Fish Physiol Biochem DOI 10.1007/s10695-013-9904-5

Enzymatic properties and primary structures of hyaluronidases from two species of lionfish (Pterois antennata and Pterois volitans) Aya Kiriake • Mihoko Madokoro • Kazuo Shiomi

Received: 5 October 2013 / Accepted: 24 December 2013 Ó Springer Science+Business Media Dordrecht 2014

Abstract Lionfish are representative venomous fish, having venomous glandular tissues in dorsal, pelvic and anal spines. Some properties and primary structures of proteinaceous toxins from the venoms of three species of lionfish, Pterois antennata, Pterois lunulata and Pterois volitans, have so far been clarified. Our recent survey established the presence of hyaluronidase, presumably a toxin-spreading factor, in the venoms of P. antennata and P. volitans. This prompted us to examine enzymatic properties and primary structures of lionfish hyaluronidases. The hyaluronidases of P. antennata and P. volitans were shown to be optimally active at pH 6.6, 37 °C and 0.1 M NaCl and specifically active against hyaluronan. These enzymatic properties are almost the same as those of stonefish hyaluronidases. The primary structures (483 amino acid residues) of the lionfish hyaluronidases were elucidated by a cDNA cloning strategy using degenerate primers designed from the reported amino acid sequences of the stonefish hyaluronidases. Both lionfish hyaluronidases share as high as 99.6 % of sequence identity with each other and also A. Kiriake  M. Madokoro  K. Shiomi (&) Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Konan-4, Minato-ku, Tokyo 108-8477, Japan e-mail: [email protected] Present Address: K. Shiomi 5092-3-1-703 Naruse, Machida, Tokyo 194-0044, Japan

considerably high identities (72–77 %) with the stonefish hyaluronidases but rather low identities (25–40 %) with other hyaluronidases from mammals and venomous animals. In consistent with this, phylogenetic tree analysis revealed that the lionfish hyaluronidases, together with the stonefish hyaluronidases, form a cluster independently of other hyaluronidases. Nevertheless, the lionfish hyaluronidases as well as the stonefish hyaluronidases almost maintain structural features (active site, glyco_hydro_56 domain and cysteine location) observed in other hyaluronidases. Keywords cDNA cloning  Hyaluronidase  Lionfish  Pterois antennata  Pterois volitans

Introduction Hyaluronidases are a group of enzymes that degrade predominantly hyaluronan, a high molecular mass polysaccharide found in the extracellular matrix of all vertebrates and in the capsule of some bacteria, and limitedly chondroitin and chondroitin sulfate. Based on their biochemical features and reaction products, they are divided into the following three families: hyaluronoglucosaminidase or hyaluronate 4-glycanohydrolase (EC 3.2.1.35) present in mammalian spermatozoa and plasma and animal venoms, hyaluronoglucuronidase or hyaluronate 3-glycanohydrolase (EC 3.2.1.36) in

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leeches and hookworms and hyaluronate lyase (EC 4.2.2.1) in various microorganisms (Stern and Jedrzejas 2006; EI-Safory et al. 2010). Since hyaluronidases regulate the metabolism of hyaluronan that is implicated in many biological processes such as fertilization, cell differentiation, inflammation and growth and metastasis of tumor cells, they have attracted the interest of many researchers. Hyaluronidases (hyaluronoglucosaminidase) are widely distributed in the venoms of a variety of animals such as lizards, snakes, fish, scorpions, spiders and bees (EI-Safory et al. 2010). Venom hyaluronidases are particularly interesting in that they act as spreading factors of venom toxins, thereby enhancing their toxic effects, as demonstrated with some venomous animals such as lizard Heloderma horridum horridum (Tu and Hendon 1983), Indian cobra Naja naja (Girish et al. 2004), stonefish Synanceia verrucosa (Madokoro et al. 2011), Brazilian yellow scorpion Tityus serrulatus (Pessini et al. 2001) and funnel web spider Hippasa partita (Nagaraju et al. 2007). A number of hyaluronidases have already been purified from the venoms of terrestrial animals and characterized to varying extents for enzymatic properties, chemical properties and structures. In contrast, information about hyaluronidases in the fish venom is very limited. At present, only the following five species of venomous fish are known to contain hyaluronidase in their venoms: two species of freshwater stingray, Potamotrygon falkneri (Haddad et al. 2004; Barbaro et al. 2007) and Potamotrygon motoro (Magalha˜es et al. 2008), and three species of stonefish, Synanceia horrida (Poh et al. 1992; Sugahara et al. 1992; Ng et al. 2005), Synanceia trachynis (Hopkins and Hodgson 1998) and S. verrucosa (Austin et al. 1965; Shiomi et al. 1993; Garnier et al. 1995). Hyaluronidases from P. motoro (Magalha˜es et al. 2008), S. horrida (Poh et al. 1992; Sugahara et al. 1992) and S. verrucosa (Madokoro et al. 2011) have so far been purified and enzymatically and chemically characterized. In addition, the hyaluronidases from two species of stonefish have already been elucidated for their primary structures by cDNA cloning. It should be noted that the stingray hyaluronidase is distinguishable in molecular mass and optimum pH from the stonefish hyaluronidases. The stingray hyaluronidase has a molecular mass of 79 kDa and is optimally active at pH 4.2 (Magalha˜es et al. 2008), while the stonefish hyaluronidases are lower in molecular mass (62 kDa for S. horrida

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and 59 kDa for S. verrucosa) and higher in optimum pH (6.0 for S. horrida and 6.6 for S. verrucosa) (Poh et al. 1992; Madokoro et al. 2011). Lionfish, belonging to the same order Scorpaeniformes as stonefish, are well known to be venomous, having venomous glandular tissues in dorsal, pelvic and anal spines. Some properties and primary structures of proteinaceous toxins from the venoms of three species of lionfish, Pterois antennata, Pterois lunulata and Pterois volitans, have been reported (Kiriake and Shiomi 2011; Kiriake et al. 2013). Recently, we found hyaluronidase activity in the venoms of P. antennata and P. volitans. Since lionfish hyaluronidases are also considered to act as toxin-spreading factors, their characterization is essential to understand the lionfish envenomation at a molecular level. In this study, therefore, enzymatic properties of hyaluronidases from two species of lionfish, P. antennata and P. volitans, were first clarified and then their primary structures elucidated by a cDNA cloning technique.

