A new phospholipase A2 isolated from the sea anemone Urticina crassicornis – its primary structure and phylogenetic classification

Abstract

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.

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A new phospholipase A2isolated from the sea anemone
Urticina crassicornis – its primary structure and
phylogenetic classification
Andrej Razpotnik1, Igor Kriz ˇaj2, Jernej Sˇribar2, Dus ˇan Kordis ˇ2, Peter Mac ˇek1, Robert Frangez ˇ3,
William R. Kem4and Tom Turk1
1 Biotechnical Faculty, Department of Biology, University of Ljubljana, Slovenia
2 Department of Molecular and Biomedical Sciences, Joz ˇef Stefan Institute, Ljubljana, Slovenia
3 Faculty of Veterinary Medicine, University of Ljubljana, Slovenia
4 Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, Gainesville, FL, USA
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.
currently dividedinto15
Recently, a new PLA2from adipose tissue was charac-
terized 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
These
structural
enzymes
groups
are
[1].
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
Keywords
enzymatic activity; phospholipase A2;
phylogenetic classification; sea anemone;
sequence; venom
Correspondence
T. Turk, Biotechnical Faculty, Department of
Biology, University of Ljubljana, Vec ˇna pot
111, SI-1000 Ljubljana, Slovenia
Fax: +386 1 257 33 90
Tel: +386 1 423 33 88
E-mail: tom.turk@bf.uni-lj.si
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)
doi:10.1111/j.1742-4658.2010.07674.x
Disulfide pairings and active site residues are highly conserved in secretory
phospholipases A2(PLA2s). However, secretory PLA2s of marine inverte-
brates display some distinctive structural features. In this study, we report
the isolation and characterization of a PLA2from 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 experi-
ments testing the influence of ionic strength on UcPLA2enzymatic 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 PLA2clade.
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, whereasViperidae
group II PLA2s. Several PLA2s from both groups bind
with high specificity to presynaptic membranes. These
enzymes, also known as b-neurotoxins, block neuro-
muscular transmission by impairing the cycling of syn-
aptic 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 enzy-
matic activities vary significantly between different
species. There are only a few reports on isolated cni-
darian 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 glycopro-
tein with a pI of 8.8. The b-form contributes about
70% of the total phospholipase activity of the venom
[16,17]. A similar PLA2probably 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 Brazil-
ian sea anemone species Bunodosoma caissarum [19].
The N-terminal amino acid sequence of the B. caissa-
rum 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]. NestedRT-PCR
primers and RACE was used to clone the PLA2from
this animal. AcPLA2 contains a putative prepropep-
tide 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.
snakevenomsharbor
with degenerate
Here we report on the isolation, cloning and charac-
terization of a novel PLA2from 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
rolein snakeand cnidarian
UcPLA2 has some unusual structural features, most
notably an asparagine at position 27 instead of cyste-
ine, which is present in the majority of known group I
and group II PLA2s. This replacement is rare in inver-
tebrate PLA2s, and has not been found yet in verte-
brate toxic and nontoxic PLA2s of group I and
group II, with a single exception, a sea lamprey PLA2.
Also, in UcPLA2there is a C-terminal truncation of
six amino acids, including a cysteine, so the usual pair-
ing 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 PLA2might
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 PLA2super-
family [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 PLA2family.
venoms.However,
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 mem-
brane. The concentrated sample was applied to a gel
filtration column in order to desalt the sample and
exchange sea waterfor
(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
whichexhibitedhemolytic
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
anappropriatebuffer
activity.SDS⁄PAGE
New PLA2from Urticina crassicornis
A. Razpotnik et al.
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was homologous to cytolytic proteins of the actinopo-
rin family, which are probably responsible for the
hemolytic activity observed in fractions containing
UcPLA2. The20 kDaprotein
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,
UcPLA2was only barely detectable.
was removedby
