raia

1. Short communication
Potamotrygon cf. henlei stingray mucus: Biochemical features of a novel
antimicrobial protein
Katia Conceição a,*, Juliane Monteiro-dos-Santos c
, Carla Simone Seibert c
,
Pedro Ismael Silva Jr. b
, Elineide Eugênio Marques c
, Michael Richardson d
,
Mônica Lopes-Ferreira a
a
Immunoregulation Unit, Special Laboratory of Applied Toxinology (CEPID/FAPESP), Butantan Institute, Avenida Vital Brazil 1500, Sao Paulo – SP 05503-900, Brazil
b
Functional Proteomic Units, Special Laboratory of Applied Toxinology (CEPID/FAPESP), Butantan Institute, Sao Paulo, Brazil
c
Federal University of Tocantins, Brazil
d
FUNED (Fundação Ezequiel Dias – Belo Horizonte, MG), Brazil
a r t i c l e i n f o
Article history:
Received 9 April 2012
Received in revised form 4 May 2012
Accepted 24 May 2012
Available online 6 June 2012
Keywords:
Stingray
Potamotrygon
Mucus
Hemoglobin
Antimicrobial
a b s t r a c t
Antimicrobial molecules are important components of the innate immune system in
vertebrates. They have been studied widely in several fishes, but little is known about
these defence factors in stingrays, which are thought to have less sophisticated adaptive
immune systems when compared to other teleosts. Stingrays from the specie Potamo-
trygon cf. henlei are distributed throughout the rivers of central-west Brazil, being the
cause of numerous envenomations occurring in the dry seasons.
In a previous study, we reported that the mucus of the stingray P. cf. henlei shows
antimicrobial effects. Here, to analyze the antimicrobial compounds from the mucus of P. cf.
henlei, we employed solid-phase extraction, chromatographic separation followed by ESI-
MS, and Edman degradation. A protein similar to the b-chain of hemoglobin was identi-
fied, isolated and partially sequenced by Edman degradation. This protein has a molecular
weight of 16072.8 Da, and was shown to be active against bacteria (Micrococcus luteus and
Escherichiacoli) and yeast (Candida tropicalis) without hemolytic activity. Effects of this new
protein in the microcirculation environment were also evaluated. The results obtained
provide fundamental information for future basic research, clinical diagnosis and develop-
ment of new therapies to accident treatment. To the best of our knowledge, this is the first
description of a bioactive polypeptide from the mucus of a stingray.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
The skin of fish constitutes a pivotal immunological
protection against the external environment. The layer of
mucus on the fish surface, considered the first line of
defence, participates in a number of functions including
disease resistance, respiration, ionic and osmotic regula-
tion, locomotion, reproduction, communication, feeding
and nest building (Negus, 1963; Ingram, 1980; Shephard,
1994; Zhao et al., 2008). The mucus, such as that
produced by the skin of the stingrays, has a complex set of
components, which may include amino acid residues,
peptides, complex carbohydrates, glycopeptides, glyco-
lipids and other chemicals (Klesius et al., 2008; Alexander
and Ingram, 1992; Birkemo et al., 2003).
Fish epidermal mucus was found to display antimicro-
bial activity against broad range of infectious pathogens
(Mozumder, 2005; Hellio et al., 2002). We recently
described the antimicrobial activity of catfish Cathorops
spixii mucus (Ramos et al., 2012). Moreover, histone H2B
and two ribosomal proteins are examples of proteins with
antimicrobial activity that have been isolated from
* Corresponding author. Tel.: þ55 11 3726 1024.
E-mail address: conceicaokatia@butantan.gov.br (K. Conceição).
Contents lists available at SciVerse ScienceDirect
Toxicon
journal homepage: www.elsevier.com/locate/toxicon
0041-0101/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.toxicon.2012.05.025
Toxicon 60 (2012) 821–829

2. epidermal mucus of Atlantic cod (Bergsson et al., 2005).
Members of some families of antimicrobial peptides (AMPs)
were also found to be important innate defence components
in the epidermal mucosal layer of Moses sole fish (Parda-
chirus marmoratus) (Oren and Shai, 1996), winter flounder
(Pleuronectes americanus) (Cole et al., 2000), Atlantic halibut
(Hippoglossus hippoglossus) (Birkemo et al., 2003), Hagfish
(Myxine glutinosa L.) (Subramanian et al., 2009) and catfish
(Pelteobagrus fulvidraco) (Su, 2011). These observations
suggest that mucus is a good source of novel molecules for
fish and human health-related applications.