Materials and methods Preparation of crude extract Live specimens of P. antennata and P. volitans were purchased from retail aquarium shops. Twelve dorsal spines (including surrounding tissues) were collected from each live specimen, homogenized in 10 volumes of 0.01 M phosphate buffer (pH 7.0) and centrifuged. The supernatant obtained was used as a crude extract in this study. Assay of hyaluronidase activity Hyaluronidase activity was estimated by the turbidimetric method of Tolksdorf et al. (1949). In brief, 0.1 ml of the reaction mixture (prepared in 0.1 M NaCl–0.1 M phosphate buffer, pH 6.6) containing a given sample and 0.02 % hyaluronan (from human umbilical cord; Sigma-Aldrich Japan, Tokyo) was incubated at 37 °C for 10 min. The mixture was then added with 0.9 ml of albumin reagent solution (0.25 % bovine serum albumin in 0.5 M sodium acetate buffer, pH 3.0), allowed to stand at room temperature for 10 min and measured for turbidity at 540 nm. Enzymatic activity was expressed as United States Pharmacopeia/National Formulary (USP/NF) units, which

Fish Physiol Biochem Table 1 Primers designed in this study

Experiment

Designation of primera

Nucleotide sequence of primer

RT-PCR

DegF

50 -GAYTTYGARGARTGGCGICC-30

DegR

50 -GTRTTCACCAGRTCRAAYTC-30

V-GspF V-GspR

50 -TTTGGACAGCAGGAAGTTGG-30 50 -GCTGCCCCAGTTTCGGATCC-30

AV-GspF1

50 -ACTACTCCATCCCCATCCAT-30

AV-GspF2

50 -ACATTCGCCCCTTGTACAAG-30

A-GspR1

50 -GGGATGGAGTAGTTGCTGTT-30

A-GspR2

50 -TAGTAGCCCCAGAGTCTGTT-30

A-GspR3

50 -AACCCGTCTGTAGATGTCTTT-30

a

Meaning of letters: Deg degenerate primer, Gsp gene-specific primer, F forward primer, R reverse primer, A primer for the P. antennata hyaluronidase, V primer for the P. volitans hyaluronidase

30 RACE 50 RACE

Table 2 Hyaluronidase activity of the crude extracts from P. antennata and P. volitans Species

P. antennata P. volitans

Specimen

Total length (cm)

Body weight (g)

Protein concentration of crude extract (mg/ml)

Hyaluronidase activity (USP/NF units/ml)

(USP/NF units/mg)

A

9.2

24.9

0.86

98

B

9.5

26.2

1.14

38

33

A

14.4

31.6

1.63

311

191

B

15.7

33.3

1.55

296

191

C

15.8

32.7

1.20

421

351

D

16.5

33.6

0.65

217

334

were referenced to a standard USP/NF hyaluronidase preparation (Cooper Biomedical, Malvern, PA, USA). Enzyme characterization To determine the optimum pH for enzymatic activity, 0.1 M buffers (acetate buffer for pH 3–5, phosphate buffer for pH 6–7 and Tris–HCl buffer for pH 8–9) containing 0.1 M NaCl were used. The optimum temperature for enzymatic activity was examined by assaying at different temperatures (0–60 °C). To examine the effect of NaCl on the enzymatic activity, assay was performed at various concentrations of NaCl (0–0.5 M). Besides hyaluronan, the following substances (0.02 % in the reaction mixture) were also evaluated for the possibility of substrates: chondroitin sulfate A, chondroitin sulfate B and heparin purchased from Sigma-Aldrich Japan and chondroitin sulfate C from Wako Pure Chemicals (Osaka, Japan).

114

Primer design Designations and nucleotide sequences of the primers designed in this study are summarized in Table 1. Two degenerate primers [degenerate forward (DegF) and reverse (DegR)] used in RT-PCR were, respectively, designed from the reported nucleotide sequences corresponding to the regions 139–145 and 314–320 (numbering is based on the amino acid sequence of S. horrida hyaluronidase) that are highly conserved between the amino acid sequences of the stonefish (S. horrida and S. verrucosa) hyaluronidases. One gene-specific primer (V-GspF) was designed from the 50 -untranslated regions conserved among the cDNAs coding for stonefish and P. antennata hyaluronidases. The other gene-specific primers were all prepared based on the determined nucleotide sequences of RT-PCR products.