N-terminal sequencing of wild-type UcPLA2
Wild-type UcPLA2was 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
followingsequence: NLLQFSSMIKCATGRSAW-
KYDNYGN.
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
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
? 500 bp PCR product was cloned and sequenced.
ThecompletecDNA sequence
combining the 3¢-RACE and 5¢-RACE sequences.
carriedout. Again,the
wasobtained by
Overexpression, refolding and purification of the
recombinant UcPLA2
Our first attempt was to overexpress UcPLA2by 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 pre-
ceding 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
A
C
B
D
Fig. 1. Isolation of native UcPLA2. Native UcPLA2was 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 mLÆmin)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 mLÆmin)1in the same ammonium acetate buffer as used for gel filtration. Bound proteins were eluted with a lin-
ear 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 mLÆmin)1. (D) A sam-
ple of the cation exchange chromatography peak 3 separated on a 12.5% SDS⁄PAGE gel.
A. Razpotnik et al.
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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. Followingsolubilization,
fusion UcPLA2was 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 diges-
tion, and the recombinant UcPLA2 was purified to
homogeneity.
therecombinant
Sequence analysis and protein structure
modeling
The cDNA sequence of UcPLA2(791 bp) was depos-
ited 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 wild-
type 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 prepropep-
tide. The first 19 amino acids of the prepropeptide
represent a cleavable signal peptide [26]. The prepro-
peptide sequence ends with two basic amino acids,
lysine and arginine,which
protease-cleavage site. The predicted molecular mass
and pI of the mature UcPLA2are 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 UcPLA2prob-
ably form six disulfide bridges. Highly conserved
amino acidsforming
(YGCYCGXGGXG) and the catalytic site (H⁄D) in
PLA2s are also present in UcPLA2. A blast search
revealed the highest similarity of UcPLA2with group I
PLA2s. It shares approximately 35% identity with the
porcine group I PLA2and about 50% identity with an
elapid snake(Oxyuranus scutellatus)
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 AcPLA2and vertebrate PLA2s. Align-
ment of the UcPLA2amino acid sequence with bovine
PLA2shows considerable differences between UcPLA2
and vertebrate PLA2s in the cysteine distribution pat-
tern. First, the highly conserved disulfide bond in
representacommon
the Ca2+-binding loop
neurotoxic
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 part-
ner 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 candi-
date for disulfide bond formation with Cys11. A 3D
modelof UcPLA2
confirms
(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 PLA2is a tenta-
tive Cys11–Cys80 bond in UcPLA2 (according to
Renetseder’s numbering [28], if a minimum number of
gaps is introduced into the UcPLA2 amino acid
suchaprediction
Fig. 2. The full-length cDNA sequence of UcPLA2and 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 indi-
cated by an arrow. The asterisk indicates the stop codon.
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A. Razpotnik et al.
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sequence in order to align cysteines with those in the
bovine PLA2). We would expect Cys80 in UcPLA2to
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
has the capacity to form six disulfide bridges. It pos-
sesses 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
A
B
Fig. 3. (A) Alignment of PLA2protein sequences from: the sea anemones Urticina crassicornis (Uc), Nematostella vectensis (Nv) and Adam-
sia carciniopados (Ac), the taipan snake Oxyuranus scutellatus (Os), and the boar Sus scrofa (Ss). Identical residues are on a black back-
ground, 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 UcPLA2and Bos taurus (Bt) BtPLA2, with cysteine
numbering according to Renetseder. Cysteines are on a black background, and the unique asparagine in UcPLA2is on a gray background.
Cysteine positions, numbered according to Renetseder, are shown above the alignment, with the positions of the cysteines involved in disul-
fide bonding in parentheses.
AB
N27
C126–C27
C44–C105
C51–C98
C96–C84
C61–C91
C44–C105
C51–C98
C61–C91
C96–C84
C11–C80
Fig. 4. Models of UcPLA2and 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 yel-
low. The unique asparagine of UcPLA2is in
magenta.
A. Razpotnik et al.
New PLA2from Urticina crassicornis
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