerate primer was designed by utilizing a part
of the sequence showing the least degeneracy and
incorporating an inosine at the position where any of

Overexpression, refolding and purification of the
recombinant UcPLA2
Our first attempt was to overexpress UcPLA2 by using
the pT7-7 vector [23] and cloning the mature UcPLA2
sequence directly after the start codon. After successful
expression, isolation of inclusion bodies, and refolding
of the recombinant protein, we subjected the protein
to amino acid sequencing. At the N-terminus of the
recombinant UcPLA2, we discovered a methionine preceding the first amino acid of the mature UcPLA2 (not
shown). As addition of amino acids at the N-terminus
interferes with the enzymatic activity of secreted PLA2s

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New PLA2 from Urticina crassicornis

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[24], we decided to clone the UcPLA2 sequence into
the pTolX plasmid [25], encoding an N-terminal fusion
protein that enhances solubility of the product and
contains the factor Xa protease restriction site (IEGR).
Nevertheless, expression of the protein in Escherichia
coli BL21(DE3) resulted in the formation of inclusion
bodies. Following solubilization, the recombinant
fusion UcPLA2 was successfully refolded and purified.
The yield of the 24 kDa construct was approximately

10 mg per liter of medium. The recombinant fusion
protein was successfully cleaved by factor Xa digestion, and the recombinant UcPLA2 was purified to
homogeneity.
Sequence analysis and protein structure
modeling
The cDNA sequence of UcPLA2 (791 bp) was deposited in GenBank under GenBank accession number
EU003992 (Fig. 2). The UcPLA2 cDNA consisted of
an ORF of 468 bp that translated into a 155 amino
acid protein. Taking into account the N-terminal
sequence obtained by Edman degradation of the wildtype protein and sequence data of related secretory
PLA2s, we concluded that the mature protein of 111
amino acids is preceded by a 44 amino acid prepropeptide. The first 19 amino acids of the prepropeptide
represent a cleavable signal peptide [26]. The prepropeptide sequence ends with two basic amino acids,
lysine and arginine, which represent a common
protease-cleavage site. The predicted molecular mass
and pI of the mature UcPLA2 are 12.4 kDa and 8.5,
respectively. A molecular mass of 12.422 kDa was
obtained by a MALDI-TOF experiment (not shown).
The 12 cysteines in the mature form of UcPLA2 probably form six disulfide bridges. Highly conserved
amino acids forming the Ca2+-binding loop
(YGCYCGXGGXG) and the catalytic site (H ⁄ D) in
PLA2s are also present in UcPLA2. A blast search
revealed the highest similarity of UcPLA2 with group I
PLA2s. It shares approximately 35% identity with the
porcine group I PLA2 and about 50% identity with an
elapid snake (Oxyuranus scutellatus) neurotoxic
group I PLA2. In comparison with other sea anemone
PLA2s, UcPLA2 shares about 50% identity with
AcPLA2 [20] and 62% identity with a putative PLA2
from the sea anemone N. vectensis [21]. It also shares

some interesting structural features with the latter that
are not seen in AcPLA2 and vertebrate PLA2s. Alignment of the UcPLA2 amino acid sequence with bovine
PLA2 shows considerable differences between UcPLA2
and vertebrate PLA2s in the cysteine distribution pattern. First, the highly conserved disulfide bond in
2644

Fig. 2. The full-length cDNA sequence of UcPLA2 and translation
of the ORF into protein. The prepropeptide is underlined; the signal
peptide part is in italics. The Ca2+-binding loop is in bold, and the
catalytic dyad (H ⁄ D) is in bold italics. The unique asparagine is indicated by an arrow. The asterisk indicates the stop codon.

PLA2s, Cys27–Cys126, is missing in UcPLA2, because
of the rare C27N substitution and the absence of
Cys126 resulting from the truncation of six amino
acids at its C-terminus, including Cys126 (Fig. 3B).
Second, although Cys11 is present in UcPLA2, its partner for the formation of a disulfide bond, Cys77, is
replaced by histidine. In UcPLA2, the missing cysteine
is shifted to the right, so its Cys80 is a plausible candidate for disulfide bond formation with Cys11. A 3D
model of UcPLA2 confirms such a prediction
(Fig. 4A).
In order to determine which cysteines are paired in
UcPLA2, we built a 3D model of UcPLA2 (Fig. 4A),
based on the crystal structure of the porcine (Sus scrofa) group I PLA2 [27], which exhibits 43% identity
and 53% sequence similarity with UcPLA2. From the
model, it is evident that the Cys27–Cys126 bond is
missing in UcPLA2. However, the equivalent of the
Cys11–Cys77 bond in porcine ⁄ bovine PLA2 is a tentative Cys11–Cys80 bond in UcPLA2 (according to
Renetseder’s numbering [28], if a minimum number of
gaps is introduced into the UcPLA2 amino acid

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New PLA2 from Urticina crassicornis