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Fish Physiol Biochem

B

A Relative activity (%)

Fig. 1 Enzymatic activity of the P. antennata (filled triangle) and P. volitans (filled circle) hyaluronidases at various pHs (a), temperatures (b) and concentrations of NaCl (c). a Estimated at 37 °C in the presence of 0.1 M NaCl. b Estimated at pH 6.6 in the presence of 0.1 M NaCl. c Estimated at pH 6.6 and 37 °C

100

100

100

80

80

80

60

60

60

40

40

40

20

20

20

0

0 3

4

5

6

7

8

pH

RT-PCR Total RNA was extracted from 1 g of dorsal spines (including surrounding tissues) of each lionfish specimen with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized from 5 lg of total RNA using the 30 RACE system for rapid amplification of cDNA ends (Invitrogen) according to the manufacturer’s instructions and used in RT-PCR as a template. In RT-PCR, amplification was performed using a pair of the DegF and DegR primers and Ex Taq polymerase (Takara, Otsu, Japan) under the following conditions: preincubation at 94 °C for 5 min; 35 cycles consisting of denaturation at 94 °C for 30 s, annealing at 46 °C for 30 s and extension at 72 °C for 2 min; and final extension at 72 °C for 5 min. Amplified products were subcloned into the pT7Blue2 T-vector (Novagen, Darmstadt, Germany) and sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and an ABI PRISM 310 genetic analyzer (Applied Biosystems). As for the P. volitans hyaluronidase, RT-PCR was further performed using a pair of gene-specific primers (V-GspF and V-GspR) under the same amplification conditions as described above, with the exception that annealing temperature was changed to 53 °C. RACE 30 RACE was carried out using the 30 RACE system for rapid amplification of cDNA ends (Invitrogen). The first-strand cDNA used as a template was the same as

123

C

9

0 0

10

20

30

40

50

Temperature (˚C)

60

0

0.1

0.2

0.3

0.4

0.5

Concentration of NaCl (M)

in the case of RT-PCR. Amplification was performed using the gene-specific forward primer (AV-GspF1) and abridged universal amplification primer (AUAP, 50 -GGCCACGCGTCGACTAGTAC-30 ), under the following conditions: 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 2 min; and 72 °C for 5 min. Furthermore, nested PCR was performed using the gene-specific primer (AV-GspF2) and AUAP. PCR products were subcloned into the pT7Blue-2 T-vector and sequenced as described above. 50 RACE was carried out only for the P. antennata hyaluronidase using the 50 RACE system for rapid amplification of cDNA ends (Invitrogen). A cDNA template was synthesized from 5 lg of total RNA using the gene-specific reverse primer (A-GspR1). The gene-specific reverse primer (A-GspR2) and abridged anchor primer (AAP, 50 -GGCCACGCGTC GACTAGTACGGGGGGGGGGGGGGGG-30 ) were used for the first PCR and the gene-specific primer (A-GspR3) and AUAP for the nested PCR. Amplification conditions were the same as adopted for 30 RACE. The nested PCR products were subcloned and sequenced as described above. Phylogenetic analysis Multiple amino acid sequence alignment of hyaluronidases including those from P. antennata and P. volitans was performed by the ClustalW algorithm (Thompson et al. 1994). Based on the multiple amino acid sequence alignment, a phylogenetic tree was constructed by the neighbor-joining method using a

Fish Physiol Biochem A GA GC GA GGT T GGC GT T T A C GC C T C A C A GGT A A T A A T T T C A C GA C T C A C A GA GT GC GC T GT T T GGA C GGC A GGA A GT T GGA C C C C C GGA GC A GA T T A C T GT T T C A A GA T GA A C C A C C T C A A GC T GC T C GA T GA C C A A C A C T T C T T C T C T A T A T T C A T C C C C A T GT T GGC C C T GA T GT GG M N H L K L L D D Q H F F S I F I P M L A L M W

89 179 24

T C C GGA T GGGC C C T A C C A A GGA C A GA C C C T C C C C T C T GT C C C GGC C A C C A C T T C A T GT T C A T GT GGA A C GC C C C GA C C GA GC T C T GC GA A S G W A L P R T D P P L C P G H H F M F M W N A P T E L C E

269 54

A C C C GC T T C A GC A T GC C GC T C GA C C T C T C C T A C T T C C A A C T GA T C A GC A GC A C GC T GA A GA C GGC GA C C A A C C A A A C C A T C A GC C T A T T C T R F S M P L D L S Y F Q L I S S T L K T A T N Q T I S L F

359 84

T A C A T C GA C C GC T T C GGC A T C T T C C C C C A C A T A A A C GA A A A A A C T GGC A A A A T A T A C GA C A A GGGC C T GC C GC A GC T GA T A GT C A T GC A G Y I D R F G I F P H I N E K T G K I Y D K G L P Q L I V M Q DegF GA GC A C T A C A A C C T C GC C GA GGA T GA C A T C A A GT A C T A C A T GC C T T C C A A C C A GC C GGGC C T C GC C GT GC T T GA T T T T GA A GA GT GGA GG E H Y N L A E D D I K Y Y M P S N Q P G L A V L D F E E W R A-GspR3 C C A C A GT GGA T C C GA A A C T GGGGC A GC A A A GA C A T C T A C A GA C GGGT T T C C A T C GA GA C GGT C A A GA A C A A GA A C GC C T C A T T GT C C GA C P Q W I R N W G S K D I Y R R V S I E T V K N K N A S L S D

449 114

A A C GA GGC T GA GA A C C A GGC GA A GA T T GA GT T T GA A C GT GC A GC C A GA A GGT A C T T T GT C C A T T C C A T C C GC A T T GGGA GGA GGC T GC GG N E A E N Q A K I E F E R A A R R Y F V H S I R I G R R L R A-GspR2 C C C A A C A GA C T C T GGGGC T A C T A C C T GT T C C C C GA C T GC T A C A A C T A C GA C T T C A A C A A GA A C A T GGC GA GC T T C A C T GGA C A GT GT C C C P N R L W G Y Y L F P D C Y N Y D F N K N M A S F T G Q C P GC C C T C GA GA A GA A A A GGA A C GA C GA A C T GA T GT GGC T C T GGA GA GA GT C C A C GGC A C T C T T T C C GT C C A T C T A C C T GGA GC T GGT GA T C A L E K K R N D E L M W L W R E S T A L F P S I Y L E L V I A-GspR1 AV-GspF1 A GA GA C T C C C A GC A GGC C C GGC A GT A T GT C C GGC A T C GT A T C C A GGA GGC C A T GA GGGT GT C GA T GC T C C C C A A C A GC A A C T A C T C C A T C R

