Toxicon 95 (2015) 67e71

Contents lists available at ScienceDirect

Toxicon
journal homepage: www.elsevier.com/locate/toxicon

Short communication

Identification of C-type isolectins in the venom of the scorpionfish

Scorpaena plumieri
F. Andrich a, b, M. Richardson c, G.B. Naumann a, c, M.N. Cordeiro c, A.V. Santos d,
D.M. Santos d, J.S. Oliveira d, M.E. de Lima d, S.G. Figueiredo a, *
a
Departamento de Ci^ gicas, Centro de Ci^
encias Fisiolo encias da Saúde, Universidade Federal do Espírito Santo, Avenida Marechal Campos, 1468, Maruípe,
ria, ES 29043-900, Brazil
Vito
b
Departamento de Fisiologia e Biofísica, Instituto de Ci^ gicas, Universidade Federal de Minas Gerais, Avenida Anto
encias Biolo ^nio Carlos, 6627, Pampulha,
Belo Horizonte, MG 31270-901, Brazil
c
Diretoria do Centro de Pesquisa e Desenvolvimento, Fundaça ~o Ezequiel Dias, Rua Conde Pereira Carneiro, 80, Gameleira, Belo Horizonte, MG 30510-010,
Brazil
d
Departamento de Bioquímica e Imunologia, Instituto de Ci^ gicas, Universidade Federal de Minas Gerais, Av. Anto
encias Biolo ^nio Carlos, 6627, Pampulha,
Belo Horizonte, MG 31270-901, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Chemical analyses of the hemagglutinating fraction from Scorpaena plumieri venom revealed that it
Received 29 September 2014 contains five components (Sp-CL 1-5) with similar chromatographic elution profiles (35e38% of aceto-
Received in revised form nitrile), molecular masses (16,800e17,000 Da) and N-terminal sequences, suggesting that they are iso-
18 December 2014
forms of the same protein. The amino acid sequence of Sp-CL4 was determined and shown to have
Accepted 6 January 2015
Available online 7 January 2015
homology with fish C-type lectins. These data demonstrate for the first time the presence of C-type
isolectins in a scorpionfish venom.
© 2015 Elsevier Ltd. All rights reserved.
Keywords:
Fish venom
Scorpaena plumieri
Scorpionfish
C-type lectin
Hemagglutination
Isolectins

Among the venomous fish, the scorpionfish Scorpaena plumieri
is considered the most dangerous in the southwestern Atlantic
Ocean and is responsible for many accidents on the Brazilian coast,
affecting tourists, bathers and fishermen (Haddad Jr. et al., 2003).
The venom of this species is highly hemolytic for rabbit erythro-
cytes (Carrijo et al., 2005), a consequence of the action of a potent
pore-forming toxin, Sp-CTx (Gomes et al., 2013). Besides cell
membrane disruption activity, the fractionation of this venom by
gel filtration chromatography also revealed a hemagglutinating
Abbreviations: SpV, Scorpaena plumieri Venom; FV, Fraction V with hemag-
glutinating activity; SDS-PAGE, Sodium Dodecyl Sulfate Polyacrylamide Gel Elec-
trophoresis; MALDI-TOF, Matrix Assisted Laser Desorption Ionization Time-Of-
Flight; MS, Mass Spectrometry; RP-HPLC, Reversed Phase High Performance Liquid
Chromatography; TFA, Trifluoroacetic Acid; ACN, Acetonitrile; CTLD, C-Type Lectin-
like Domain; CRD, Carbohydrate Recognition Domain; Sp-CL, S. plumieri C-type
Lectin.
* Corresponding author.
E-mail address: suelygfi@cbm.ufes.br (S.G. Figueiredo).
fraction (unpublished data). Agglutination of hemocytes induced
by extracts from vegetable or animal sources, like venoms, has been
associated with the activity of molecules with carbohydrate-
binding properties: the lectins (Kilpatrick, 2002; Sharon and Lis,
2004). Despite the extensive data about lectins in snake venoms
and the great interest of researchers on their action upon the he-
mostatic system, cell proliferation and adhesion processes
(Markland, 1998; Eble et al., 2001), up to now, just two lectins have
been characterized in piscine venoms: (i) Plumieribetin, a protein
homologous to B-type lectins with integrin-binding inhibitory ac-
tivity was purified from S. plumieri venom (de Santana Evangelista
et al., 2009); and (ii) Nattectin, a galactose-binding pro-inflam-
matory C-type lectin was isolated from toadfish Thalassophryne
nattereri venom (Lopes-Ferreira et al., 2011). Probably the scarcity
of data about lectins in fish venoms is due to the high hemolytic
activity of the crude venom, which masks the agglutinating effect
evoked by the lectins and hinders their identification by hemag-
glutination assay. Therefore, the aim of the present work was to