A. Razpotnik et al.

A

B

Fig. 3. (A) Alignment of PLA2 protein sequences from: the sea anemones Urticina crassicornis (Uc), Nematostella vectensis (Nv) and Adamsia carciniopados (Ac), the taipan snake Oxyuranus scutellatus (Os), and the boar Sus scrofa (Ss). Identical residues are on a black background, and similar residues are on a gray background. The sea anemone unique asparagine replacing the cysteine at position 27 is marked
with an asterisk. The cysteine at position 80 is marked with an arrow. (B) Alignment of UcPLA2 and Bos taurus (Bt) BtPLA2, with cysteine
numbering according to Renetseder. Cysteines are on a black background, and the unique asparagine in UcPLA2 is on a gray background.
Cysteine positions, numbered according to Renetseder, are shown above the alignment, with the positions of the cysteines involved in disulfide bonding in parentheses.

A

B

C1

26

N27

–C

27

C44

C4

–C1

4–C

05

105

Fig. 4. Models of UcPLA2 and AcPLA2.
UcPLA2 (A) was modeled on the basis of
the crystal structure of the porcine PLA2
(PDB 5p2p) and AcPLA2 (B) on the basis of
the crystal structure of D. r. russelli PLA2
(PDB 1vip). Amino acids involved in catalysis
are in blue, and disulfide bridges are in yellow. The unique asparagine of UcPLA2 is in
magenta.

C5

C5

1–C

0

C8

1–C

98

1–

98

C1

C9

6–C

84

C9

6–C

84

C6

1–C

C6

91

sequence in order to align cysteines with those in the
bovine PLA2). We would expect Cys80 in UcPLA2 to
adopt a spatial position similar to that of Cys77 in the
porcine and bovine PLA2s.
As the amino acid sequence alignment of UcPLA2
and AcPLA2 revealed substantial differences between
the two proteins, a 3D model of AcPLA2 (Fig. 4B)
was also constructed. The model was built on the basis
of the crystal structure of Daboia russelli russelli PLA2
[29], which shares 40% identity with AcPLA2. AcPLA2

1–C

91

has the capacity to form six disulfide bridges. It possesses the Cys27–Cys126 pairing, but because it lacks
Cys11, the disulfide bond between Cys11 and Cys77 is
missing.
Hemolytic activity
Purified recombinant and wild-type UcPLA2 did not
exhibit hemolytic activity on bovine red blood cells
(not shown). The hemolytic activity of the UcPLA2

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New PLA2 from Urticina crassicornis

A. Razpotnik et al.

preparation observed in the earlier stages of purification was found to be due to traces of a coeluting actinoporin-like cytolytic protein. This protein was
removed by RP-HPLC, and the purified UcPLA2 was
devoid of hemolytic activity.

Neuromuscular effects of UcPLA2
Neuromuscular toxicity in a mouse hemidiaphragm
neuromuscular preparation was tested only with
recombinant UcPLA2. A neuromuscular effect was not
observed with UcPLA2 after up to 120 min of incubation and with more than 10-fold the concentration of
AtxC, a neurotoxic PLA2 from Vipera ammodytes ammodytes that significantly decreased indirectly stimulated muscle contractions after only several minutes of
exposure to the toxin (not shown).

UcPLA2 enzymatic activity
The phospholipase activities of the wild-type and the
recombinant UcPLA2 were tested on two different substrates, anionic 1-hexadecanoyl-2-(1-pyrenedecanoyl)sn-glycero-3-phosphoglycerol (PyPG) and zwitterionic
1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (PyPC) vesicles. Their phospholipase activities were practically identical, so in all subsequent
measurements only the recombinant enzyme was used.
In comparison with ammodytoxin C (AtxC), UcPLA2
was found to be approximately five-fold and 10-fold
less active on PyPG and PyPC vesicles, respectively.
UcPLA2 is a Ca2+-dependent enzyme. In the presence
of EGTA, it showed no enzymatic activity on either of
the substrates (Fig. 5). In the presence of NaCl, its
enzymatic activity followed the same tendency as that
of the group II AtxC, which possesses a Cys27–Cys126
bond, when either PyPG or PyPC was used as substrate. At 600 mm NaCl, the phospholipase activity of
UcPLA2 was identical to that measured in the absence

of NaCl in the case of both substrates.