D

S

Q

Q A R Q Y V R H R I Q E A M R V S M L P N S N Y S I AV-GspF2 DegR C C C A T C C A T GC C T A C A T T C GC C C C T T GT A C A A GGA C A C C A C C GA C A T C T A C A T GT C A GA GT T C GA T C T GGT GA A C A C C A T C GGA GA GGC T P

I

H

A

Y

I

R

P

L

Y

K

D

T

T

D

I

Y

M

S

E

F

D

L

V

N

T

I

G

E

A

539 144 629 174 719 204 809 234 899 264 989 294 1079 324

GC T GC T C T C GGC GC C GC T GGC GT C A T T T C C T GGGGA GA C A T GA A C GT C GT A A A GA C A GA GGA C T C C T GC T T T GA C GC T A A A A GC C A C C T G A A L G A A G V I S W G D M N V V K T E D S C F D A K S H L

1169 354

GA C A A GGT C A T GA A C C C GT A C A T C C T GA A C GT C A C C A C A GC GA C GC A A C T C T GC A GC GA GGC GC T C T GC C A GGGC C T A GGT C GC T GC GT G D K V M N P Y I L N V T T A T Q L C S E A L C Q G L G R C V

1259 384

A GGA A GC GC T GGGA C GA C GA C GT C T T C C T C C A C C T C GA C C C GC GC C GT T A C C GGA T C GA GC GGA A A C A C C GC GGC GGC C C GC T GA C T GT G R K R W D D D V F L H L D P R R Y R I E R K H R G G P L T V

1349 414

A GC GGA GGC C T GT C GC A GGA T GA C GT C A A C T GGT T C GA C C GC A GT T T C GA C T GC A T GT GC T A C A C C GA GC A GC C GT GT C GA T C GGT C A T G S G G L S Q D D V N W F D R S F D C M C Y T E Q P C R S V M

1439 444

A T T T T C A A C GT C A T C A A C A A GA C T GT C T T C A C C T C A A A GA A T GGA GGC GC T C GC GGGC C A C GC C C C C T C C T GC T GGT GA T GA A A C T GA T C I F N V I N K T V F T S K N G G A R G P R P L L L V M K L I

1529 474

T GT C T C A T GT A T GT C GT GA T GT GGA A GT A A A GT GA T C A C A C GA A GC T GA T A T C T GT GA C A C A GGA C GT GC GA T T A C A GA A A A A A GGA C A T C L M Y V V M W K

1619 483

GGT T T T A T T A T T T C T GA C T GT T T T T A C C T GC A T T T T C A T A A A T T T C T T T A C T GA C T T T C T C C A A A T A GC T C T GC C T C A T T C A C A C A A C T T C C T T T T A GA A T A A GC T T GT T T T A A A C T A A A C C A A GGT T T A T A T T A T GA T T A T T A T C A A T GA T A T A A GGT C T C A A C A T A A GGT T T T GA C T G AATAAA

1709 1799 1805

Fig. 2 Nucleotide sequence of the cDNA encoding the P. antennata hyaluronidase. Deduced amino acid sequence is denoted below the nucleotide sequence. Nucleotide and amino acid numbers are shown on the right. Asterisks indicate an inframe stop codon. Primer sequences are indicated by single

underlines and designations. Putative signal sequence is doubly underlined. The determined nucleotide sequence can be seen in the DDBJ/EMBL/GenBank databases under the accession number AB759697

software of molecular evolution genetics analyses (MEGA) version 5.2 (Tamura et al. 2011).

activity was somewhat higher in P. volitans (217-421 USP/NF units/ml or 191-351 USP/NF units/mg) than in P. antennata (38-98 USP/NF units/ml or 33-114 USP/ NF units/mg). As shown in Fig. 1, no significant differences in enzymatic properties were observed between P. antennata and P. volitans hyaluronidases. Both P. antennata and P. volitans hyaluronidases were optimally active at pH 6.6, 37 °C and 0.1 M NaCl. Furthermore, both hyaluronidases were active against hyaluronan but exhibited no degrading

Results Enzymatic properties of lionfish hyaluronidases Hyaluronidase activity was found in all the crude extracts from two specimens of P. antennata and four specimens of P. volitans (Table 2). The estimated

123

Fish Physiol Biochem V-GspF T T T GGA C A GC A GGA A GT T GGA C C C C C A GA GA GC A GA T T A C T GT T T C A A GA T GA A C C A C C T C A A A C T GC T C GA T GA C C A A C A C T T C T T C M N H L K L L D D Q H F F

88 13

T C T A T A T T C A T C C C C A T GT T GGC C C T GA T GT GGT C C GGA T GGGC C C T A C C A A GGA C A GA C C C T C C C C T C T GT C C C GGC C A C C A C T T C A T G S I F I P M L A L M W S G W A L P R T D P P L C P G H H F M

178 43

T T C A T GT GGA A C GC C C C GA C C GA GC T C T GC GA A A C C C GC T T C A GC A T GC C GC T C GA C C T C T C C T A C T T C C A A C T GA T C A GC A GC A C GC T G F M W N A P T E L C E T R F S M P L D L S Y F Q L I S S T L

268 73

A A GA C GGC GA C C A A C C A A A C C A T C A GC C T A T T C T A C A T C GA C C GC T T C GGC A T C T T C C C C C A C A T A A A C GA A A A A A C T GGC A A A A T A T A C K T A T N Q T I S L F Y I D R F G I F P H I N E K T G K I Y