We also recently reported that the mucus of the stingray
Potamotrygon. cf. henlei shows antimicrobial effects and
a pro-inflammatory response (Monteiro-Dos-Santos et al.,
2011). The aim of the present study was therefore to
identify and characterize the major component(s) with
antimicrobial activity in the mucus of P. cf. henlei, which is
a very common stingray found in northern and central-
western rivers from Brazil (Carvalho et al., 2003). Their
spines are hard, sharp, bilaterally retroserrated and covered
by an integumentary sheath with a ventrolateral glandular
groove containing venom glands along both edges
(Halstead, 1970) and the mucus of biological importance
that covers the entire body of these animals. This study
employed a screening approach on mucus components that
were purified by RP-HPLC, and characterized by ESI-MS and
Edman degradation. By this approach, several compounds
including peptides were obtained and a protein similar to
Hemoglobin b-chain was identified, isolated and charac-
terized. Following antimicrobial and hemolytic assays,
intravital microscopy was used to image the effects of the
protein on the microcirculation.
2. Materials and methods
2.1. Collection of stingray mucus
Specimens of adult female and male (n ¼ 15) P. cf. henlei
fish were collected from the Manoel Alves River in the state
of Tocantins, Brazil. Mucus dispersed all over the body was
collected by scraping the skin with a glass slide, and
immediately stored on ice, then diluted in 0.15 M
phosphate-buffered sterile saline, pH 7.4, homogenized,
and centrifuged (5000 Â g for 20 min at 4
C) for collection
of the supernatant. The supernatant was collected and
stored at À20
C. Protein content was determined by the
method of Bradford (1976) using bovine serum albumin
(Sigma Chemical Co., St Louis, MO) as standard protein.
2.2. Purification of mucus
The supernatants were loaded onto solid phase extrac-
tion cartridges Sep-Pak, C18 (Waters Corporation, Taunton,
MA, USA) equilibrated in acidified water (0.1% trifluoro-
acetic acid (TFA)). A single aliquot of 3 mg diluted in 3 mL of
0.1% TFA was loaded and the elution was performed
sequentially with 40 and 80% acetonitrile. These fractions
were further concentrated by a vacuum centrifugation.
Aliquots of 1 mg of the samples were dissolved in 1 mL of
deionized water in 0.1% TFA and centrifuged at 5000 Â g for
20 min (10
C). The supernatants were applied to a system
of RP – HPLC (Äkta basic, Amersham Biosciences – Sweden)
for the sample separation. The sample was loaded in
a Jupiter C18 column (4.6 mm  150 mm, 5 mm, Phenomenex,
USA) in a two-solvent system: (A) TFA/H2O (1:1000) and (B)
TFA/Acetonitrile (ACN)/H2O (1:900:100). The column was
eluted at a flow rate of 1.0 mL/min with a 10–80% gradient of
solvent B over 40 min. For protein purification, further
additional steps of chromatography were necessary, using
a Bio Basic C8 column (4.6 mm  250 mm, 5 mm, Thermo,
USA) with optimized gradients. The HPLC column eluates
were monitored by their absorbance at 214 nm.
2.3. Sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE)
SDS-PAGE was carried out according to Laemmli (1970).
Proteins (10 mg) from the mucus of P. cf. henlei were
analyzed by SDS-PAGE 4–20% acrylamide gradient under
reducing conditions. Prior to electrophoresis, the samples
were mixed 1:1 (v/v) with sample buffer. The gels were
stained with the Silver method.
2.4. Mass spectrometry (MS) and peptide mass fingerprint
(PMF)
The fractions were analyzed by electrospray, with direct
injection in an LC–MS Surveyor MSQ Plus (Thermo Elec-
tron, USA) under positive ionization mode. The needle and
cone potential were set to 3.1 kV and 40 V, respectively. The
aqueous sample solutions (10 mL) were directly injected at
a 50 mL/min constant flow rate of acetonitrile H2O/0.1%
formic acid (1:1). External calibration was performed with
NaI (Sigma) over m/z 100–2000.
Protein band was excised and in-gel trypsin digestion
was performed according to Hellman et al. (1995). Nano-
spray MS/MS analysis was performed on tryptic digests of
SDS-Page band of purified PcfHb using Q-Tof mass spec-
trometry (Q-TOF Ultima API Waters/Micromass, Man-
chester, United Kingdom). An aliquot (5 mL) of the resulting
peptide mixture were injected into Symmetry C18 trapping
column (5 mm particles, 180 mm i.d. Â 20 mm, Waters, USA)
to desalt the peptide mixture. The nano UPLC (Waters)
conditions were 0.1% formic acid in water (solvent A) and
acetonitrile with 0.1% formic acid as solvent B. The sepa-
rations were performed at a flow rate of 0.6 ml/min using
a 0–80% gradient of solvent B over 45 min. The LC system
was coupled to a nano ESI source of the Q-ToF instrument
using a BEH130C18 column (75 mm  100 mm, 1.7 mm
particles; Waters, MA, USA). Typical conditions were
a capillary voltage of 3.1 kV, a cone voltage of 50 V, and
source temperature of 70
C. Data dependent acquisition
(parent ions with 2, 3 and 4 charges) were automatically
recognized by the charge state recognition software Mas-
sLynx 4.1 (Waters, USA). The peptide ions were detected by
scanning from m/z 200 to m/z 2000 at a rate of 1 scan/s, and
were subjected to collision-induced dissociation with
argon in the 13–55 eV collision energy range. Product ions
from MS/MS experiments were detected by scanning from
m/z 50 to m/z 2000 at a rate of 1 scan/s. External calibration
was performed using phosphoric acid (Merck, Darmstadt,
Germany).
