Báo cáo khoa học: Apolipoproteins A-I and A-II are potentially important effectors of innate immunity in the teleost fish Cyprinus carpio pot

Apolipoproteins A-I and A-II are potentially important effectors
of innate immunity in the teleost fish
Cyprinus carpio
Margarita I. Concha
1
, Valerie J. Smith
2
, Karina Castro
1
, Adriana Bastı
´
as
1
, Alex Romero
1
and Rodolfo J. Amthauer
1
1
Instituto de Bioquı
´
mica, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile;
2
Gatty Marine Laboratory,
School of Biology, University of St. Andrews, Fife, UK
We have previously shown that high d ensity lipoprotein is
the most abundant protein in the carp plasma and dis-
plays bactericidal activity in vitro. Therefore the aim of
this study was to analyze the contribution of its principal
apolipoproteins, apoA-I and apoA-II, in defense. Both
apolipoproteins were isolated by a two step procedure
involving affinity and gel filtration chromatography and

were shown to display bactericidal and/or bacteriostatic
activity in the micromolar range against Gram-positive
and Gram-negative bacteria, including some fish patho-
gens. In addition, a cationic peptide derived from the
C-terminal region of carp apoA-I was synthesized and
shown to posses antimicrobial activity (EC
50
¼ 3– 6 l
M
)
against Planococcus citreus. This peptide was also able to
potentiate the inhibitory effect of lysozyme in a radial
diffusion assay at subinhibitory concentrations of both
effectors. Finally, limited proteolysis o f HDL-associated
apoA-I with chymotrypsin in vitro was shown to generate
a major tr uncated fragment, which indicates that apoA-I
peptides liberated in vivo through a regulated prot eolysis
couldalsobeinvolvedininnateimmunity.
Keywords: antimicrobial cationic peptide; carp; HDL;
innate immunity; synergism.
The innate immune system is essential to prevent infections
during t he fir st critical hours a nd days of exposure to a
pathogen. A lthough innate immunity is not specific to a
particular pathogen in the way that the adaptive immune
system is, it is of critical relevance in lower vertebrates such
as teleost fish, where the acquired immunity is not well
developed [1]. A ntibacterial proteins and peptides have been
recognized as important effectors of the innate immune
system in most animals, however, the i mportance o f these
molecules in the primary defense of fish h as been only

recently demonstrated by several studies [2,3]. Most of these
antimicrobial macromolecules have been isolated from fish
skin that constitutes a first line barrier against microbial
invasion. Surprisingly several of thes e antimicrobial com-
pounds seem to correspond to proteins or protein fragments
previously considered nonimmune, e.g. histones H1 and
H2A [4–7].
In a previous study we demonstrated that high-density
lipoprotein (HDL) locally produced in the carp (Cyprinus
carpio) epidermis is secreted to the mucus and displays
antimicrobial activity aga inst Esche richia c oli i n v itro [8].
This lipoprotein is c on stituted by two major apolipopro-
teins (apoA-I and a poA-II) and corresponds to the most
abundant plasma protein in sever al teleost fish [9,10], with
a concentration as high as 1 gÆdL
)1
in the carp [11].
Although the main role of HDL and i ts principal
apolipoproteins has long been considered to be its
participation in reverse chol esterol transport and its anti-
atherogenic effect [12], more recent studies have involved
these proteins in other defensive functions in mammals,
such as antiviral, antimicrobial and anti-inflammatory
activities [13–15].
Multiple alignments of apolipoprotein A-I deduced
amino acid sequence shows that the primary structure of
this protein is poorly conserved among vertebrates, how-
ever, the predicted secondary structure of t hese proteins is
surprisingly similar (high content of amphipathic a-helix).
Therefore we h ypothesized that in sp ite of the l ow sequence