A

120

Relative enzymatic activity

Relative enzymatic activity

Phylogenomic analysis has demonstrated that group I
PLA2s are present in all major metazoan taxonomic
groups. Cnidarians, placozoans, protostomes and basal
deuterostome lineages (echinoderms, cephalochordates,
and urochordates) possess highly divergent group I
PLA2 multigene families in their genomes. Members of
the group I PLA2 family are much more conserved
within vertebrates than in numerous invertebrate lineages. In contrast to those in vertebrates, invertebrate
group I PLA2s seem to have undergone much more
complex and dynamic evolution by numerous gene
duplications (forming diverse multigene families),

B
120
100
80
60
40
20

100
80
60
40
20
0

0
AtxC

1 m M CaCl 2

UcPLA 2

EGTA

D

C
120

Relative enzymatic activity

Relative enzymatic activity

Evolutionary classification of the group I PLA2
family – no orthologous group I PLA2s exist in
invertebrates

100

80
60
40
20
0
0

150

NaCl (mM)

2646

600

200
180
160
140
120
100
80
60
40
20
0
0

150

NaCl (mM)

600

Fig. 5. Enzymatic activity of UcPLA2 on
anionic (PyPG) and zwitterionic (PyPC) vesicles. Phospholipase activity assays were
performed in 50 mM Tris ⁄ HCl (pH 8.0) buffer supplemented with 50 mM KCl, 1 mM
CaCl2 or 1 mM EGTA, and 0.09% (w ⁄ v) fatty
acid-free BSA. The final concentration of
lipid vesicles in assays was 4.2 lM, that of
UcPLA2 was 8 ng, and that of AtxC was
1 ng. Fluorescence was measured with a
SAFIRE microplate monochromator reader.
All measurements were taken at the room
temperature. The results are normalized and
displayed as relative values. (A) Comparison
between the phospholipase activity of AtxC
and UcPLA2 on PyPG (gray) and PyPC
(black) vesicles. (B) The enzymatic activity
of UcPLA2 on either PyPG (gray) or PyPC
(black) is Ca2+-dependent. (C) Dependence
of hydrolysis of PyPC vesicles by AtxC
(gray) and UcPLA2 (black) on NaCl concentration. (D) Dependence of hydrolysis of
PyPG vesicles by AtxC (gray) and UcPLA2
(black) on NaCl concentration.

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A. Razpotnik et al.

resulting in the greatest diversity of group I PLA2s in
invertebrate genomes (Fig. S1). This is not surprising
in view of the early appearance of these diverse phyla
during metazoan evolution relative to phylum Chordata.
Previously, the oldest known group I PLA2 representative was from cnidarians, but recently an even
older metazoan group I PLA2 sequence was found in
the Sycon sponge (AM765083). Pfam HMM logo
(PF00068) shows that the vertebrate-specific group II
PLA2s represent just a minor variant of the older
metazoa-specific group I PLA2s. This HMM Logo was
made from 37 group I, group II and group V PLA2
sequences originating from diverse mammals and
snakes. In the above HMM model of group I and
group II PLA2s, a number of absolutely conserved
invariant cysteines are evident (Fig. S2). This HMM
model is based mostly on vertebrate PLA2 sequences.
Surprisingly, the disulfide bonding pattern in the Sycon
sponge group I PLA2 is the same as in vertebrate
group I PLA2s, indicating that the ancestral cysteine
position pattern is as follows: Cys11, Cys27, Cys29,
Cys44, Cys45, Cys51, Cys61, Cys77, Cys84, Cys91,
Cys96, Cys98, Cys105, and Cys126 (Fig. S3). However,
our analysis shows a number of exceptions in the
highly divergent invertebrate group I PLA2s, when
otherwise absolutely conserved cysteine positions are
mutated or lost (Fig. S4).
By searching all available metazoan genomic, proteomic and transcriptomic databases, we found that Asn27
PLA2s are not limited to the sea anemone U. crassicornis. Instead, we found that such variants are also present
in other anthozoans (N. vectensis and Anemonia), but

not in hydrozoans (Hydra). The distribution of Asn27
PLA2s in metazoans is quite interesting, because they
are present in diverse marine organisms, such as anthozoans, placozoans (Trichoplax), and mollusks (Crassostrea and Mytilus), and also in the only known
vertebrate PLA2, that of the sea lamprey (Petromyzon marinus). Furthermore, the Asn27 PLA2s can also
be found in some freshwater organisms, such as crustaceans (Daphnia pulex) and planarians (Schmidtea mediterranea), and even in few terrestrial invertebrates, such
as centipedes (Scolopendra viridis) and tardigrades
(Milnesium tardigradum) (Fig. S4).

Discussion
A novel PLA2 was purified from the milking exudates
of several U. crassicornis specimens. On the basis of
the N-terminal sequence of the isolated protein, its
cDNA was isolated, sequenced, and recombinantly
overexpressed in order to obtain a sufficient amount
of enzyme for detailed studies of its biochemical