358 103

GA C A A GGGC C T GC C GC A GC T GA T A GA C A T GC A GGA GC A C T A C A A C C T C GC C GA GGA T GA C A T C A A GT A C T A C A T GC C T T C C A A C C A GC C G D K G L P Q L I D M Q E H Y N L A E D D I K Y Y M P S N Q P DegF V-GspR GGC C T C GC C GT GC T T GA T T T T GA A GA GT GGA GGC C A C A GT GGA T C C GA A A C T GGGGC A GC A A A GA C A T C T A C A GA C GGGT T T C C A T C GA G G L A V L D F E E W R P Q W I R N W G S K D I Y R R V S I E

448 133 538 163

A C GGT C A A GA A C A A GA A C GC C T C A T T GT C C GA C A A C GA GGC T GA GA A C C A GGC GA A GA T T GA GT T T GA A C GT GC A GC C A GA A GGT A C T T T T V K N K N A S L S D N E A E N Q A K I E F E R A A R R Y F

628 193

GT C C A T T C C A T C C GC A T T GGGA GGA GGC T GC GGC C C A A C A GA C T C T GGGGC T A C T A C C T GT T C C C C GA C T GC T A C A A C T A C GA C T T C A A C V H S I R I G R R L R P N R L W G Y Y L F P D C Y N Y D F N

718 223

A A GA A C A T GGC GA GC T T C A C T GGA C A GT GT C C C GC C C T C GA GA A GA A A A GGA A C GA C GA A C T GA T GT GGC T C T GGA GA GA GT C C A C GGC A K N M A S F T G Q C P A L E K K R N D E L M W L W R E S T A

808 253

C T C T T T C C GT C C A T C T A C C T GGA GC T GGT GA T C A GA GA C T C C C A GC A GGC C C GGC A GT A T GT C C GGC A T C GT A T C C A GGA GGC C A T GA GG L F P S I Y L E L V I R D S Q Q A R Q Y V R H R I Q E A M R AV-GspF1 AV-GspF2 GT GT C GA T GC T C C C C A A C A GC A A C T A C T C C A T C C C C A T C C A T GC C T A C A T T C GC C C C T T GT A C A A GGA C A C C A C C GA C A T C T A C A T GT C A V S M L P N S N Y S I P I H A Y I R P L Y K D T T D I Y M S DegR GA GT T C GA T C T GGT GA A C A C C A T C GGA GA GGC T GC T GC T C T C GGC GC C GC T GGC GT C A T T T C C T GGGGA GA C A T GA A C GT C GT A A A GA C A E F D L V N T I G E A A A L G A A G V I S W G D M N V V K T

898 283

1078 343

GA GGA C T C C T GC T T T GA C GC T A A A A GC C A C C T GGA C A A GGT C A T GA A C C C GT A C A T C C T GA A C GT C A C C A C A GC GA C GC A A C T C T GC A GC E D S C F D A K S H L D K V M N P Y I L N V T T A T Q L C S

1168 373

GA GGC GC T C T GC C A GGGC C T A GGT C GC T GC GT GA GGA A GC GC T GGGA C GA C GA C GT C T T C C T C C A C C T C GA C C C GC GC C GT T A C C GGA T A E A L C Q G L G R C V R K R W D D D V F L H L D P R R Y R I

1258 403

GA GC GGA A A C A C C GC GGC GGC C C GC T GA C T GT GA GC GGA GGC C T GT C GC A GGA T GA C GT C A A C T GGT T C GA C C GC A GA T T C GA C T GC A T G E R K H R G G P L T V S G G L S Q D D V N W F D R R F D C M

1348 433

T GC T A C A C C GA GC A GC C GT GT C GA T C GGT C A T GA T T T T C A A C GT C A T C A A C A A GA C T GT C T T C A C C T C A A A GA A T GGA GGC GC T C GC GGG C Y T E Q P C R S V M I F N V I N K T V F T S K N G G A R G

1438 463

C C A C GC C C C C T C C T GC T GGT GA T GA A A C T GA T C T GT C T C A T GT A T GT C GT GA T GT GGA A GT A A A GT GA T C A C A C GA A GC T GA T A T C T GT G P R P L L L V M K L I C L M Y V V M W K

1528 483

A C A C A GGA C GT GC GA T T A C A GA A A A A A GGA C A A T GGT T T T A T T A T T T C T GA C T GT T T T T A C C T GC A T T T T C A T A A A T T T C T T T A C T GA C T T T C T C C A A A T A GC T C T GC C T C A T T C A C A C A A C T T C C T T T T A GA A T A A GC T T GT T T T A A A C T A A A C C A A GGGT T T A T A T T A T GA T T A T T A T C A T GA T A T A GGT C T C A C A T A A GGT T T T GA C T GA T A A GT GT A GT C A T T A T GA T GC T A A A A A A A A A A A A A A A A A A

1618 1708 1781

988 313

Fig. 3 Nucleotide sequences of the cDNA encoding the P. volitans hyaluronidase. Deduced amino acid sequence is denoted below the nucleotide sequence. Nucleotide and amino acid numbers are shown on the right. Asterisks indicate an inframe stop codon. Primer sequences are indicated by single

underlines and designations. Putative signal sequence is doubly underlined. The determined nucleotide sequence can be seen in the DDBJ/EMBL/GenBank databases under the accession number AB759698

activity against either chondroitin sulfate A–C or heparin.

determined nucleotide sequence of the full-length cDNA (1,805 bp) encoding the P. antennata hyaluronidase is shown in Fig. 2. An open reading frame of 1,449 bp (corresponding to 483 amino acid residues) is recognized in the cDNA.