similarities that exist between mammalian and teleost
apolipoprotein A -I, its conserved overall structur e w ould
be responsible for preserving these defensive f unctions
through evolution. The aim of this study was to evaluate i f
the antimicrobial activity observed for carp HDL resides in
its major apolipoproteins (apoA-I and apoA-II) and in
addition to determine if a syn thetic peptide derive from
apoA-I sequence could display a similar activity.
Materials and methods
Blood sample collection
Commoncarp(C. carpio L) were caught in th e C ayumapu
river ( Province of Valdivia, Chile) and maintained in an
outdoor tank with running river water. Fish weighing
Correspondence to M. I. Concha, Instituto de Bioquı
´
mica, Universi-
dad Austral de Chile, Campus Isla Teja, Valdivia, Chile.
Fax: + 56 63 221 107, Tel.: + 56 63 221 108,
E-mail:
Abbreviations: AMP, antimicrobial peptide; Apo, apolipoprotein;
EC
50
, effective inhibitory concentration; HDL, high-density
lipoprotein; MBC, minimal bactericidal concentration;
MHB, Mueller–Hinton broth.
(Received 1 3 February 2004, revised 6 April 2004,
accepted 25 May 2004)
Eur. J. Biochem. 271, 2984–2990 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04228.x
800–1200 g were acclimatized at 20 ± 2 °C with a photo-
period of 14-h light : 10-h dark, for at least 3 weeks b efore

they were used. Animals were anesthetized in a b ath
containing 50 mgÆL
)1
of benzocaine and blood samples
were collected from the caudal vein in heparinized tubes.
The ethical guidelines, from the UK Home Office, on
animal care were followed.
Bacterial strains and culture
Field i solates of t he salmonid p athogens Yersinia ruckeri
and Pseudomonas sp. were kindly p rovided by M. Fernan-
dez (Fundacio
´
n Chile, Puerto Montt, Chile) a nd were typed
with Mono-year (BIONOR, Norway) and API 20NE
(BioMerioux, France) kits, respectively. Fish bacteria,
Planococcus citreus (NCIMB 1493) and E. coli DH5a were
grown to logarithmic phase in Mueller–Hinton broth
(Merck) at the appropriate temperature (20 °C f or fish
pathogens and P. citreus and 37 °CforE. co li).
HDL and apolipoprotein isolation
Carp plasma HDL was purified from fresh plasma samples
treated with protease inhibitors (phenylmethanesulfonyl
fluoride and benzamidine) by affinity chromatography on
Affi-GelÒ Blue-Gel (Bio-Rad), essentially as described by
Amthauer and coworkers [16]. ApoA-I and A-II were
isolated from HDL particles a ccording to Amthauer a nd
coworkers [9]. Briefly, HDL was delipidated with eth-
anol:ether (3 : 2, v/v) at ) 20 °C. One milliliter of the
delipidated plasma HDL (5 mgÆml
)1

)wasloadedona
Sephacryl S-200 (Pharmacia) column (100 · 1.5 cm) equil-
ibrated with 10 m
M
Tris/HCl pH 8.6/8
M
urea/1 m
M
EDTA and eluted with the same buffer at a flow rate of
0.3 mLÆmin
)1
. Fractions corresponding to the t hree peaks
were pooled, exhaustively dialyzed in 5 m
M
Tris/HCl
pH 8.0/ 0.1 m
M
EDTA and concentrated 10-fold in a
Speed-Vac centrifuge. Prior to its use in antimicrobial
assays, p rotein concentration of apolipoprotein samples was
determined by the b icinconinic acid method [17] and its
purity and integrity was checked by S DS/PAGE according
to Laemmli [18].
Peptide synthesis
A 24-residue peptide derived from the C-terminal sequence
of carp apoA-I [AQEFRQSVKSGELRKKMNELGRRR]
was produced, using N-(9-fluorenyl)methoxycarbonyl
chemistry a nd purified to > 70% by HPLC (Global Peptide
Services LLC, Fort Collins, CO, USA).
Antiserum preparation

Antiserum to apoA-I synthetic peptide coupled to keyhole
lymphet hemocyanin was raised in rabbits by the following
procedure. Briefly, 4 mg of peptide was dissolved in 1 mL of
sterile NaCl/P
i
and mixed with 1 m L of 8 mg ÆmL
)1
hemocyanin. Twenty-five aliquots of 100 m
M
glutaralde-
hyde (20 lL each) were slowly added to the mix while
stirring at room temperature for 1 h. The reaction was
stopped by the addition of an excess of glycine and diluted
aliquots were stored at ) 20 °C until used. Rabbits were
selected for immunization after checking their preimmune
sera by Western blot. The immunization schedule consisted
of one subcutaneous injection of antigen plus Freund’s
complete adjuvant, two injections of antigen plus Freund’s
incomplete adjuvant spaced by a period of 12 d ays and a
final booster. The ethical guidelines, from the UK Home
Office, on animal care were followed.
Antimicrobial activity assays
Determination of the effective 50% reduction concentration
(EC
50
) of the purified protein/peptide against each of the
test b acteria used was performed using the microtiter broth
dilution assay [19]. One hundred microliters of each
bacterial suspension containing 10
5