New PLA2 from Urticina crassicornis

characteristics. Some differences in the primary structure between the isolated protein and the UcPLA2
cDNA are probably due to intraspecific variability, as
mRNA was obtained from a single animal, whereas
the protein was isolated from exudates of several
U. crassicornis specimens. The novel cnidarian PLA2
is composed of 111 amino acids, and possesses 12
cysteines that putatively interconnect to form six
disulfide bonds. UcPLA2 possesses a structurally
conserved Ca2+-binding loop and the catalytic dyad,
histidine and aspartic acid, at positions 48 and 99,
respectively. In agreement with this, it displayed considerable Ca2+-dependent enzymatic activity on anionic and zwitterionic phospholipid vesicles (Fig. 5).
Despite the structural homology with toxic snake

venom PLA2s, and the fact that RNA encoding
UcPLA2 was isolated from the sea anemone tentacles,
where PLA2s may participate in sting site, irritation,
and systemic envenomation syndrome resulting from
contact with nematocysts [3], UcPLA2 expresses neither cytolytic nor neurotoxic activity, at least not on
the mammalian nerve–muscle preparation. Although
UcPLA2 also possesses the so-called group I ⁄ elapid
loop, which is characteristic of group I PLA2s, from
some other structural points of view it differs significantly from the majority of group I PLA2s. For
example, Cys27, which is, with a single known exception, strictly conserved in vertebrate group I PLA2s,
is replaced by an asparagine in UcPLA2. In addition,
Cys126, which usually forms a disulfide bond with
Cys27, is missing, as UcPLA2 is shorter by several
amino acids at its C-terminus. Therefore, UcPLA2
lacks the Cys27–Cys126 bond, one of the disulfide
bonds that is typical of group I PLA2s. Substitution
of Cys27 and truncation of the C-terminus was also
observed in at least two hypothetical PLA2s from
N. vectensis and also in a significant number of predominantly marine invertebrate PLA2s (Fig. S4).
However, this property is probably not a general feature of sea anemone PLA2s, as both Cys27 and
the ‘normal’ C-terminus with Cys126 are present in
AcPLA2 [20]. In UcPLA2, the C-terminal part of the
molecule is not linked by a disulfide bond to the core
of the molecule, and it is expected to be much more
flexible than in other PLA2s, including AcPLA2.
A usual partner of Cys11 in group I PLA2s is Cys77.
In the case of UcPLA2, Cys77 is replaced by a histidine if a minimum number of gaps is introduced into
the amino acid sequence when aligned with the
bovine PLA2 and Renetseder’s system [28] is used for
cysteine numbering (Fig. 3B). As a consequence of

such alignment and numbering in UcPLA2, Cys11
should be paired with Cys80. Such pairing is also

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A. Razpotnik et al.

supported by the correct spatial proximity of both
cysteines in the 3D model of UcPLA2 (Fig. 4).
The functional significance of the above-mentioned
structural peculiarities of UcPLA2 is not clear. Experiments with bovine or porcine group I PLA2 mutants in
which certain cysteines were replaced by other amino
acids and ⁄ or C-terminal parts were truncated or
extended give us some possible answers to this question
[30–32]. A deletion mutant of the bovine group I PLA2,
in which the C-terminus was shortened by eight amino
acids and the cysteine at position 27 was replaced by an
alanine, showed negligible structural perturbations but
an interesting change in substrate specificity. The catalytic constant (kcat) of the mutated enzyme was about
100-fold lower for the zwitterionic substrate, but
remained essentially unchanged for the anionic substrate. Activity on the zwitterionic substrate was
restored in 4 m NaCl. The authors concluded that the
Cys27–Cys126 bond is not very important for the structural and functional properties of the enzyme, but might
have an important role in determining its substrate specificity and in the uncoupling between substrate and
Ca2+ binding [31]. The dependence on NaCl concentration of UcPLA2 enzymatic activity on anionic PyPG

and zwitterionic PyPC was essentially the same as that
of the group II AtxC, which possesses the Cys27–
Cys126 bond. Moreover, on both substrates at 600 mm
NaCl, the enzymatic activity of UcPLA2 was the same
as that measured in the absence of NaCl. Therefore, we
can conclude that the missing Cys27–Cys126 bond is
not particularly important for substrate recognition in
the variable salt concentrations that might exist in different marine environments. Apart from the deletion of
the Cys11–Cys77 bond, which significantly perturbed
the stability of bovine group I PLA2, site-directed mutagenesis revealed little importance of most of the disulfide bonds for the overall conformational stability of
this enzyme. Furthermore, deletion of the Cys27–
Cys126 bond even resulted in increased conformational
stability of the molecule [30]. The results obtained with
the bovine group I PLA2 were later supported by studies on the porcine group I PLA2 [32]. For example, in
this study the authors demonstrated the importance of
the Cys11–Cys77 bond for the stability of the enzyme.
UcPLA2 lacks this bond; however, according to our
model, it is probably replaced by a unique disulfide
bond between Cys11 and Cys80, which would thus be
important for UcPLA2 conformational stability.
Although homologous with mammalian [27] or snake
venom group I PLA2s [33], UcPLA2 is sufficiently structurally different for the question of its placement into
the existing PLA2 classification scheme to arise. In addition, the two cnidarian PLA2s, AcPLA2 and UcPLA2,
2648

despite possessing many common structural features,
also differ in some important structural properties.
AcPLA2, lacking the Cys11–Cys77 disulfide bond but
otherwise possessing all of the group I PLA2 characteristics, seems to be structurally much closer to group I
PLA2s than UcPLA2 (Fig. 3A). Other known cnidarian