K. Conceição et al. / Toxicon 60 (2012) 821–829822

3. All MS/MS spectra were acquired using MassLynx 4.0
software (Waters), deisotoped and converted (by Waters
ProteinLynx Global server 1.0 software) and searched using
a licensed copy of the Mascot Server 2.2 program (Matrix
Science, London, UK). In each instance, the search was
carried out against the non-redundant protein database at
the National Center for Biotechnology Information bank
(NCBI www.ncbi.nlm.nih.gov) assuming the parameters of
Iodoacetamide derivative of cysteine as fixed modification
and methionine oxidation as variable modification, mon-
oisotopic mass values, Æ 0.5 Da peptide mass tolerance, and
Æ0.5 Da fragment mass tolerance. Mascot identifications
required that at least the ion scores must be greater than
the associated identity scores, and 20, 30, 40 and 50 for
single, double, triple and quadruple charged peptides.
Furthermore, Mascot searches were followed by manual
interpretation of MS/MS spectra to eliminate false positives
with the help of the PepSeq tool (MassLynx 4.1 software,
Waters, USA).
2.5. Antimicrobial assay
The antimicrobial activities were determined using
a modified microtiter broth dilution method. The antimi-
crobial activity was monitored by a liquid growth inhibition
assay against gram positive bacteria Micrococcus luteus
A270, gram negative Escherichia coli SBS 363 and yeast
Candida tropicalis MDM8, as described by Bulet et al. (1993)
and Ehret-Sabatier et al. (1996). Pre inocula of the strains
were prepared in Poor Broth (1.0 g peptone in 100 mL of
H2O containing 86 mM NaCl at pH 7.4; 217 mOsM for M.
luteus and E.coli and 1.2 g potato dextrose in 100 mL of H2O
at pH 5.0; 79 mOsM for C. albicans) and incubated at 37
C
with shaking. The absorbance at 595 nm was determined
and one aliquot of this solution was taken to obtain cells in
logarithmic growth (A595nm w 0.6), and diluted 600 times
(A595 nm ¼ 0.0001). The venom, mucus and fractions were
dissolved in sterile Milli-Q water, at a final volume of 100 mL
(10 mL of the sample and 90 mL of the inoculum in PB broth).
After incubation for 18 h at 30
C the inhibition of bacterial
growth was determined by measuring absorbance at
595 nm.
2.6. Hemolytic studies
For hemolytic studies human red blood cells from
a healthy donor (type A) were collected in 0.15 M citrate
buffer, pH7.4, and washed 3 times by centrifugation with
0.15 M phosphate-buffered saline, pH 7.4. To determine the
hemolytic activity, protein samples were assayed in tripli-
cate and tested up to 100 mM: 1.563, 3.125, 6.250, 12.5, 25,
50 and 100 mM in a 3% suspension of erythrocytes incu-
bated for 3 h at room temperature. Hemolysis was deter-
mined by reading the absorbance at 595 nm of each well in
a plate reader. A suspension of erythrocytes incubated with
water was used as a positive control (100% hemolysis).
2.7. Mice and intravital microscopy
Male Swiss mice (5–6 weeks old) were obtained from
a colony at the Butantan Institute, São Paulo, Brazil. Animals
were housed in a laminar flow holding unit (Gelman
Sciences, Sydney, Australia) on autoclaved bedding, in
autoclaved cages, in an air-conditioned room under a 12 h
light/dark cycle. Irradiated food and acidified water were
provided ad libitum. All procedures involving animals were
in accordance with the guidelines provided by the Brazilian
College of Animal Experimentation.
The dynamics of alterations in the microcirculatory
network were determined using intravital microscopy by
transillumination of mice cremaster muscle after subcuta-
neous application 10 ml of protein dissolved in sterile saline.
Administration of the same amount of sterile saline was
used as control. In three independent experiments (n ¼ 4),
mice were injected with 0.4% Xilazine (CoopazineÒ
,
Schering-Plough) and then anaesthetized with 0.2 g/kg
chloral hydrate, and the cremaster muscle was exposed for
microscopic examination in situ as described by Conceição
et al. (2009), Baez (1973) and Lomonte et al. (1994). The
animals were maintained on a board thermostatically
controlled at 37
C, which included a transparent platform
on which the tissue to be transilluminated was placed.