colony-forming units
per mL was mixed with serial twofold dilutions of test
protein/peptide in 0.2% (w/v) bovine serum albumin in
sterile polypropylene 96-well microtiter plates (Corning
Costar, C ambridge, UK). T he positive c ontrol contained
bacteria and diluent only. P. citreus , Y. ruckeri and Pseudo-
monas sp. were incubated at 2 0 °CandE. coli at 37 °Cand
the attenuance (D) was read at 570 nm using a MRX II
microtiter plate r eader (Dynex, West Sussex, UK) against a
blank comprising d iluent only. Values for experimental wells
were recorded when the attenuance reached 0.2 in the
positive control well. The EC
50
was considered to be the
lowest concentration of p rotein that reduce s the growth b y
50% relative to the control. The minimal bactericidal
concentration (MBC) was obtained by plating out the
contents of each well showing no visible growth. MBC was
taken as the lowest concentration of protein that prevents
any r esidual c olony formation after incubation for 24 h .
Synergism between hen egg white lysozyme and the carp
apoA-I synthetic peptide was assessed using a modified
version of the two-layer radial d iffusion assay of Lehrer et al.
as described by Smith et al. using P. citreus Gram-positive
as a test bacteria [20,21]. Briefly, bacteria g rown exponen-
tially in Mueller –Hinton broth were washed, resuspended in
Mueller–Hinton broth (MHB) and adjusted to an attenu-
ance at 570 nm of 0.4. An aliquot of 100 lL of the bacterial
suspension was mixed with 15 mL of melted sterile Mueller–
Hinton agar (0.1· MHB in 1 gÆdL

)1
agar), immediately
prior to its solidification and poured into a sterile square
(100 · 100 mm) Petri dish. Once solidified for 15 min at
4 °C, 0.3-mm diameter wells were bored i nto the agar using a
sterile plastic Pasteur pipette. Three microliter aliquots of
different combinations of the peptide and lysozyme, each at
subinhibitory concentrations were loaded into each well and
allowedtodiffuseforatleast3hat4°C. After the diffusion
step, melted top agar (1· MHB in 1 gÆdL
)1
agar) was
poured onto the dishes and after 20–24 h of incubation at
20 °C the diameter of the inhibition halos were measured.
Limited proteolysis and Western blotting
To obtain a limited proteolysis of HDL-associated apoA-I;
HDL particles (200 lgÆmL
)1
in 100 m
M
ammonium bicar-
bonate buffer) was incubated with bovine pancreas chymo-
trypsin at 37 °C u sing a m olar ratio of protease to
lipoprotein (1 : 100) and t aking aliquots each 30 min o ver
4 h . The reaction was s topped by h eating the samples at
100 °C for 5 min in sample buffer [62.5 m
M
Tris/HCl;
Ó FEBS 2004 ApoA-I and apoA-II in innate immunity of the carp (Eur. J. Biochem. 271) 2985
2% w/v SDS; 10% v/v glycerol; 5% v/v 2-mercaptoethanol

and bromophenol blue). T he products of proteolys is were
analyzed by Tricine-SDS/PAGE essentially as described by
Scha
¨
gger and von Jagow [22] and then transferred to
nitrocellulose membranes using a semidry blotter unit.
Membranes w ere b locked for 1 h with 1% (w/v) bovine
serum albumin in NaCl/P
i
buffer and then alternatively
incubated a further hour with rabbit anti-carp apoA-I s erum
diluted 1 : 25 000, rabbit anti-apoA-I synthetic peptide
serum diluted 1 : 1500 or with rabbit anti-carp apoA-II
diluted 1 : 1000 in the same blocking solution. After several
washes with NaCl/P
i
, t he membranes w ere incubated for 1 h
with a 1 : 2000 dilution of alkaline phosphatase-conjugated
goat anti-rabbit IgG (Gibco BRL). T he blot was developed
by incubating the membranes for 1 0 min in phosphatase
buffer (0.1
M
Tris/HCl, pH 9 .5; 0.1
M
NaCl; 5 m
M
MgCl
2
)
containing of 0.16 mgÆmL