Cloning of P. antennata hyaluronidase cDNA A 600-bp product was amplified by RT-PCR using a pair of the DegF and DegR primers. Based on the nucleotide sequence of this product, the remaining sequence of the P. antennata hyaluronidase cDNA was elucidated by 30 RACE and 50 RACE. The

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Cloning of P. volitans hyaluronidase cDNA As in the case of P. antennata, a 600-bp product was also amplified for P. volitans by RT-PCR using the

Fish Physiol Biochem

pair of DegF and DegR. The determined nucleotide sequence of the 600-bp product led to the success of 30 RACE, by which the 30 -terminal nucleotide sequence of the P. volitans hyaluronidase cDNA was elucidated. However, no products could be amplified by 50 RACE using several gene-specific reverse primers combined with AUAP. Therefore, the 50 -terminal sequence of the P. volitans hyaluronidase cDNA was analyzed by RT-PCR using a pair of gene-specific primers (V-GspF and V-GspR). Thus, the nucleotide sequence of the P. volitans hyaluronidase cDNA (1,781 bp) was determined as shown in Fig. 3. The cDNA contains an open reading frame of 1,449 bp (corresponding to 483 amino acid residues), which is the same length as that of the P. antennata hyaluronidase cDNA. Amino acid sequences of lionfish hyaluronidases The deduced amino acid sequences of the P. antennata and P. volitans hyaluronidases are shown in Fig. 4, together with those of various animal hyaluronidases. As analyzed by the SignalP 4.1 server (Petersen et al. 2011), the N-terminal segment (up to the 28th residue) of both lionfish hyaluronidases was predicted to be a signal peptide. It is thus considered that the region 29–483 (455 amino acid residues) of the precursor protein is a mature portion for both hyaluronidases. Surprisingly, little difference is recognized between the amino acid sequences of P. antennata and P. volitans hyaluronidases; there are only two alterations at positions 102 (Val/Asp) and 429 (Ser/Arg). Accordingly, the amino acid sequence identity between both lionfish hyaluronidases is as high as 99.6 %. The lionfish hyaluronidases also share considerably high amino acid sequence identities (72–77 %) with the stonefish hyaluronidases but rather low identities with those from mammals (about 41 %), snakes (about 38 %) and honeybees (about 26 %). To shed more light on the molecular relationship between hyaluronidases from lionfish and other animals, a phylogenetic tree was generated by the MEGA server. As shown in Fig. 5, the tree includes two main branches. The first branch is composed of fish, frog, snake and mammalian hyaluronidases and the second branch arthropod and snail hyaluronidases. In the first branch, the lionfish hyaluronidases, together with the stonefish hyaluronidases, form an isolated cluster, suggesting that both lionfish and stonefish

hyaluronidases have evolved independently from other hyaluronidases.

Discussion Hyaluronidases in the animal venoms are not toxic principles by themselves but are implicated in the envenomation events by acting as toxin-spreading factors. In contrast to the accumulated knowledge on hyaluronidases from terrestrial venomous animals, fish venom hyaluronidases have not been extensively studied. So far, enzymatic and chemical information about fish venom hyaluronidases has been limited to those only from the following three species of fish: stingray P. motoro (Magalha˜es et al. 2008) and two species of stonefish, S. horrida (Poh et al. 1992; Sugahara et al. 1992) and S. verrucosa (Madokoro et al. 2011). Based on the finding of hyaluronidases in the venoms of two species of lionfish, P. antennata and P. volitans, we clarified their enzymatic properties and primary structures in this study. Both P. antennata and P. volitans hyaluronidases showed the same optimum pH (6.6), optimum temperature (37 °C) and optimum NaCl concentration (0.1 M) and also the same substrate specificity (specific only for hyaluronan). This is not surprising in consideration of the fact that only two alterations in amino acid residues are seen between the primary structures of both hyaluronidases. The determined enzymatic properties of the lionfish hyaluronidases are close to those of the stonefish hyaluronidases; only a slight difference from the lionfish hyaluronidases is observed in optimum pH (6.0) for the S. horrida hyaluronidase (Poh et al. 1992) and in optimum NaCl concentration (0.15 M) for the S. verrucosa hyaluronidase (Madokoro et al. 2011). However, the lionfish hyaluronidases are distinct from the stingray hyaluronidase with an optimum pH of 4.2 (Magalha˜es et al. 2008). Based on optimum pH, hyaluronidases can be divided into two types, acid active and neutral active types (Frost et al. 1996). Apparently, the lionfish hyaluronidases as well as the stonefish hyaluronidases belong to the neutral active type, while the stingray hyaluronidase is a member of the acid active type. It is worth mentioning that the lionfish hyaluronidases are active only against hyaluronan, showing no activity against chondroitin sulfate A–C and heparin. Similar results have also been reported for the

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stonefish hyaluronidases (Sugahara et al. 1992; Madokoro et al. 2011), although the substrate specificity of the stingray hyaluronidase is unknown. On the other hand, mammalian testicular hyaluronidases are

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active not only against hyaluronan but also against chondroitin and its sulfates (Stern and Jedrzejas 2006; EI-Safory et al. 2010). In the case of terrestrial venomous animals, some hyaluronidases such as those

Fish Physiol Biochem b Fig. 4 Alignment of the amino acid sequences of the P.