http://dx.doi.org/10.1016/j.toxicon.2015.01.004
0041-0101/© 2015 Elsevier Ltd. All rights reserved.
68 F. Andrich et al. / Toxicon 95 (2015) 67e71
 F. Andrich et al. / Toxicon 95 (2015) 67e71 69

further investigate the hemagglutinating molecules from
S. plumieri venom through a protein chemistry approach.
Scorpionfish venom was extracted from wild specimens,
collected from shallow water beaches of Fund~ ao City (Espírito
Santo, Brazil), according to a batch method (Schaeffer et al., 1971)
using chilled phosphate buffered saline (PBS, pH 7.5). The protein
content was measured according to Lowry et al. (1951) or estimated
by spectrophotometry. In order to obtain the fraction V (FV) with
hemagglutinating activity, 49.2 mg of protein of fresh S. plumieri
soluble venom extract (SpV) was fractionated by chromatography
on a Sephacryl S-200 HR column (120
2.0 cm) according to de
Santana Evangelista et al. (2009). The purified fraction repre-
sented about 9% of the total of proteins in the venom. Hemagglu-
tination was evaluated through a serial dilution method (Matsui,
1984) by using a washed suspension of rabbit erythrocytes sus-
pension (2.5% v/v in PBS). The minimum agglutinating concentra-
tion of FV, defined as the reciprocal of the highest dilution giving
visible agglutination, was 16 mg/mL.
The protein content of FV was examined on a SDS-PAGE-Tricine
gel (Schagger and Von Jagow, 1987) which showed two diffuse
bands at ~15 kDa under non-reducing conditions (Fig. 1A). In spite
of only two components being resolved by electrophoresis, five
main absorbance peaks (RP1 to RP5, Fig. 1B) with very similar
elution/retention time were detected when FV was subjected to
reversed phase high performance liquid chromatography (RP-
HPLC). Mass spectrometry analysis of the RP-HPLC fractions on a
MALDI-TOF instrument (Fig. 1C) revealed that RP2, RP4 and RP5
were obtained with high degree of homogeneity with m/z signals of
16,982, 16,841 and 16,842 respectively. The main m/z signals
detected in RP1 and RP3 were 16,981 and 16,975, albeit lesser in-
tensity signals were also found (16,200 and 16,830, respectively).
The conditions used for these analyses are detailed in the legend to
Fig. 1.
The fractions RP2, 4 and 5 were subjected, in their native form,
to automated N-terminal sequence determination on a PPSQ-21A
(Shimadzu, Kyoto, Japan) apparatus using a standard Edman
degradation program. The N-terminal sequence of RP4 could be
confidently determined from amino acid residue 1 (Asp1) to residue
35 (Phe35) (Fig. 2). A sequence of 9 residues (DAEPPXGPAE) was
obtained for RP2, which exhibits high identity to that from RP4.
However no signal was obtained when RP5 (500 pMol) was sub-
jected to the procedure, suggesting that it has a blocked N-
terminus.
In order to determine the remainder of its primary structure,
RP4 was submitted to proteolytic fragmentation. First, about 1 mg
of protein was reduced and S-alkylated with vinyl pyridine as
described by Henschen (1986). Samples of the modified proteins
were desalted and then separately digested with trypsin and Glu-
specific V8 endoproteinase from Staphylococcus aureus. Fragments
produced by these procedures were separated by RP-HPLC on a m-
Bondapak C18 column (Felicori et al., 2003) and sequenced. Over-
lapping sequences were aligned and yielded details of a 144 amino
acid residues long polypeptide chain, with only two unidentified