After the stabilization of the microcirculator, the number of
roller cells and adherent leukocytes in the postcapillary
venules were counted 10 min after venom injection. The
study of the microvascular system of the transilluminated
tissue was accomplished with an optical microscope (Axio
Imager A.1, Carl-Zeiss, Germany) coupled to a camera (IcC 1,
Carl-Zeiss, Germany) using a 10/025 longitudinal distance
objective/numeric aperture and 1.6 optovar.
2.8. N-terminal sequence analysis
To determine the amino acid sequence, HPLC purified
samples of the native proteins were subjected to Edman
degradation using a Shimadzu PPSQ-21 automated protein
sequencer, following manufacturer’s instructions.
2.9. Statistical analysis
All results were presented as means Æ SEM of at least
four animals in each group. Differences among data were
determined by ne way analysis of variance (ANOVA) fol-
lowed by Dunnett’s test. Differences between two means
were determined using unpaired Student’s t-test. Data
were considered significant at p 0.05.
3. Results
3.1. PcfHb structural characterization
PcfHb mucus was partially purified by solid-phase
extraction to identify the mucus component(s) respon-
sible for the antimicrobial activity (Monteiro-Dos-Santos
et al., 2011). Three fraction eluates containing 0, 40 and
80% of acetonitrile were obtained. The eluate sample con-
taining 80% acetonitrile reported an enhanced antimicro-
bial activity against M. luteus, E. coli and C. Tropicalis. When
the 80% acetonitrile eluate active factor was purified,
a fraction with antimicrobial activity against the microor-
ganisms tested was detected (Fig. 1A). The antimicrobial
fraction 8 was subjected to further purification by the C8
K. Conceição et al. / Toxicon 60 (2012) 821–829 823

4. RP-HPLC where four peaks were eluted as illustrated in
Fig. 1B. A peak (indicated by an arrow in Fig. 1B), which was
found to contain antimicrobial activities, was eluted out at
the acetonitrile concentration of 43%.
The peak F8 after 12% SDS-PAGE gel analysis presented
a single band with a molecular weight of approximately
16 kDa (Fig. 1C). Furthermore, ESI-MS analysis of F8 peak
revealed that only the last fraction (Fig. 1 arrow) was pure
enough to be chemically characterized. Thus, ESI-MS
spectrum of the compound present in peak 8 revealed an
observed mass of 16072.8 [M þ H]þ1
(Fig. 2A and B). The
purified antimicrobial protein indicated by an arrow in
Fig. 1 was named PcfHb. In order to carry out the structural
elucidation of the purified compound, automated Edman
N-terminal sequencing was performed on a sample of the
native PcfHb and internal peptides were characterized by
peptide mass fingerprinting of the excised 16 kDa band
(Hellman et al., 1995). Due to the low concentration of the
sample, only the fragmentation pattern for LVAEALSSNYH
(parent mass of 1203.6004 – doubly charged peptide) was
obtained by PMF and is presented in Fig. 3.
To determine the resemblance of the partial N-terminal
sequence of the protein PcfHb, with the amino acid
sequences of other proteins deposited in Gen- Bank, we
used ClustalX program (Thompson et al., 1997) to align the
amino acid sequences. The amino acid residues of the
partial N-terminus sequence of PcfHb were found to be
highly similar to the sequences of the beta subunits of
hemoglobins (see Table 1). Overall, there are amino acid
residues conserved throughout the sequences showing
high similarity among the species.
3.2. Antimicrobial, hemolytic and microcirculatory activities
Purified PcfHb exhibited potential antimicrobial activity
against E. coli, M. luteus and C. tropicalis, being more potent
against the last two microorganisms with a minimum
inhibitory concentration (MIC) of 4 mM. It had the weakest
antimicrobial activity (MIC of 12 mM) against E. coli. As
negative and positive controls, deionized water and tetra-
cycline (10 mL, 10 mg/mL) were used. The toxicity of PcfHb
for eukaryotic cells was evaluated through hemolytic assays
in human RBCs. In concentrations up to 100 mM PcfHb had
no hemolytic activity on human red blood cells.
To further evaluate a possible role of PcfHb as
pro inflammatory agent, intravital microscopy was
Fig. 1. Purification of PcfHb from P. cf. henlei mucus (A) Chromatographic profile of P. cf. henlei. The samples were applied to a Phenomenex C18 column
(4.6 mm  150 mm, 5m, California, USA) connected to a RP-HPLC in a two-solvent system: (a) TFA/H2O (1:1000) and (b) TFA/ACN/H2O (1:900:100). The column
was eluted at a flow rate of 1.0 mL/min with a 5–90% gradient of solvent B over 40 min. The HPLC column eluates were monitored by their absorbance at 214 nm.