antennata and P. volitans hyaluronidases with those of various animal hyaluronidases. Accession numbers for hyaluronidases (DDBJ/EMBL/GenBank databases): AB759697 for lionfish P. antennata; AB759698 for lionfish P. volitans; AY232496 for stonefish S. horrida; AB607856 for stonefish S. verrucosa; S67798 for human PH-20; AK005638 for mouse PH-20 (sperm adhesion molecule); DQ840256 for puff adder Bitis arietans; DQ840250 for horn viper Cerastes cerastes; AF351623 for Eastern honeybee Apis cerana cerana; L10710 for Western honeybee Apis mellifera. The residues identical with those of P. antennata hyaluronidase are indicated by dots. Signal peptides are boxed. The residues essential for the hyaluronan binding of hyaluronidases are indicated by closed circles. Putative N-glycosylation sites are shown by asterisks. Cysteine residues are highlighted in white on a black background

from Indian cobra N. naja (Girish et al. 2004) and funnel web spider H. partita (Nagaraju et al. 2007), like the fish hyaluronidases, are specific only against hyaluronan and some hyaluronidases such as those from rattlesnake Crotalus durissus terrificus (Bordon et al. 2012) and brown spider Loxosceles intermedia (da Silveira et al. 2007), like the mammalian testicular hyaluronidases, show a wider substrate specificity. It is thus plausible that the absolute specificity against hyaluronan is a common property of hyaluronidases in fish venoms, although future study is needed with many species of venomous fish. The lionfish hyaluronidases are almost identical in primary structure with each other and also exhibit high sequence identities (72–77 %) with the stonefish hyaluronidases. However, their sequence identities with those from mammals and other venomous animals are rather low (mostly in the range of 25–40 %). In consistent with this, the phylogenetic tree showed that the lionfish hyaluronidases, together with the stonefish hyaluronidases, form a cluster, being independent from other hyaluronidases. Nevertheless, the lionfish hyaluronidases almost maintain some structural features observed in other hyaluronidases. Firstly, one catalytic residue (Glu-141) and four substrate positioning residues (Asp-139, Tyr-212, Tyr-259 and Trp-335), which have been identified for human hyaluronidase (Stern and Jedrzejas 2006), are conservatively recognized in the lionfish hyaluronidases as well as in other hyaluronidases. Secondly, a database search by the Pfam (Bateman et al. 2002) revealed that the lionfish hyaluronidases have a glyco_hydro_56 domain in the region 34–444, which is seen at almost the same position in other hyaluronidases (e.g., region 33–370 for S. verrucosa

hyaluronidase and region 38–374 for human hyaluronidase). Thirdly, 10 of the 12 cysteine residues included in the lionfish hyaluronidases are mostly located at the same positions as other animal hyaluronidases, except for honeybee hyaluronidases lacking about 100 residues in the C-terminal region. It is, therefore, assumed that the lionfish hyaluronidases contain disulfide bridges at the same position as other hyaluronidases, that is, they show a similar conformation to other hyaluronidases. The stonefish S. horrida hyaluronidase has been demonstrated to be a glycoprotein (Poh et al. 1992). Importantly, its cDNA can be expressed in an active form in insect cells, but not in Escherichia coli (Ng et al. 2005), suggesting that its carbohydrate moieties are required for the correct folding and/or for the catalytic activity of the enzyme. Another stonefish hyaluronidase from S. verrucosa is also assumed to be a glycoprotein, because it has a high sequence identity with the S. horrida hyaluronidase and contains three putative N-glycosylation sites at the same positions (78–80, 179–181 and 364–366) as the S. horrida hyaluronidase. In view of these circumstances, it is interesting to note that the lionfish hyaluronidases include three N-glycosylation sites at the same positions as the stonefish hyaluronidases and two additional N-glycosylation sites at positions 281–283 and 460–462, at which Asn-281 and Thr-462 are replaced by Ala in the case of the stonefish hyaluronidases. This allows us to assume that the molecules of lionfish hyaluronidases include carbohydrate moieties essential for conformation and/or enzymatic activity. Hyaluronidases in the animal venoms function as toxin-spreading factors, as previously demonstrated with a number of venom hyaluronidases including the S. verrucosa hyaluronidase. This is presumably the case with the lionfish hyaluronidases. In addition to toxin-spreading factors, some venom hyaluronidases, especially those from bees, hornets and wasps, are known to act as allergens (King and Wittkowski 2011). Allergenic hyaluronidases induce adverse reactions in sensitive individuals, such as urticaria, angioedema, nausea, vomiting and shivers, through the binding with specific IgE on the surface of mast cells; in severe cases, even death occurs due to anaphylactic shock. As far as we know, no allergic symptoms have previously been described in the envenomations by venomous fish. However, in view of the considerable sequence identity between

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Fish Physiol Biochem 100 100 76 100 98 99

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Fig. 5 Phylogenetic tree of animal hyaluronidases. The tree was generated using a ClustalW algorithm combined with a MEGA 5.2 tool. Accession numbers for hyaluronidases (DDBJ/EMBL/GenBank databases) are shown in parentheses

hyaluronidases from fish venoms and other animal venoms, fish venom hyaluronidases may act as potential allergens. To clarify this, it should be examined whether individuals previously stung by a certain venomous fish (e.g., lionfish) have significant levels of serum IgE specific for its hyaluronidase. In this study, the venom hyaluronidases from two species of lionfish were enzymatically and structurally characterized. However, knowledge about fish venom hyaluronidases is still poor, being limited to those from stingray, stonefish and lionfish. It is possible that hyaluronidases are distributed in a number of venomous fish other than the above three kinds of fish. For the

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better understanding on the pathophysiology of fish envenomation, future study is needed to characterize hyaluronidases as well as toxins in a variety of venomous fish. Acknowledgments Financial support from Tokyo University of Marine Science and Technology is greatly appreciated.