PLA2s, such as those from the hydrozoan Hydra magnipapillata [34] and the sea anemones A. pallida [16,17]
and B. caissarum [19], as well as the PLA2 from the jellyfish Rhopilema nomadica [35], are structurally only distantly related to UcPLA2. However, the starlet sea
anemone N. vectensis [21] genome project revealed the
existence of at least two putative PLA2s that share
about 60% identity and 70% homology with UcPLA2
(Fig. 3A). Like UcPLA2, they possess the C27N replacement, and Cys77 is replaced by either histidine or tyrosine. Both enzymes have truncated C-termini, meaning
that they do not possess Cys126. It seems that UcPLA2type PLA2s are more widespread within the Cnidaria,
especially among sea anemones.
Although orthologous relationships can be easily
reconstructed for vertebrate group I PLA2s, our phylogenetic analysis failed to obtain any evidence for
orthologous groups within invertebrate group I PLA2s.
However, this analysis has shown that group I PLA2s
underwent numerous gene duplication events within
the following groups: Cnidaria, Protostomia, and basal
Deuterostomia (echinoderms, cephalochordates, and
urochordates). Phylogenetic analysis therefore provides
evidence that, in invertebrates, a large number of species-specific multigene families evolved from a single
ancestral group I PLA2 and became highly diversified
by adaptive evolution, like group II PLA2s in snake
venoms [36].
Also, a long-standing assumption has been the conservation of disulfide bridges [37]. Although this conservation is apparent in mammals and other
vertebrates, it is less certain in the case of invertebrates
and basal metazoans. Nevertheless, the amino acid
sequence of the Sycon group I PLA2 demonstrates that
the ancestor of group I PLA2s possessed the same pattern of disulfide bridges as observed in vertebrate
group I PLA2s (Fig. S3).
The analysis of the conservation of disulfide bridges
shows that invertebrate group I PLA2s possess numerous mutations at otherwise absolutely conserved cysteine positions, as observed in Urticina and Adamsia
PLA2s. Despite the presence of the sequence motif
NWC or its variants (NYC, NFC, NHC, HYC and

RYC), the level of sequence conservation between
Asn27 PLA2s is quite low (Fig. S4). Asn27 PLA2s are
structurally diverse; they contain 10–14 cysteines, some
of them have a shortened C-terminal part (Urticina),

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A. Razpotnik et al.

and others may possess C-terminal extensions. Some
Asn27 PLA2s possess unique positions of cysteines,
whereas others have lost the Cys11–Cys77 disulfide
bridge in addition to the characteristically lost Cys27–
Cys126 bond. In Schmidtea sp., we observed a triple
natural PLA2 mutant that had lost both the Cys11–
Cys77 and Cys27–Cys126 disulfide bridges, and also
the C-terminal part of the molecule. In the group of
currently available representatives of Asn27 PLA2s, we
can observe structural plasticity in the surface
(group I) loop (e.g. insert of a 16 amino acid sequence
after Cys77), the C-terminal part of the molecule might
be shortened or extended, and, as far as disulfide
bridges are concerned, the Cys27–Cys126 pair is
always missing, and sometimes the Cys11–Cys77 pair
is also missing. Although these structural mutants
might have evolved as structural and functional adaptations for the optimal digestion of dietary lipids in
marine, freshwater and terrestrial environments, our
findings concerning enzymatic activities obtained with
different substrates and different salt concentrations

do not support this hypothesis.
The evolutionary (orthologous) relationships in the
group I PLA2s can be reconstructed for vertebrates,
but not for invertebrates. Because of their highly divergent sequences, the recognition of orthologous relationships among group I PLA2s of invertebrates is not
possible at present. In the phylogenetic tree, the
Asn27-containing PLA2 sequences do not form a
monophyletic clade, and are instead scattered among
the invertebrate group I PLA2s, indicating that they
evolved independently several times. We can conclude
that neither Asn27 PLA2s nor any other invertebrate
group I PLA2s can presently be classified as a new
subfamily or subgroup of group I PLA2s.