The fractions highlighted presented antimicrobial activity against E. coli, M. luteus and C. tropicalis; (B) Chromatography of the proteins obtained from the fraction
8, the arrow indicates the peak containing the pure protein; (C) SDS–polyacrylamide gel electrophoresis of PchH80_8. Purified PchH80_8 was electrophoresed in
12% polyacrylamide gels containing 0.1% SDS under nonreducing condition with 0.1 M b-mercaptoethanol. Lane 1: purified protein and Mw: molecular weight
markers.
K. Conceição et al. / Toxicon 60 (2012) 821–829824

5. employed to assess the effects of the protein in cellular
recruitment on microcirculation, as well to detect
eventually a toxic effect of the protein. The topical
application of 4 mM of the protein induced an increase of
cellular recruitment characterized by an increase in the
number of leukocyte rolling (Fig. 4). The cellular
recruitment was more pronounced 5 min after topical
application and decreased continuously, returning to
normal after 30 min of experiment. No change in
numbers of adhered cells was observed.
Fig. 2. ESI-MS spectrum of charge distribution from the fraction PcfHb. Molecular weight of the PcfHb fraction that was performed by mass spectrum decon-
volution using the program Mag Tran 1.0 beta 8.
Fig. 3. MS/MS spectrum. The fragmentation pattern for LVAEALSSNYH with a parent mass of 1203.6004 (a doubly charged peptide) is shown. The peptide is
a fragment found in chain b form of hemoglobin from Dasyatis akajei (UniProt ID: P56692).
K. Conceição et al. / Toxicon 60 (2012) 821–829 825

6. 4. Discussion
Although the basic features of the adaptive immune
system have been described in fish, the limited classes of
immunoglobulin present and functional variants of fish
immunoglobulins show the lack of a well-developed
adaptive immune system in fish (Rajanbabu and Chen,
2011).
To overcome this limitation, epithelial surfaces of fish,
like mucus and skin have a wide variety of host defences,
characterizing an efficient innate immune system which
acts as the first line of defense against the broad spectrum
of pathogens encountered in the aquatic environment
(Subramanian et al., 2009; Robinette et al., 1998).
Antimicrobial activity is most commonly found in
specialized peptides with a number of unusual structures
(Rajanbabu and Chen, 2011). However, an increasing
number of antimicrobials are found to be larger proteins or
fragments obtained thereof. Among the most common of
these are histone-like proteins: 13.5–21 kDa proteins iso-
lated from the skin, gill, liver and spleen of various fish.
Highly homologous to histones, they have potent, broad-
spectrum activity against Gram-negative bacteria, water
molds and parasites (Richards et al., 2001; Fernandes et al.,
2002). Another example is the antimicrobial peptide hip-
posin from the skin mucus of Atlantic halibut (Hippoglossus
hippoglossus L.) derived from the histone H2A (Birkemo
et al., 2003). Other antimicrobial proteins isolated from
fish and having other primary functions include apolipo-
proteins A-I and A-II, present in skin or serum of carp
(Cyprinus carpio) and active against some fish bacterial
pathogens (Concha et al., 2004). These proteins with other
well established functions appear to be recruited to
a second antimicrobial role in nature.
In the present work we purified and identified the
fraction of the P. cf henlei mucus responsible for antimi-
crobial activity against E. coli, M. luteus and C. tropicalis. The
purified PcfHb exhibited a lower MIC against gram-
Table 1
Sequence alignment between PcfHb and three stingray hemoglobin b-chain performed by Clustal (Thompson et al., 1997). To optimize maximal alignment,
gaps were introduced in the sequences indicated as dots. Proteins: HBB_DASAK (P56692) – hemoglobin subunit beta (beta-globin), [Dasyatis akajei (red
stingray) (Akaei)]; HBB2_TORMA (P20247): Hemoglobin subunit beta-2 (beta-2-globin) (hemoglobin beta-2 chain) [HBB2] [Torpedo marmorata (Marbled
electric ray)]; Q4JDG3_BATEA – beta 1 globin OS ¼ Bathyraja eatonii; HBB1_TORMA (P20246) – hemoglobin subunit beta-1 (beta-1-globin) (hemoglobin
beta-1chain) [HBB1] [T. marmorata (Marbled electric ray)]; HBB_BATEA hemoglobin subunit beta OS ¼ Bathyraja eatonii. Amino acid sequences that are
conserved are highlighted with asterisk.
K. Conceição et al. / Toxicon 60 (2012) 821–829826

7. negative bacteria and higher against gram-positive bacteria
and fungi. The MIC values were in the same range as well-
characterized peptide fragments from bovine hemoglobin
(Adje et al., 2011) and antimicrobial peptides including
pardaxins and hipposins (Oren and Shai, 1996; Birkemo
et al., 2003). Interestingly, the partial sequence alignment
of PcfHb with several hemoglobin b-chain of different
species, demonstrated a high degree of conservation of
certain amino acids (Table 1).