residues (Fig. 2, residues 105 and 106). RP4 displays a high level
(20%) of acidic amino acids with an estimated isoelectric point of
4.4. The calculated molecular mass (16,135 Da) is 706 Da lower than
that determined by MALDI-TOF-MS (16,841 Da), and it suggests
that the protein may suffer posttranslational modifications (e.g.,
glycosilation).
In order to investigate the presence of glycans in FV components
a panel of lectins (DIG Glycan Differentiation Kit, Roche Diagnostics,
Mannhein, Germany) with different capacities to recognize sugar
residues typical for N- and O-glycoconjugates was used. It was
demonstrated, after blotting onto a nitrocellulose membrane, that
FV reacted only with lectin DSA (Datura stramonium agglutinin),
which recognizes the sugar motif galactose-b(1 / 4)-N-acetyl-
glucosamine (Fig. 1D). Interestingly, the molecular mass of the
protein bands recognized are 2e4 fold higher than that observed in
the SDS-PAGE-Tricine profile (Fig. 1A). This fact suggested that FV
proteins are able to oligomerize and this is in accordance with the
m/z signals of 16,800-17,000 multiples (i.e., 34,000, 51,000 and
68,000 Da) observed in FV MALDI-TOF-MS spectrum at a higher m/
z range (20e70 kDa, Supplementary Data). The molecular mass of
the carbohydrate moiety linked (383 Da) taken together with the
two non-identified amino acid residues (probably W and K or E,
according to the CRD signature and the cleavage sites of the pro-
teinases used) may explain the mass difference found between the
MS and the sequence analyses.
Searches for similarities between the RP4 amino acid sequence
with those of related proteins stored on the Swiss-prot/Trembl data
bases using the FASTA 3 and BLAST programs revealed homology
(24e32 % of identity) with various fish C-type lectins or proteins
containing C-type lectin-like domains, CTLDs (Fig. 2). The family of
the proteins containing CTLDs is a large group of extracellular
Metazoan proteins with diverse function. Many CTLDs have evolved
to specifically recognize protein, lipid and inorganic ligands,
including those found in snake venom proteins, bird egg-shell
proteins and fish antifreeze proteins (Zelensky and Gready, 2005).
However, Caþ2-dependent carbohydrate binding is the most com-
mon CTLD function in vertebrates, and usually they are referred as
(true) C-type lectins. These proteins have a compact domain of
approximately 110e130 amino acid residues, named the carbohy-
drate recognition domain, CRD (Drickamer, 1993).
The RP4 amino acid sequence exhibits 11 of the 14 invariant and
17 of the 18 conserved residues found in the C-type lectins CRD
signature (Fig. 2). Another feature observed in the primary struc-
ture of RP4 is the presence of a QPD motif (residues 109e111),
which is reported in the galactose-binding C-type lectins. These
structural data about RP4, taken together with the agglutinating
property exhibited by FV, are suggestive that it is a carbohydrate-
binding protein or true C-type lectin. The galactose-binding spec-
ificity of FV proteins was confirmed by affinity chromatography on
immobilized D-galactose gel column (Thermo Scientific e data not
shown).
It is noteworthy that the RP fractions exhibited similar chemical
characteristics: (i) the amount of acetonitrile necessary for elution
(35e38 %) in RP-HPLC, (ii) molecular mass (16,800e17,000 Da) and
(iii) N-terminal sequences. It is well known that the development of