)1
5-bromo-4-chloroindolyl
phosphate and 0.33 mgÆmL
)1
nitroblue tetrazolium.
Results
Purification of HDL and apolipoproteins A-I and A-II
As shown in Fig. 1 (insert), the plasma HDL particles
isolated by affinity chromatography display essentially two
protein b ands on SDS/PAGE (lane 1), that correspond to
apoA-I and A -II. Delipidation of the concentrated HDL
fractions and separation on Sephacryl S-200 gel filtration
chromatography resulted in three major peaks. T he fi rst
peak was s hown t o contain aggr egates of both apolipopro-
teins, while peaks 2 and 3 contained isolated apoA-I and
apoA-II, respectively (Fig. 1, insert, lanes 2 and 3). The
identity of both apolipoproteins was d emonstrated not only
by their expected molecular mass (27.5 and 12.5 kDa,
respectively) but also by Western blot analysis using
previously characterized antibodies specific for each apo-
lipoprotein (data not shown) [8].
Antimicrobial activity of purified apolipoproteins
Quantification of antimicrobial activity using the microtiter
broth dilution assay showed that apoA-I is active at
submicromolar concentrations, with an EC
50
and a MBC
of approximately 0.4 l
M
against P. citreus (Gram-positive)

and at micromolar concentrations (2.6–4.0 l
M
)againsttwo
Gram (–) fish pathogens Pseudomonas sp. and Yersinia
ruckeri (Table 1). In addition, purified apoA-II a lso dis-
played bacteriostatic activity against the Gram-positive and
-negative b acteria a t m icromolar concentrations (Table 1).
These results clearly s how that although apoA-I seems to be
more active than apoA-II, both major apolipoproteins
contribute significantly to the antimicrobial activity dis-
played by carp plasma HDL.
Design and evaluation of apoA-I synthetic peptide
Based o n our observations and on several studies that have
shown that mammalian apoA-I associated to HDL parti-
cles suffers limited proteolysis in vitro by several potentially
relevant insult-activated p roteases (e.g. t ryptase, chyma se
and several matrix metalloproteases) [23,24], we hypothes-
ized that during acute in flammation one or more peptides
could be released from the HDL particle by proteolysis
either from th e N- o r C-terminal r egion of a poA-I. In this
context, we postulate that these putative peptides could
also contribute to the systemic and mucosal innate
immunity. Initially we analyzed the carp apoA-I amino
acid sequence deduced from the sequence of a partial
cDNA clone isolated in a previous study [8] and we found
that this sequence is predicted to posses a high content o f
amphipathic a-helix (Fig. 2B). In p articular, a peptide
corresponding to the last 24 residues (Fig. 2A) would be a
highly cationic helix (net charge + 5) although not amphi-
pathic. Thus, this peptide should share some important

Fig. 1. Purification of apolipoproteins A -I and A-II from isolated carp
plasma HDL. HDL-associated apolipoproteins were purified by gel
filtration chromatography on Sephacryl S-200. Dialyzed a nd concen-
trated fractions of each peak w ere separated by SDS/PAGE. ( Insert)
Carp plasma (lan e P), lanes 1–3 c orrespond to p eaks 1 –3, respe ctively
(50 lg p rotein per lane). Arrows indicate the migration of c arp apoA-I
(27.5 k Da ) and A-II (12.5), respectivel y.
Table 1. Bacteriostatic and bactericidal a ctivities o f carp apoA-I and A-II. Each value in the table represents the mean ± SE of experiments
performed in triplicate. Similar results we re obtained with different preparations of apolipoproteins. ND, not determined.
Bacterium Gram staining
ApoA-I ApoA-II
EC
50
(l
M
) MBC (l
M
)EC
50
(l
M
)
Planococcus citreus + 0.3 ± 0.06 0.4 ± 0.2 1.8 ± < 0.001
Pseudomonas sp. – 2.6 ± 0.01 ND 3.5 ± 0.04
Yersinia ruckeri – 2.6 ± < 0.001 4.0 ± 0.5 3.7 ± 0.15
Escherichia coli – 5.2 ± 0.85 8.5 ± 0.5 7.1 ± < 0.001
2986 M. I. Concha et al.(Eur. J. Biochem. 271) Ó FEBS 2004
structural features with a known group of antimicrobial
peptides (AMPs) also released by regulated proteolysis
from larger precursor polypeptides (e.g. cathelicidins)