Some factors could explain the surprising antimicrobial
activity of fragments of hemoglobin. One possibility is that
the heme moiety could act either as an iron chelator or as
an oxidant, leading to damage of the bacterial and fungal
cell walls. Parish et al. (2001) working with isolated chains
of hemoglobin identified that the isolated b chain without
heme exhibited activity on tested organisms, supporting
the hypothesis that the heme plays no role in the antimi-
crobial activity and that subunit separation leads to
enhanced activity. Thus, although the hemoglobin tetramer
is only negligibly active against two gram-positive
organism, the activity of the isolated b globin chain is
greatly enhanced. In the case of bþheme, antimicrobial
activity was observed against two of the bacterial targets
but not on C. albicans. The results with isolated subunits
indicate that tetramer dissociation exposes additional
bioactive peptidic surfaces.
Even though the tested microorganisms do not affect
freshwater fish such as stingrays, proteins homologous to
hemoglobin are also present in the microsomes of gill cells
from a number of teleosts including Mozambique tilapia,
Oreochromis mossambicus (Peters), rainbow trout, common
carp, Cyprinus carpio L., European eel, Anguilla anguilla (L.),
elephant fish, Gnathonemus petersii (Günther) (Stekhoven
et al., 2004) as well the presence of a family of AMPs
derived from Hb-b present in the skin and gill epithelium of
channel catfish (Ullal and Noga, 2010). Our study further
suggests that PcfHb may protect against freshwater
microorganisms as well as having a role in innate immunity
to microorganisms. The known three-dimensional struc-
ture of human hemoglobin shows an alpha-helical region
within the C-terminal part of the hemoglobin b-chain (PDB
ID: 2HHB). The structure of this polypeptide has given rise
to the hypothesis that the antimicrobial mechanism of
action resembles that of known peptides such as the
magainins and defensins which permeabilize bacterial
membranes (Oren and Shai, 1998; Brogden, 2005).
The cytotoxicity of many antimicrobial peptides to
mammalian cells greatly limits their use as therapeutics
(Rajanbabu and Chen, 2011). When tested on red blood
cells and on microcirculation, PcfHb showed no hemolytic
activity or tissue damage even at peptide concentrations of
up to 100 mM. Moreover, a small pro-inflammatory
response at the microcirculatory environment was seen
indicating that PcfHb may have a potential protective
activity being immunogenic to humans, not necessarily in
terms of antibody generation but as inflammation
promoters and recruitment agents or immune enhancers
(Otero-González et al., 2010). PcfHb could also be expected
Fig. 4. (A) Intravital micrograph of cremaster venule presenting normal venular flow before topical application of PcfHb; (B and C) Intravital micrograph of
cremaster venule showing leukocyte rolling and (circles); (D) Representative graph showing leukocyte rolling after topical application of 20 mL (4 mM) of PcfHb in
different times. Photographs were obtained from digitized images on the computer monitor. These data are representative of observations in three mice analyzed
on different days. Each point represents mean Æ SEM. *p 0.05 compared with control-group of three independent experiments.
K. Conceição et al. / Toxicon 60 (2012) 821–829 827

8. to function in conjunction with the histone-like proteins
that were found in the same epithelial mucus in this
stingray (data not shown), providing a strong line of innate
host defence against eukaryotic as well as prokaryotic
pathogens.
Although innate immunity to microbial infection is
a property common to almost all forms of life, it was quite
unexpected that hemoglobin, one of the most well-char-
acterized proteins due to its function in oxygen transport,
should contribute to innate immunity. However, recent
studies have identified Hb-derived AMPs from humans and
other animals; some of which were more inhibitory to
eukaryotes than bacteria (Ullal et al., 2008; Ullal and Noga,
2010). In view of the fact that different hemoglobin-derived
peptide fragments exhibit diverse antibiotic activities, it is
conceivable that, in addition to its role in oxygen transport
hemoglobin functions as an important multi-defense agent
against a wide range of microorganisms. In conclusion, we
have shown for the first time that a protein with high
sequence similarity to the hemoglobin b chain is an
antimicrobial polypeptide naturally occurring in the mucus
of stingrays. This finding is in accordance with the data of
Parish et al. (2001) which identified the region containing
the antimicrobial fragments at the amino acid sequences of
free b -hemoglobin chain with a greater activity on gram-
positive bacteria. Due to the broad antimicrobial action of
PcfHb against Gram-positive and Gram-negative bacteria
and yeast and its pro-inflammatory action, it may be sug-
gested that this antimicrobial polypeptide could play
a significant role in the innate immune response of this and
other fishes. Beyond that, it might ultimately lead to new
therapies with novel engineered antimicrobial molecules
that support an integrative and complementary action
against pathogens related to infectious diseases in
vertebrates.
Acknowledgement
This work has been supported by a FAPESP (2007/
55148-9), CNPq and FAPEMIG. Alessandra Cardoso is
acknowledged for technical assistance, Marta Maria Batista
Ribeiro and Vera Luisa Neves for helpful discussions.