Fig. 1. Chemical analyses and fractionation of the hemagglutinating fraction (FV) of S. plumieri venom. (A) Non-reducing SDS-PAGE-Tricine profile of FV (40 mg). The gel (10% T, 3% C)
was stained with Serva blue G. M, molecular mass markers. (B) RP-HPLC profile of FV (100 mg) on Waters Symmetry300 C18 column (250
4.6 mm). The column was equilibrated
using 0.1% TFA (v/v) in ultrapure water and eluted using a double gradient from 0 to 50 % of TFA 0.1% (v/v) in ACN (1 mL/min flow rate and fractions of 0.4 mL). Elution was
monitored by light absorbance at 214 nm e mAU, milli Absorption Units. (C) MS spectrum obtained respectively for fractions RP1, 2, 3, 4 and 5 on a MALDI-TOF AutoFlex III in-
strument (Bruker Daltonics, Billerica, USA) in positive/linear mode (laser frequency 50e60%, m/z range 5e20 kDa) and using a-Cyano-4-hydroxycinnamic acid as matrix. A.U.,
arbitrary units. (D) Glycoconjugates detection. FV and N/O-glycosilated control proteins (denotedþ) were transferred to a nitrocellulose membrane after SDS-PAGE and stained with
digoxigenin-labeled lectins and enzyme conjugated anti-digoxigenin-antibodies. Lectins and their target structures: PNA e Peanut agglutinin (galactose-b(1e3)-N-acetylga-
lactosamine of O-Glycans); GNA e Galanthus nivalis agglutinin (mannose in highmannose-type N-glycoconjugates); SNA e Sambuca nigra agglutinin ((2e6)-linked sialic acid in
complex type N-glycoconjugates); MAA e Maackia amurensis agglutinin (a(2e6)-linked sialic acid in complex type N-glycoconjugates and O-glycans); DSA e Datura stramonium
agglutinin (galactose-b(1e4)-N-acetylglucosamine in complex and hybrid N-glycans, in O-glycans and N-acetylglucosamine in O-glycans).
70 F. Andrich et al. / Toxicon 95 (2015) 67e71

Fig. 2. RP4 (Sp-CL4) amino acid sequence and alignment with fish C-type lectins. Arrows (>) indicate residues determined by N-terminal sequencing of the native protein,
discontinuous lines () e e /) denote sequences of peptides obtained from digestion with trypsin (T) and Staphylococcus aureus V8 protease (V) of the reduced and alkylated
protein. Dark gray and light gray shading denotes respectively identical and conserved amino acid residues in all sequences. Four Cys residues that form two conserved disulfide
bonds (Drickamer, 1993) are indicated. The conserved QPD motive for galactose binding is shown in bold characters. Gaps (-) have been introduced to optimize identity. In the
sequence motif characteristic of C-type CRD, absolutely conserved residues are indicated by the one-letter amino acid code, while residues conserved in character are indicated by O,
oxygen-containing; F, aromatic; q, aliphatic; and U, aromatic or aliphatic. The identity percentages between Sp-CL4 sequence and related proteins are indicated in parentheses.
CTL1_Tf (Uniprot K9MQ70), C-type lectin from Trachidermus fasciatus; eCL2_Aj (Uniprot Q8AXR8) and eCL1_Aj (Uniprot Q8AXR7); C-type lectins 2 and 1, respectively, from Anguilla
japonica; Nattectin_Tn (Genbank AAU11827), C-type lectin from the venom of Thalassophryne nattereri; AFP_Ec (Uniprot R4TGG3), Antifreeze protein from Epinephelus coioides;
Ntc_Eb (Uniprot G1FKK1), Nattectin from Epinephelus bruneus; CTLP_Ec (Uniprot F6KMP1), C-type lectin-like protein from Epinephelus coioides; LEC_Pw (Uniprot Q02988), Lectin
from Pleurodeles waltl; CRD, Carbohydrate Recognition Domain signature (Drickamer, 1993). A multiple sequence alignment was made with CLUSTAL W. For better alignment, the
sequences determined by genomic analyses had its signal peptide predicted by SignalP 4.1 server and removed afterward. X, unidentified residues.