Experimental procedures
Native UcPLA2 isolation
Live U. crassicornis sea anemones were collected by Westwind SeaLab Supplies (Victoria, British Columbia, Canada)
and shipped on ice to the University of Ljubljana (Slovenia), where they were held in a fish tank at 10 °C.
The exudate obtained by milking, i.e. gentle squeezing of
fully expanded sea anemones until totally contracted, was
filtered through a 0.45 lm filter membrane (Sartorius,
Goettingen, Germany) and concentrated in a 200 or 50 mL
Amicon Stirred Cell (Millipore, Billerica, MA, USA), using
the YM-3 membrane with a molecular mass cutoff of
3000 Da, to a volume of approximately 2 mL.
A three-step chromatography procedure was required to
purify the protein sufficiently for chemical analysis. The

New PLA2 from Urticina crassicornis

ă

initial two steps were performed on an Akta FPLC
apparatus (GE Healthcare, Uppsala, Sweden). First, an
aliquot of the concentrated exudate was separated on a
Superdex 75 HR 10 ⁄ 30 gel filtration column using 10 mm
ammonium acetate (pH 5.3) running buffer at a flow rate
of 0.5 mLỈmin)1. This procedure was repeated several times
with 0.5 mL aliquots in order to maintain optimal resolution. Then, fractions displaying hemolytic activity were
pooled and applied to a Mono S HR 5 ⁄ 5 cation exchange
column. After the sample had been loaded onto the column, the column was eluted with a linear gradient from 0.0
to 1.0 m NaCl in the same ammonium acetate buffer used
for gel filtration, at a flow rate of 1 mLỈmin)1. The last
chromatographic step was performed on a Millipore-Waters
chromatography apparatus (Millipore), using a Vydac
218TP C18, 4.6 · 250 mm, 7 lm reverse phase column
(Grace, Deerfield, IL, USA), with a 0–70% (v ⁄ v) acetonitrile
gradient in 0.1% (v ⁄ v) trifluoroacetic acid at 1 mLỈmin)1.
All fractions were collected manually.

SDS/PAGE
Key fractions obtained by different types of chromatography were analyzed and checked for purity with the MiniProtean II SDS ⁄ PAGE apparatus (BioRad, Hercules, CA,
USA).

MALDI-TOF spectra
MALDI-TOF spectra of UcPLA2 were obtained using a
Proteomics 4700 analyzer (Applied Biosystems, Foster City,
CA, USA). The spectra were obtained in positive mode,
using a-cyano-4-hydroxycinnamic acid (Sigma-Aldrich,
St Louis, MO, USA) as a matrix.

N-terminal protein sequencing

The N-terminal Edman sequence analyses of protein samples were performed on an Applied Biosystems Model 492A
Procise Protein Sequencing System (Applied Biosystems).
Following RP-HPLC purification, separated proteins were
applied to glass-fiber discs and sequenced with a pulsedliquid sequencing protocol. PTH amino acid derivatives
were analyzed on-line on a microbore HPLC system 140C
(Applied Biosystems), using an RP C18 Brownlee Spheri-5
column (PerkinElmer, Waltham, MA, USA). Cysteines
were alkylated before sequencing. All reagents and solvents
were of sequencing grade (Applied Biosystems).

Total RNA isolation
A few tentacles of a living U. crassicornis sea anemone were
quickly cut off and homogenized on ice with a Teflon pestle. Total RNA was isolated from the excised tentacles with

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the SV Total RNA Isolation System (Promega, Madison,
WI, USA), and stored at )80 °C. The concentration and
purity of the total RNA were evaluated spectrophotometrically (UV-2101PC; Shimadzu, Kyoto, Japan).

RACE
5¢-RACE and 3¢-RACE were performed on the RACEready cDNA prepared from previously isolated total RNA
with the GeneRacer Kit (Invitrogen, Carlsbad, CA, USA).

For the 3¢-RACE reaction, the GeneRacer 3¢ Primer and
the degenerate primer ATGATHAARTGYGCIACIGG,
corresponding to the amino acid sequence MIKCATG
obtained by Edman degradation of the isolated UcPLA2,
were used. Amplification was performed in the Primus 25
Thermal Cycler (MWG Biotech, Ebersberg, Germany) with
the PCR Master Mix (· 2) (Fermentas, Vilnius, Lithuania),
to which the following components were added: the
degenerate primer and the GeneRacer 3¢ Primer to final
concentrations of 5 and 0.5 lm, respectively, and 1 lL of the
RACE-ready cDNA in a total volume of 50 lL. The following PCR program was used for amplification: 5 min at
95 °C, 30 cycles of 30 s at 95 °C, 30 s at 50 °C, and 60 s at
72 °C, and 10 min at 72 °C. The products of the PCR reaction were separated with HE 33 Mini agarose electrophoresis
apparatus (Hoefer, Holliston, MA, USA), and the band of
interest was excised from the 1% agarose gel. DNA was
extracted from the gel with the QIAquick Gel Extraction kit
(Qiagen, Hilden, Germany), and cloned with the Zero Blunt
TOPO PCR Cloning Kit (Invitrogen), using E. coli TOP10
chemically competent cells. Positive clones were determined
by restriction analysis and sequenced. Primers used for
5¢-RACE were the gene-specific reverse primer GTTAGG
GTGGTAGGTGTTCCTTGC corresponding to the amino
acid sequence ARNTYHPN of UcPLA2 and the GeneRacer
5¢ Primer, both at a final concentration of 1 lm. PCR was
performed with the same polymerase master mix and thermal
cycler as the 3¢-RACE reaction, under following conditions:
5 min at 95 °C, 35 cycles of 30 s at 95 °C, 60 s at 65 °C, and
60 s at 72 °C, and 10 min at 72 °C. The subsequent procedure was the same as for 3¢-RACE. After both 5¢-RACE and
3¢-RACE sequences had been obtained, the full-length
UcPLA2 cDNA was amplified using primers annealing at the

beginning and end of the cDNA and sequenced.