Conflict of interest statement
None to declare.
References
Adje, E.Y., Balti, R., Kouach, M., Dhulster, P., Guillochon, D., Nedjar-
Arroume, N., 2011. Obtaining antimicrobial peptides by controlled
peptic hydrolysis of bovine hemoglobin. Int. J. Biol. Macromol. 49,
143–153.
Alexander, J.B., Ingram, G.A., 1992. Noncellular nonspecific defence
mechanism of fish. Annu. Rev. Fish. Dis. 2, 249–279.
Baez, S., 1973. An open cremaster muscle preparation for the study of
blood vessels by in vivo microscopy. Microvasc. Res. 5, 384–396.
Bergsson, G., Agerberth, B., Jornvall, H., Gudmundsson, G.H., 2005. Isola-
tion and identification of antimicrobial components from the
epidermal mucus of Atlantic cod (Gadus morhua). FEBS J. 272, 4960–
4969.
Birkemo, G.A., Lüders, T., Andersen, Ø, Nes, I.F., Nissen-Meyer, J., 2003.
Hipposin, a histone-derived antimicrobial peptide in Atlantic halibut
(Hippoglossus hippoglossus L.). Biochim. Biophys. Acta 1646, 207–215.
Bradford, M.M., 1976. A rapid and sensitive method for quantitation of
microgram quantities of protein utilizing the principle of protein dye
binding. Anal. Biochem. 72, 248–254.
Brogden, K.A., 2005. Antimicrobial peptides: pore formers or metabolic
inhibitors in bacteria? Nat. Rev. Microbiol. 3, 238–250.
Bulet, P., Dimarcq, J.L., Hetru, C., Lagueux, M., Charlet, M., Hegy, G., Van
Dorsselaer, A., Hoffmann, J.A., 1993. A novel inducible antimicrobial
peptide of Drosophila carries an O-glycosylated substitution. J. Biol.
Chem. 268, 14893–14897.
Carvalho, M.R., Lovejoy, N.R., Rosa, R.S., 2003. Family Potamotrygonidae.
In: Reis, R.E., Kullander, S.O., Ferraris, C.J. (Eds.), Checklist of the
Freshwater Fishes of South and Central America (CLOFFSCA). Edi-
pucrs, Porto Alegre, pp. 22–29.
Cole, A.M., Darouiche, R.O., Legarda, D., Connell, N., Diamond, G., 2000.
Antimicrob. Agents Chemother. 44, 2039–2045.
Conceição, K., Santos, J.M., Bruni, F.M., Klitzke, C.F., Marques, E.E.,
Borges, M.H., Melo, R.L., Fernandez, J.H., Lopes-Ferreira, M., 2009.
Characterization of a new bioactive peptide from Potamotrygon gr.
orbignyi freshwater stingray venom. Peptides 30, 2191–2199.
Concha, M.I., Smith, V.J., Castro, K., Bastias, A., Romero, A., Amthauer, R.J.,
2004. Apolipoproteins A-I and A-II are potentially important effectors
of innate immunity of the teleost fish Cyprinus carpio. Eur. J. Biochem.
271, 2984–2990.
Ehret-Sabatier, L., Loew, D., Goyffon, M., Fehlbaum, P., Hoffmann, J.A., Van
Dorsselaer, A.V., Bulet, P., 1996. Characterization of novel cysteine-rich
antimicrobial peptides from scorpion blood. J. Biol. Chem. 271,
29537–29544.
Fernandes, J.M.O., Kemp, G.D., Molle, M.G., Smith, V.J., 2002. Anti-
microbial properties of histone H2A from skin secretions of rainbow
trout, Oncorhynchus mykiss. Biochem. J. 368, 611–620.
Halstead, B.W., 1970. Poisonous and Venomous Marine Animals of the
World. The Darwin Press, Princeton, New Jersey, NJ. 1988:1168.
Hellio, C., Pons, A.M., Beaupoil, C., Bourgougnon, N., Gal, Y.L., 2002.
Antimicrobial, antifungal and cytotoxic activities of extracts from
fish epidermis and epidermal mucus. Int. J. Antimicrob. Agents 20,
214–219.
Hellman, U., Wernstedt, C., Góñez, J., Heldin, C.H., 1995. Improvement of
an “In-Gel” digestion procedure for the micropreparation of internal
protein fragments for amino acid sequencing. Anal. Biochem. 224,
451–455.
Ingram, G.A., 1980. Substances involved in the natural resistance of fish to
infection – a review. J. Fish Biol. 16, 23–60.
Klesius, P.H., Shoemaker, C.A., Evans, J.J., 2008. Flavobacterium columnare
chemotaxis to channel catfish mucus. FEMS Microbiol. Lett. 288, 216–220.
Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly
of the head of bacteriophage T4. Nature 227, 680–685.