[25,26]. Therefore we synthesized this C-ter minal peptide
and evaluated its antimicrobial activity in vitro.The
synthetic peptide was active against P. citreus displaying
an EC
50
of 3–6 l
M
.
Considering that other cationic proteins and peptides
have been shown to e xhibit synergism w ith hen egg white
lysozyme [27–29], we attempted to ascertain if the carp
apoA-I peptide w ould be also able t o synergize with
lysozyme. As shown i n Fig. 3 , the synthetic peptide
enhanced the activity of lysozyme when both compounds
were used at subinhibitory concentrations in a radial
diffusion assay against P. citreus. Maximal synergism was
observed at c oncentrations of 6 lgÆmL
)1
and 0.8 m
M
of
lysozyme and peptide, respectively (Fig. 3).
Limited proteolysis of HDL-associated apoA-I
in vitro
To determine if one or more peptides could be liberated
after limited proteolysis of apoA-I in vitro, we a nalyzed the
kinetics of carp H DL digestion with chymotrypsin by SDS/
PAGE (Fig. 4A). Under the conditions used, two major
truncated apoA-I fragments were generated; one of them
seemed to be short-lived while the third band remained

stable far more t han 3 h. Duplicate gels were transferred to
nitrocellulose membranes and analyzed by Western blot
using specific antiserum against the intact carp apoA-I or
the C-terminal apoA-I synthetic peptide. As shown on
Fig. 2. Prediction of a-helicity of apoA-I p eptide and three-dimensional
model of carp a poA-I. (A) Helical wheel projection o f the synthetic
peptide performed with
ANTHEPROT
V.5. program (http://
www.antheprot-pbil.ibcp.fr). A discontinuous line was used to separ-
ate the helix in two f aces. The prefe ren tial localization of the positively
charged residu es o n t he up per f ac e o f t he he lix is depicted. The amino
acid sequence of the peptide is shown at the top of the figure ; basic
residues are underlined. (B) The three-dimensional model of the p artial
carp a poA-I sequence wa s generated b y
SWISS
-
MODEL
(http://
www.expasy.org/swissmod/SWISS-MODEL.html) based on the
crystallographic d ata for human apoA-I. The N-terminal residue (N)
corresponds to the first residue of t he carp apoA-I partial sequence
(GenBank a ccession number AJ308993) and ( C) corresponds to the
C-terminal residue. Hydrophilic residues are in dark gray and
hydrophobic residues in light gray.
Fig. 3. Synergy of the apoA-I synthetic peptide with lysozyme.
(A) B acterial growth in the presenceofdifferentcombinationsofthe
peptide and lysozyme, each at subinhibitory concentrations, was
analyzed by radial diffusion assay using P. citreus as test bacterium.
Variable concentrations of lysozyme without peptide ( d); plus 0.2 m

M
(h); 0.4 m
M
(m); 0.6 m
M
(e)or0.8m
M
(r) of the synthetic peptide.
The experiments were performed i n triplicate and the error b ars c or-
respond to the standard error around the mean. (B) Depicts the
increased inhibitory h alo observed with increasing concentrations of
peptide were used in c ombination with 6 lgÆmL
)1
of lysozyme. W ells
1–5 correspond to t he same pe ptide concentration as in (A), ranging
from 0 to 0 .8 m
M
, respectively.
Ó FEBS 2004 ApoA-I and apoA-II in innate immunity of the carp (Eur. J. Biochem. 271) 2987
Fig. 4 B, t he inta ct a poA-I (b and a), an intermedia ry
fragment (band b) and a more stable third band (band c)
were recognized by the specific antiserum against carp
apoA-I. However, when incubated with the antiserum
against the synthetic carp apoA-I peptide, only bands a and
b were i mmunodetected wh ile t he th ird band, which was the
most ab undant after 30 m in of digestion w as not detected
by this antiserum, indicating that it would c orrespond to a
fragment truncated both at t he N- and C-terminal e nd of
the protein. The detection of the larger fragment (band b) of
apoA-I in Fig. 4B indicates that it still contains at least part