Sequence analysis and homology modeling
DNA and amino acid sequences were processed, analyzed
and aligned with the vector nti software package (Invitrogen). Amino acid numbering and cysteine positions are
used in accordance with the standard numbering for PLA2s
introduced by Renetseder et al. [28]. A blast search for
homologous sequences was performed by querying UcPLA2
and AcPLA2 sequences against the Protein Data Bank
(PDB) database. The sequence of the porcine group I PLA2

2650

exhibited the highest similarity with that of UcPLA2, and
an anticoagulant PLA2 from the venom of Russell’s viper
(D. r. russelli) exhibited the highest similarity with that of
AcPLA2. Hence, we have modeled the structures of
UcPLA2 and AcPLA2 on the basis of the crystal structures
of the porcine group I PLA2 (PDB 5p2p [27]) and
D. r. russelli PLA2 (PDB 1vip [29]), respectively. Structure
modeling and validation was performed using modeller
[38], and visualization was performed using discovery
studio visualizer (Accelrys, San Diego, CA, USA).

Construction of the expression vector for
heterologous expression of UcPLA2
The expression vector pTolX–UcPLA2 was used for overexpression of the recombinant UcPLA2. In brief, a derivative
of pET8c, the pTolX vector [25], contained, under a
T7 promoter, sequences for a His6-tag, the fusion protein
TolIIIA from E. coli, and the factor Xa protease recognition site (IEGR). The sequence of the mature form of

UcPLA2 was ligated directly after the factor Xa cleavage
site. Following transformation into E. coli DH5a, several
clones were checked by sequencing, and the constructed
plasmid pTolX–UcPLA2 was isolated with the Wizard Plus
SV Kit (Promega).

Bacterial overexpression, refolding and
purification of recombinant UcPLA2
One colony of freshly transformed E. coli BL21(DE3) with
pTolX–UcPLA2 was transferred into LB medium with
100 lgỈmL)1 ampicillin (LBA), and cultivated overnight at
37 °C, with shaking. The overnight culture was used to
inoculate the M9 LBA medium. When the broth D600 nm
reached 0.8–1, isopropyl thio-b-d-galactoside was added to
a final concentration of 1 mm. The broth was then left on a
shaker to incubate at 37 °C for another 3 h. Cells were then
pelleted, and inclusion bodies were isolated. Refolding of
UcPLA2 was performed following the protocol used for
refolding of ammodytoxins [39]. The refolded TolX–UcPLA2 was further purified on an Ni2+–nitrilotriacetic acid
agarose column (Qiagen) and Mono S HR 5 ⁄ 5 cation
exchange column (Pharmacia Biotech, Sweden). The pure
fusion protein was subjected to factor Xa cleavage according to the manufacturer’s instructions (Qiagen). The final
step in obtaining the pure recombinant UcPLA2 was the
separation of the cleavage mixture on a Vydac 214TP C4,
4.6 · 250 mm, 5 lm RP-HPLC column (Grace). Elution
was achieved with a linear acetonitrile gradient from 5% to
60% (v ⁄ v) in 0.1% (v ⁄ v) trifluoroacetic acid at 1 mLỈmin)1.
Purity and molecular mass were confirmed by SDS ⁄ PAGE
and MALDI-TOF MS, using standard protocols. The correct N-terminal amino acid sequence was confirmed by five
steps of Edman degradation.

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A. Razpotnik et al.

UcPLA2 hemolytic activity
Hemolytic activity was assessed by means of a turbidimetric
method, as previously described [40]. Briefly, an erythrocyte
suspension in 130 mm NaCl, 1 mm CaCl2 and 20 mm
Tris ⁄ HCl (pH 7.4) was prepared, with an apparent initial D
of 0.5 at 630 nm measured in a UV–visible microplate
reader (MRX; Dynex Technologies, Berlin, Germany).
After addition of the samples, activity was monitored until
the D dropped to half of its initial value (t50).

