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 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: +386 257 33 90 Tel: +386 423 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 April 2010, accepted 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 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 FEBS Journal 277 (2010) 2641–2653 Journal compilation ª 2010 FEBS No claim to original US government works 2641 New PLA2 from Urticina crassicornis A Razpotnik et al 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 FEBS Journal 277 (2010) 2641–2653 Journal compilation ª 2010 FEBS No claim to original US government works New PLA2 from Urticina crassicornis A Razpotnik et al A B C D Fig 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 ⁄ cation exchange column at a flow rate of 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 fractions were subjected to RP-HPLC, using a Vydac 218TP C18, 4.6 · 250 mm, lm reverse phase column Proteins were eluted with an acetonitrile gradient of 0–70% (v ⁄ v) in 0.1% trifluoroacetic acid (v ⁄ v) at mLỈmin)1 (D) A sample of the cation exchange chromatography peak 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 FEBS Journal 277 (2010) 2641–2653 Journal compilation ª 2010 FEBS No claim to original US government works 2643 New PLA2 from Urticina crassicornis A Razpotnik et al [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 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 FEBS Journal 277 (2010) 2641–2653 Journal compilation ª 2010 FEBS No claim to original US government works New PLA2 from Urticina crassicornis A Razpotnik et al A B Fig (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 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 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 FEBS Journal 277 (2010) 2641–2653 Journal compilation ª 2010 FEBS No claim to original US government works 2645 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 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 AtxC m M CaCl UcPLA 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 0 150 NaCl (mM) 2646 600 200 180 160 140 120 100 80 60 40 20 0 150 NaCl (mM) 600 Fig 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, mM CaCl2 or 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 ng, and that of AtxC was 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 FEBS Journal 277 (2010) 2641–2653 Journal compilation ª 2010 FEBS No claim to original US government works 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 FEBS Journal 277 (2010) 2641–2653 Journal compilation ª 2010 FEBS No claim to original US government works 2647 New PLA2 from Urticina crassicornis 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 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 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), FEBS Journal 277 (2010) 2641–2653 Journal compilation ª 2010 FEBS No claim to original US government works 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 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 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 mL A three-step chromatography procedure was required to purify the protein sufficiently for chemical analysis The New PLA2 from Urticina crassicornis ¨ 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 ⁄ 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 mLỈmin)1 The last chromatographic step was performed on a Millipore-Waters chromatography apparatus (Millipore), using a Vydac 218TP C18, 4.6 · 250 mm, lm reverse phase column (Grace, Deerfield, IL, USA), with a 0–70% (v ⁄ v) acetonitrile gradient in 0.1% (v ⁄ v) trifluoroacetic acid at 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 FEBS Journal 277 (2010) 2641–2653 Journal compilation ª 2010 FEBS No claim to original US government works 2649 New PLA2 from Urticina crassicornis A Razpotnik et al 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 and 0.5 lm, respectively, and lL of the RACE-ready cDNA in a total volume of 50 lL The following PCR program was used for amplification: at 95 °C, 30 cycles of 30 s at 95 °C, 30 s at 50 °C, and 60 s at 72 °C, and 10 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 lm PCR was performed with the same polymerase master mix and thermal cycler as the 3¢-RACE reaction, under following conditions: at 95 °C, 35 cycles of 30 s at 95 °C, 60 s at 65 °C, and 60 s at 72 °C, and 10 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 mm The broth was then left on a shaker to incubate at 37 °C for another 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 ⁄ 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, 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 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 FEBS Journal 277 (2010) 2641–2653 Journal compilation ª 2010 FEBS No claim to original US government works 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, 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, 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 mm CaCl2 or 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 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 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 mL Rhodorsil-covered organ bath A physiological solution containing 154 mm NaCl, mm KCl, mm CaCl2, mm MgCl2, 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 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 (http://www.ncbi.nlm.nih.gov) In addition, we searched the Ensembl (http://www.ensembl.org) and the DOE Joint Genome Institute (http://www.jgi.doe.gov) 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 (http://www.expasy.org/tools/ 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 (http://www.ebi.ac.uk/ 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 FEBS Journal 277 (2010) 2641–2653 Journal compilation ª 2010 FEBS No claim to original US government works 2651 New PLA2 from Urticina crassicornis A Razpotnik et al (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] References Burke JE & Dennis EA (2009) Phospholipase A2 biochemistry Cardiovasc Drugs Ther 23, 49–59 Duncan RE, 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Comput Appl Biosci 13, 227–230 Tamura K, Dudley J, Nei M & Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0 Mol Biol Evol 24, 1596– 1599 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 Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 2641–2653 Journal compilation ª 2010 FEBS No claim to original US government works 2653 ... 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... Pacific sea anemone Urticina crassicornis (UcPLA2) This new PLA2 (UniProt UcPLA2 A7 LCJ2) is homologous with AcPLA2, and, with regard to many structural features, is also similar to Elapidae snake... N27 –C 27 C44 C4 –C1 4–C 05 105 Fig 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