of the epitope(s) r ecognized by the antipeptide antibodies.
Using the same antiserum we could not detect a band in the
range of m olecular m asses expected for the peptide ( 3 kDa).
However with the antiserum against intact apoA-I we
observed during the first minutes of digestion a very faint
band that could correspond to this peptide (data not
shown). In t he same experiment, no degradation was
observed for apoA-II neither by direct staining (Fig. 4 D)
nor by Western blot (Fig. 4E), reflecting that in the HDL
particle, apoA-II should be much less exposed to the
protease than apoA-I.
Discussion
Although there are a few s tudies of mammalian HDL and
its principal apolipoproteins A-I a nd A-II in antimicrobial
or antiviral activities in vitro [13,14,30], these proteins have
not been yet recognized as important effectors in innate
immunity. Moreover, only recently we reported that this
defensive function could also b e relevant for teleost fish [8].
Here we clearly demonstrate the i mportant contribution of
both apolipoproteins A-I and A-II in the in vitro anti-
microbial activity of carp HDL. Both proteins inhibit the
growth of Gram-positive and -negative bacteria, including
fish pathogens, at micromolar concentrations. These find-
ings indicates that HDL and its apolipoproteins could
constitute important effectors in the systemic innate defense
mechanisms of the carp, especially taking in consideration
that the plasma concentration of HDL-associated apolipo-
proteins reaches values as high a s 1 gÆdL
)1
irrespectiv e, of

the acclimatization condition of the fish [11]. Although the
relative abundance of HDL varies among different teleosts,
it is generally accepted that this lipo protein is clearly more
abundant in fish plasma than in h igher vertebrates [10]. This
situation probably reflects among other things, the need of
teleost fish to rely more on their innate immunity for
survival. As we described previously [8], apoA-I and
apparently also apoA-II are locally synthesized and secreted
in the carp epidermis as a nascent HDL particle. Although
as yet we cannot state unequivocally that this particle or
even plasma HDL contribute significantly to innate defense,
the p resent study, together with the previous work described
by Concha et al. [8], offers promising evidence that they
might. Certainly there is no reason why apolipoproteins in
the skin secretion should function independently from
apolipoproteins an d HDL in the plasma. In both, apoA-I
and apoA-II are de rived f rom HDL particles. While the size
of skin nascent HDL is different from that of plasma HDL,
it contains both apoA-I and a poA-II, molecules shown by
the present paper to have potent antimicrobial properties
in v itro. W ork is currently underway to investigate the
precise mechanism by which HDL and the associated
apolipoproteins act. The results of these s tudies should help
to confirm the biological role of these proteins.
Although the primary structure of apoA-I is poorly
conserved among different species, the overall secondary and
tertiary structure of HDL-associated apoA-I is remarkably
similar, displaying an arrangement of s everal amphipatic a-
helices in a horseshoe-shape structure [12]. In fact, i t has been
demonstrated that various HDL functions (e.g. activation of

lecithin-cholesterol acyltransferase or lipid binding) a re
dependent on these structural features of apoA-I [31]. In
view of the fact t hat an important group of antimicrobial
peptides (cationic peptides) also have a-helical structure, in
the present study we demonstrate that a cationic peptide
analog to the C-terminus of carp apoA-I exhibits in vitro
antimicrobial activity at micromolar concentration. This
peptide was susceptible t o salt as no activity was detected at
150 m
M
NaCl. This is a rather common feature among
antimicrobial peptides, for example magainins and cecropins
which also correspond to a-helical peptides are inhibited at
100 m
M
NaCl [19]. In the particular case o f carp apoA-I, it
could be argued that i f a C-terminal peptide would be
released in vivo it would b e expected to be more active in a
low-salt environment like the mucus of this f reshwater fish
than in its blood stream. Another i nteresting feature of t his
C-terminal peptide is i ts ability to s ynergize with lysoz yme.
Synergy of several antimicrobial peptides and proteins with
lysozyme has been previously described [27,28]. I n teleosts,
the b lood and skin mucosa a re particularly rich in lyso zymes
[32,33], so as HDL is also very abundant in these tissues it
could assist in pathogen killing. At this point we cannot
assure that such a synergism observed in vitro would be
physiologically relevant, neither can w e rule out a p ossible
synergism between intact apolipoproteins and l ysozyme.
Fig. 4. Limited proteolysis of HDL-associated apoA-I. (A) Tricine-