References Austin L, Gillis RG, Youatt G (1965) Stonefish venom: some biochemical and chemical observations. Aust J Exp Biol Med Sci 43:79–90

Fish Physiol Biochem Barbaro KC, Lira MS, Malta MB, Soares SL, Neto DG, Cardoso JL, Santoro ML, Haddad V Jr (2007) Comparative study on extracts from the tissue covering the stingers of freshwater (Potamotrygon falkneri) and marine (Dasyatis guttata) stingrays. Toxicon 50:676–687 Bateman A, Birney E, Cerruti L, Durbin R, Etwiller L, Eddy SR, Griffiths-Jones S, Howe KL, Marshall M, Sonnhammer ELL (2002) The Pfam protein families database. Nucleic Acids Res 30:276–280 Bordon KCF, Perino MG, Giglio JR, Arantes EC (2012) Isolation, enzymatic characterization and antiedematogenic activity of the first reported rattlesnake hyaluronidase from Crotalus durissus terrificus venom. Biochimie 94:2740–2748 Da Silveira RB, Chaim OM, Mangili OC, Gremski W, Dietrich CP, Nader HB, Veiga SS (2007) Hyaluronidases in Loxosceles intermedia (brown spider) venom are endo-b-Nacetyl-D-hexosaminidases hydrolases. Toxicon 49:758–768 EI-Safory NS, Fazary AE, Lee C (2010) Hyaluronidases, a group of glycosidases: current and future perspectives. Carbohydr Polym 81:165–181 Frost GI, Csoka T, Stern R (1996) The hyaluronidase: a chemical, biological and clinical overview. Trends Glycosci Glycotechnol 8:419–434 Garnier P, Goudey-Perriere F, Breton P, Dewulf C, Petek F, Perriere C (1995) Enzymatic properties of the stonefish (Synanceia verrucosa Bloch and Schneider, 1801) venom and purification of a lethal, hypotensive and cytolytic factor. Toxicon 33:143–155 Girish KS, Shashidharamurthy R, Nagaraju S, Gowda TV, Kemparaju K (2004) Isolation and characterization of hyaluronidase a ‘‘spreading factor’’ from Indian cobra (Naja naja) venom. Biochimie 86:193–202 Haddad V Jr, Neto DG, de Paula Neto JB, de Luna Marques FP, Barbaro KC (2004) Freshwater stingrays: study of epidemiologic, clinic and therapeutic aspects based on 84 envenomings in humans and some enzymatic activities of the venom. Toxicon 43:287–294 Hopkins BJ, Hodgson WC (1998) Enzyme and biochemical studies of stonefish (Synanceja trachynis) and soldierfish (Gymnapistes marmoratus) venoms. Toxicon 36:791–793 King TP, Wittkowski KM (2011) Hyaluronidase and hyaluronan in insect venom allergy. Int Arch Allergy Immunol 156: 205–211 Kiriake A, Shiomi K (2011) Some properties and cDNA cloning of proteinaceous toxins from two species of lionfish (Pterois antennata and Pterois lunulata). Toxicon 58:494–501 Kiriake A, Suzuki Y, Nagashima Y, Shiomi K (2013) Proteinaceous toxins from three species of scorpaeniform fish (lionfish Pterois lunulata, devil stinger Inimicus japonicus and waspfish Hypodytes rubripinnis): close similarity in properties and primary structures to stonefish toxins. Toxicon 70:184–193

Madokoro M, Ueda A, Kiriake A, Shiomi K (2011) Properties and cDNA cloning of a hyaluronidase from the stonefish Synanceia verrucosa venom. Toxicon 58:285–292 Magalha˜es MR, da Silva Jr NJ, Ulhoa CJ (2008) A hyaluronidase from Potamotrygon motoro (freshwater stingrays) venom: isolation and characterization. Toxicon 51:1060–1067 Nagaraju S, Devaraja S, Kemparaju K (2007) Purification and properties of hyaluronidase from Hippasa partita (funnel web spider) venom gland extract. Toxicon 50:383–393 Ng HC, Ranganathan S, Chua KL, Khoo HE (2005) Cloning and molecular characterization of the first aquatic hyaluronidase, SFHYA1, from the venom of stonefish (Synanceja horrida). Gene 346:71–81 Pessini AC, Takao TT, Cavalheiro EC, Vichnewski W, Sampaio SV, Giglio JR, Arantes EC (2001) A hyaluronidase from Tityus serrulatus scorpion venom: isolation, characterization and inhibition by flavonoids. Toxicon 39:1495–1504 Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786 Poh CH, Yuen R, Chung MC, Khoo HE (1992) Purification and partial characterization of hyaluronidase from stonefish (Synanceja horrida) venom. Comp Biochem Physiol B 101:159–163 Shiomi K, Hosaka M, Kikuchi T (1993) Properties of a lethal factor in stonefish Synanceia verrucosa venom. Nippon Suisan Gakkaishi 59:1099 Stern R, Jedrzejas MJ (2006) Hyaluronidases: their genomics, structures, and mechanisms of action. Chem Rev 106:818–839 Sugahara K, Yamada S, Sugiura M, Takeda K, Yuen R, Khoo HE, Poh CH (1992) Identification of the reaction products of the purified hyaluronidase from stonefish (Synanceja horrida) venom. Biochem J 283:99–104 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680 Tolksdorf S, McCready MH, McCullagh DR, Schwenk E (1949) The turbidimetric assay of hyaluronidase. J Lab Clin Med 34:74–89 Tu AT, Hendon RR (1983) Characterization of lizard venom hyaluronidase and evidence for its action as a spreading factor. Comp Biochem Physiol B 76:377–383

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Enzymatic properties and primary structures of hyaluronidases from two species of lionfish (Pterois antennata and Pterois volitans).

Lionfish are representative venomous fish, having venomous glandular tissues in dorsal, pelvic and anal spines. Some properties and primary structures...
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