UcPLA2 enzymatic activity
Phospholipase activity was measured using a modification
of the method of Radvanyi et al. [41] on phospholipid vesicles prepared from PyPG and PyPC as substrates. In a
96-well plate, 8 ng of UcPLA2 was added to 200 lL of
0.09% (w ⁄ v) BSA (fatty-acid free) in 50 mm KCl and
50 mm Tris ⁄ HCl (pH 8.0), with the addition of either 1 mm
CaCl2 or 1 mm EGTA, followed by 100 lL of either PyPG
or PyPC (4.2 lm) in the same buffer. Fluorescence was
measured in 10 kinetic cycles on a SAFIRE microplate
monochromator reader (Tecan, Mannedorf, Switzerland),
ă
using the following parameters: excitation wavelength,
342 nm; emission wavelength, 395 nm; number of flashes,
10; and integration time, 40 ls. In control experiments,

either no PLA2 was added, to determine the background
fluorescence, or 1 ng of AtxC [42] was added, to establish
the relative enzymatic activity of UcPLA2. To determine
the relative enzymatic activity of UcPLA2, the slopes of the
curves were calculated from the obtained measurements,
the background fluorescence was subtracted, and the
resulting value was compared with that of the AtxC
sample. The influence of high ionic strength on the enzymatic activity of UcPLA2 was tested by using the abovedescribed 1 mm CaCl2 assay buffer containing 150 or
600 mm NaCl.

Neuromuscular effects of UcPLA2
The neuromuscular toxicity of UcPLA2 was tested using a
mouse hemidiaphragm nerve–muscle preparation. Left
hemidiaphragm muscles, with their associated phrenic
nerves, were isolated from male BALB ⁄ C mice weighing
20–25 g (4–6 months old) and killed by cervical dislocation
followed by immediate exsanguinations. The hemidiaphragm was mounted in a 2 mL Rhodorsil-covered organ
bath. A physiological solution containing 154 mm NaCl,
5 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 5 mm Hepes and
11 mm glucose was bubbled with pure oxygen, and had a
pH of 7.4. One tendon was pinned to the Rhodorsil-lined
bath, and another tendon was attached with a silk thread
via a stainless steel hook to an isometric mechanoelectrical
transducer (Itis, Ljubljana, Slovenia). The motor nerve of
the isolated neuromuscular preparations was stimulated

New PLA2 from Urticina crassicornis

with pulses of 0.1 ms in duration, using a suction microelectrode. Supramaximal voltage pulses (typically 3–5 V)
supplied by an S-44 stimulator (Grass Instruments, West

Warwick, RI, USA) were used for phrenic nerve stimulation at a frequency of 0.1 Hz. The resting tension for each
muscle preparation was adjusted in order to achieve maximal contractile responses upon indirect muscle stimulation.
Electrical signals from the mechanoelectrical transducer
were amplified and digitized at a sampling rate of 1 kHz,
using a data acquisition system (Digidata 1440A; Molecular
Devices, Sunnyvale, CA, USA). The final concentrations of
UcPLA2 and AtxC in the bath were 3.2 and 0.28 lm,
respectively.

Data mining and phylogenetic analysis
All database searches were performed online and were completed in January 2009. The databases analyzed were the
nonredundant (NR), EST, GSS, HTGS, WGS and genome
databases at the National Center for Biotechnology
Information (). In addition, we
searched the Ensembl () and the
DOE Joint Genome Institute () databases. Taxon-specific genome databases were searched for
through the Ensembl and Joint Genome Institute websites,
and diverse taxon-specific transcriptomic databases were
searched for at National Center for Biotechnology Information for all metazoan lineages. To detect all available representatives of the group I PLA2 family, database searches
were performed iteratively. Comparisons were performed
using the tblastn program [43], with the E-value cutoff set
to 10)5 and default settings for other parameters. Highly
divergent invertebrate group I PLA2s were used as queries.
The translate program ( />dna.html) was used to translate DNA sequences. All of the
nonredundant metazoan representatives of the group I
PLA2 family were included in the analysis. The PLA2
domain in the newly discovered representatives of the
group I PLA2 family was identified by using the SMART
(smart.embl-heidelberg.de), InterPro ( />interpro/) and Pfam (pfam.janelia.org) domain databases.
The protein sequences were aligned using clustal w2 [44].

All of the available correction models were tested, but the
complex models were outperformed by the simple correction models. We therefore used uncorrected p-distances for
deduced amino acid sequences to measure the extent of
sequence divergence. When many divergent sequences are
being analyzed, and the number of positions used is relatively small, the uncorrected distances are more efficient for
obtaining reliable topology than more complicated correction models, owing to their smaller variance [45]. Phylogenetic trees were reconstructed using the neighbor-joining
method [46] and the maximum likelihood method [47]. The
reliability of the resulting topologies was tested by the
bootstrap method. Diverse metazoan secretory PLA2s

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(group III, group IX and group XII PLA2s) were used as
outgroups. Phylogenetic analyses were performed with the
computer programs treecon [48], mega 4.0 [49], and
raxml [47].

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Supporting information
The following supplementary material is available:
Fig. S1. Evolutionary relationships between the representatives of the GI PLA2 family in the Metazoa.
Fig. S2. Pfam HMM Logo.
Fig. S3. Alignment of Sycon GI PLA2 and Sus GIB
PLA2.
Fig. S4. Alignment of metazoan N27 GI PLA2s.
This supplementary material can be found in the
online version of this article.
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