multiple similar proteins (isoforms) is a common feature exhibited
by several venomous animal toxins, probably as a result of an
evolution mechanism in order to deceive the defense system of the
prey. In addition, isoforms were also reported when analyzing
proteins with ice-binging CTLDs in the serum of the sea raven
Hemitripterus americanus by RP-HPLC (Ng et al., 1986). These data,
together with the similarities found between RP4 amino acid
sequence and those from C-type lectins, let us suppose that RP1, 2,
3, 4 and 5 are C-type lectins isoforms or isolectins. Therefore, these
fractions were named as Sp-CL (Scorpaena plumieri C-type Lectin 1,
2, 3, 4 and 5).
Furthermore, the Sp-CL-4 lectins homologues presented rele-
vant biological activities: Nattectin induces inflammatory response
and acts as an immunomodulatory agent (Saraiva et al., 2011;
Ishizuka et al., 2012) promoting the increase of cell adhesion and
apoptosis resistance through the binding to b1-integrins (Komegae
et al., 2011); whereas AJL-2 exhibited agglutinating activity against
Escherichia coli (Tasumi et al., 2002). These studies show the
pharmacological potential of fish lectins and raise the question
about the biological functions of those found in S. plumieri venom.
In summary, this work reported the chemical characterization of
new proteins from S. plumieri venom. In spite of the hemolysis
induced by the crude venom, a fraction with hemagglutinating
activity was revealed/separated after gel filtration chromatography.
Our data demonstrated that C-type isolectins are the main com-
ponents of this fraction, nevertheless further studies are required to
unveil the function of such molecules in scorpionfish venom and its
role during human envenomation provoked by this fish. Further-
more, the potential for these new molecules to possible therapeutic
applications, e.g. as immunomodulating or antimicrobial agents,
amongst others, remains to be studied.
Ethical statement
No sentient animals were used in the current study.
Acknowledgments
This work was supported by CNPq (Conselho Nacional de
Desenvolvimento Científico e Tecnolo  gico), FAPEMIG (Fundaça~o de
Amparo  a Pesquisa do Estado de Minas Gerais), CAPES (Coor-
denaça~o de Aperfeiçoamento de Pessoal de Nível Superior),
INCTTOX (Instituto Nacional de Cie ^ncia e Tecnologia em Toxinas)
and FACITEC (Fundo de Apoio a  Cie
^ncia e Tecnologia do Município
ria). The authors are indebted to M.L.B. Gozze for capturing
de Vito
the fish used to extract the venom.
 F. Andrich et al. / Toxicon 95 (2015) 67e71
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.toxicon.2015.01.004.
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.toxicon.2015.01.004.
References
Carrijo, L.C., Andrich, F., de Lima, M.E., Cordeiro, M.N., Richardson, M.,
Figueiredo, S.G., 2005. Biological properties of the venom from the scorpionfish
(Scorpaena plumieri) and purification of a gelatinolytic protease. Toxicon 45,
843e850.
de Santana Evangelista, K., Andrich, F., Figueiredo de Rezende, F., Niland, S.,
Cordeiro, M.N., Horlacher, T., Castelli, R., Schmidt-Hederich, A., Seeberger, P.H.,
Sanchez, E.F., Richardson, M., Gomes de Figueiredo, S., Eble, J.A., 2009. Plu-
mieribetin, a fish lectin homologous to mannose-binding B-type lectins, in-
hibits the collagen-binding alpha1beta1 integrin. J. Biol. Chem. 284,
34747e34759.
Drickamer, K., 1993. Caþ2 dependent carbohydrate-recognition domains in animal
proteins. Curr. Opin. Struct. Biol. 3, 393e400.
Eble, J.A., Beermann, B., Hinz, H.J., Schmidt-Hederich, A., 2001. a2b1 integrin is not
recognized by rhodocytin but is the specific high affinity target of rhodocetin,
an RGD-independent disintegrin and potent inhibitor of cell adhesion to