Lomonte, B., Lundgren, J., Johansson, B., Bagge, U., 1994. The dynamics of
local tissue damage induced by Bothrops asper snake venom and
myotoxin II on the mouse cremaster muscle. An intravital and elec-
tron microscopic study. Toxicon 32, 41–55.
Monteiro-Dos-Santos, J., Conceição, K., Seibert, C.S., Marques, E.E., Ismael
Silva Jr., P., Soares, A.B., Lima, C., Lopes-Ferreira, M., 2011. Studies on
pharmacological properties of mucus and sting venom of Potamo-
trygon cf. henlei. Int. Immunopharmacol. 11, 1368–1377.
Mozumder, M.M.H., 2005. Antimicrobial Activity in Fish Mucus from
Farmed Fish. Norwegian College of Fishery Science, University of
Tromsø, Norway.
Negus, V.E., 1963. The functions of mucus. Acta Otolaryngol. 56, 204–214.
Oren, Z., Shai, Y., 1996. A class of highly potent antimicrobial peptides
derived from pardaxin, a pore-forming peptide isolated from Moses
sole fish Pardachirus marmoratus. Eur. J. Biochem. 237, 303–310.
Oren, Z., Shai, Y., 1998. Mode of action of linear amphipathic alpha-helical
antimicrobial peptides. Biopolymers 47, 451–463.
Otero-González, A.J., Magalhães, B.S., Garcia-Villarino, M., López-
Abarrategui, C., Sousa, D.A., Dias, S.C., Franco, O.L., 2010. Antimicrobial
peptides from marine invertebrates as a new frontier for microbial
infection control. FASEB J. 24, 1320–1334.
Parish, C.A., Jiang, H., Tokiwa, Y., Berova, N., Nakanishi, K., McCabe, D.,
Zuckerman, W., Xia, M.M., Gabay, J.E., 2001. Broad-spectrum antimi-
crobial activity of hemoglobin. Bioorg. Med. Chem. 9, 377–382.
Rajanbabu, V., Chen, J.Y., 2011. Applications of antimicrobial peptides from
fish and perspectives for the future. Peptides 32, 415–420.
Ramos, A.D., Conceição, K., Silva Jr., P.I., Richardson, M., Lima, C., Lopes-
Ferreira, M., 2012 Feb 21. Specialization of the sting venom and skin
mucus of Cathorops spixii reveals functional diversification of the
toxins. Toxicon.
Richards, R.C., O’Neil, D.B., Thibault, P., Ewart, K.V., 2001. Histone H1. An
antimicrobial protein of Atlantic salmon (Salmo salar). Biochem.
Biophys. Res. Commun. 284, 549–555.
K. Conceição et al. / Toxicon 60 (2012) 821–829828

9. Robinette, D., Wada, S., Arroll, T., Levy, M.G., Miller, W.L., Noga, E.J., 1998.
Antimicrobial activity in the skin of channel catfish Ictalurus punc-
tatus: characterization of broad-spectrum histone-like antimicrobial
proteins. Cell. Mol. Life Sci. 54, 467–475.
Shephard, K.L., 1994. Functions for fish mucus. Rev. Fish Biol. Fish 4,
401–429.
Stekhoven, F., Bonga, S., Flik, G., 2004. Extranuclear histones in teleost
gills: an evolutionary study. Fish Physiol. Biochem. 30, 201–211.
Su, Y., 2011. Isolation and identification of pelteobagrin, a novel antimi-
crobial peptide from the skin mucus of yellow catfish (Pelteobagrus
fulvidraco).Comp.Biochem. Physiol. B Biochem. Mol. Biol.158,149–154.
Subramanian, S., Ross, N.W., MacKinnon, S.L., 2009. Myxinidin, a novel
antimicrobial peptide from the epidermal mucus of hagfish, Myxine
glutinosa L. Mar. Biotechnol. (NY) 11, 748–757.
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G.,
1997. The CLUSTAL_X windows interface: flexible strategies for
multiple sequence alignment aided by quality analysis tools. Nucleic
Acids Res. 25, 4876–4882.
Ullal, A.J., Litaker, R.W., Noga, E.J., 2008. Antimicrobial peptides derived
from hemoglobin are expressed in epithelium of channel catfish
(Ictalurus punctatus, Rafinesque). Dev. Comp. Immunol. 32, 1301–
1312.
Ullal, A.J., Noga, E.J., 2010. Antiparasitic activity of the antimicrobial
peptide HbbetaP-1, a member of the beta-haemoglobin peptide
family. J. Fish. Dis. 33, 657–664.
Zhao, X., Findly, R.C., Dickerson, H.W., 2008. Cutaneous antibody-
secreting cells and B cells in a teleost fish. Dev. Comp. Immunol. 32,
500–508.
K. Conceição et al. / Toxicon 60 (2012) 821–829 829