SDS/PAGE and C oomassie b lue s taining were used to analyze the
progress of HDL-associated apoA-I proteolysis w ith chymotrypsin.
(B,C) Western blot analyses o f the g el in (A) immunodetected with a
specific anti-apoA-I and anti-peptide serum, respectively. Arrows
indicate the different bands of apo A-I: (a) intact form; (b) intermediary
fragment and (c) stable t runcated apo A-I. Incubation tim e is shown
above each gel. (D) Tricine-SDS/PAGE and Coomassie blue staining
of HDL-associated apoA-II i ncubated with chymotrypsin under the
same conditions as in (A). (E) Western blot analysis of the gel in
(D) using a s pecific anti-apoA-II serum.
2988 M. I. Concha et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Once the antimicrobial mechanism o f action of apolipopro-
teins has been established it would be very interesting to
evaluate these and several other possibilities of synergistic
and additive effects between effecto rs.
The above results raise t he possibility that not only t he
intact apolipoproteins but also putative fragments derived
from their limited proteolysis could participate in innate
defense. Additional support for this idea comes from
previous studies and our results that show that HDL-
associated apoA-I is susceptible to limited proteolysis by
physiologically relevant proteases, such as those liberated by
neutrophils and mast cells after an insult [23,24]. In the
present study, chymotrypsin was used because it has the
same specificity than chymase, a protease released by mast
cells, w hich has previously been shown to produce human
apoA-I truncated either at the N- or a t the C-terminus [23].
In this same study it was a lso demonstrated that apoA-II is
resistant to degradation under the conditions used. Our
results are in close a greement with these data as following the

digestion with c hymotrypsin, a stable a poA-I fragment that
seems t o l ack both N- and C-termini, is generated. We also
observed negligible degradation of apoA-II associated to
HDL. Although we could not detect the C -terminal peptide
released from apoA-I by Western blot utilizing the specific
antipeptide serum, it must be considered that under the
in vitro conditions of protease digestion, the peptide could
be very short-lived and t herefore extremely h ard to dete ct.
Based on these preliminary results we postulate that besides
the constitutive contribution of HDL and i ts apolipoproteins
in teleost fish innate immunity, an additional mechanism
might involve the r elease of one or more antimicrobial
peptides by limited proteolysis of HDL-associated apoA-I
possibly t riggered by one or more insult-regulated proteases,
e.g. elastase or chymase. Such a mechanism has already been
described f or another nonimmune protein, histone H2A, in
catfish skin, where a complex cascade of injury-induced
proteases is involved in the regulation of the A MP parasin I
production [5]. Therefore further studies will attempt to
evaluate the p resence of t he peptide in t he mucus and plasma
of pathogen-challenged fish.
Given that anti-inflammatory, antiviral, antibacterial
activities have been reported for mammalian HDL and its
apolipoproteins [13–15,30], the findings described in the
present study showing antimicrobial activity for teleost
apolipoproteins A-I and A-II and for a synthetic peptide
derived from apoA-I, further confirm t he multifunctionality
of these proteins. Moreover the synergism observed between
the a poA-I synthetic peptide and lysozyme suggests th at a
mechanism i nvolving the regulated release of peptides f rom

the HDL-associated apoA-I present in plasma a nd mucus
could be very important in the context of innate defense in
fish.
Acknowledgements
This research was supported by grant (S-2002–11) from the D ireccio
´
n
de Investigacio
´
n y Desarrollo, Universidad Austral d e Chile. We are
also grateful for grants MECESUP AUS 0006 and AUS 0005 that
supported the research visits of M.I.C. to the Gatty Marine Laboratory
University of St. A ndrews, S cotland, UK and of V.J.S. to the Institut e
of Biochemistry, Faculty of S ciences, Universidad Austral de Chile,
Valdivia , Chile.
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