collagen. J. Biol. Chem. 276, 12274e12284.
Felicori, L.F., Souza, C.T., Velarde, D.T., Magalhaes, A., Almeida, A.P., Figueiredo, S.,
Richardson, M., Diniz, C.R., Sanchez, E.F., 2003. Kallikrein-like proteinase from
bushmaster snake venom. Protein Expr. Purif. 30, 32e42.
Gomes, H.L., Andrich, F., Fortes-dias, C.L., Perales, J., Teixeira-ferreira, A.,
Vassallo, D.V., Cruz, J.S., Figueiredo, S.G., 2013. Molecular and biochemical
characterization of a cytolysin from the Scorpaena plumieri (scorpionfish)
venom: evidence of pore formation on erythrocyte cell membrane. Toxicon 74,
92e100.
Haddad Jr., V., Martins, I.A., Makyama, H.M., 2003. Injuries caused by scorpionfishes
(Scorpaena plumieri Bloch, 1789 and Scorpaena brasiliensis Cuvier, 1829) in the
Southwestern Atlantic Ocean (Brazilian coast): epidemiologic, clinic and ther-
apeutic aspects of 23 stings in humans. Toxicon 42, 79e83.
71
Henschen, A., 1986. Analysis of cysteine residues, disulfide bridges, and sulfhydryl
groups in proteins. In: Wittmann-Liebold, B., Salnikow, J., Erdmann, V.A. (Eds.),
Advanced Methods in Protein Microsequence Analysis. Springer-Verlag, Berlin,
pp. 244e255.
Ishizuka, E.K., Ferreira, M.J., Ground, L.Z., Coutinho, E.M.M., Komegae, E.N.,
Cassado, A.A., Bortoluci, K.R., Lopes-Ferreira, M., Lima, C., 2012. Role of interplay
between IL-4 and IFN-g in the in regulation M1 macrophage polarization
induced by Nattectin. Int. Immunopharmacol. 14, 513e522.
Kilpatrick, D.C., 2002. Animal lectins: a historical introduction and overview. Bio-
chim. Biophys. Acta 1572, 187e197.
Komegae, E.N., Ramos, A.D., Oliveira, A.K., Serrano, S.M., Lopes-Ferreira, M., Lima, C.,
2011. Insights into the local pathogenesis induced by fish toxins: role of nat-
terins and nattectin in the disruption of cell-cell and cell-extracellular matrix
interactions and modulation of cell migration. Toxicon 58, 509e517.
Lopes-Ferreira, M., Magalh~ aes, G.S., Fernandez, J.H., Junqueira-de-Azevedo, I.L.,
Ho, P.L., Lima, C., 2011. Structural and biological characterization of Nattectin, a
new C-type lectin from the venomous fish Thalassophryne nattereri. Biochimie
93, 971e980.
Lowry, O.H., Rosenbrough, N.I., Faar, A.L., Randall, R.J., 1951. Protein measurement
with the Folin phenol reagent. J. Biol. Chem. 193, 265e275.
Markland, F.S., 1998. Snake venoms and the hemostatic system. Toxicon 36,
1749e1800.
Matsui, T., 1984. D-Galactoside specific lectins from coelomocytes of the echiuran,
Urechis unicinctus. Biol. Bull. 166, 178e188.
Ng, N.F., Trinh, K.Y., Hew, C.L., 1986. Structure of an antifreeze polypeptide precursor
from the sea raven, Hemitripteruc arnericanus. J. Biol. Chem. 261, 15690e15695.
Saraiva, C.T., Grund, L.Z., Komegae, E.N., Ramos, A.D., Conceiç~ ao, K., Orii, N.M., Lopes-
Ferreira, M., Lima, C., 2011. Nattectin a fish C-type lectin drives Th1 responses
in vivo: licenses macrophages to differentiate into cells exhibiting typical DC
function. Int. Immunopharmacol. 11, 1546e1556.
Schaeffer Jr., R.C., Carlson, R.W., Russell, F.E., 1971. Some chemical properties of the
venom of the scorpionfish Scorpaena guttata. Toxicon 9, 69e78.
Schagger, H., Von Jagow, G., 1987. Tricine-sodium dodecyl sulfate-polyacrylamide
gel electrophoresis for the separation of proteins in the range from 1 to
100KDa. Anal. Biochem.. 166, 368e379.
Sharon, N., Lis, H., 2004. History of lectins: from hemagglutinins to biological
recognition molecules. Glycobiology 14, 53Re62R.
Tasumi, S., Ohira, T., Kawazoe, I., Suetake, H., Suzuki, Y., Aida, K., 2002. Primary
structure and characteristics of a lectin from skin mucus of the Japanese eel
Anguilla japonica. J. Biol. Chem. 277, 27305e27311.
Zelensky, A.N., Gready, J.E., 2005. The C-type lectin-like domain superfamily. FEBS J.
272, 6179e6217.