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

In addition, a cationic peptide derived from the C-terminal region of carp apoA-I was synthesized and shown to posses antimicrobial activity EC50¼ 3–6 lM against Planococcus citreus.. Fi

Apolipoproteins A-I and A-II are potentially important effectors

Margarita I Concha1, Valerie J Smith2, Karina Castro1, Adriana Bastı´as1, Alex Romero1

and Rodolfo J Amthauer1

1

Instituto de Bioquı´mica, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile;2Gatty Marine Laboratory,

School of Biology, University of St Andrews, Fife, UK

We have previously shown that high density 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 (EC50¼ 3–6 lM) 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 of HDL-associated apoA-I with chymotrypsin in vitro was shown to generate

a major truncated fragment, which indicates that apoA-I peptides liberated in vivo through a regulated proteolysis could also be involved in innate immunity

Keywords: antimicrobial cationic peptide; carp; HDL; innate immunity; synergism

The innate immune system is essential to prevent infections

during the first critical hours and days of exposure to a

pathogen Although 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] Antibacterial proteins and peptides have been

recognized as important effectors of the innate immune

system in most animals, however, the importance of these

molecules in the primary defense of fish has 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 these 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 against Escherichia coli in vitro [8]

This lipoprotein is constituted by two major apolipopro-teins (apoA-I and apoA-II) and corresponds to the most abundant plasma protein in several teleost fish [9,10], with

a concentration as high as 1 gÆdL)1 in the carp [11] Although the main role of HDL and its principal apolipoproteins has long been considered to be its participation in reverse cholesterol 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 these proteins is surprisingly similar (high content of amphipathic a-helix) Therefore we hypothesized that in spite of the low sequence similarities that exist between mammalian and teleost apolipoprotein A-I, its conserved overall structure would

be responsible for preserving these defensive functions through evolution The aim of this study was to evaluate if the antimicrobial activity observed for carp HDL resides in its major apolipoproteins (apoA-I and apoA-II) and in addition to determine if a synthetic peptide derive from apoA-I sequence could display a similar activity

Materials and methods Blood sample collection Common carp (C carpio L) were caught in the Cayumapu 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: mconcha@uach.cl

Abbreviations: AMP, antimicrobial peptide; Apo, apolipoprotein;

EC 50 , effective inhibitory concentration; HDL, high-density

lipoprotein; MBC, minimal bactericidal concentration;

MHB, Mueller–Hinton broth.

(Received 13 February 2004, revised 6 April 2004,

accepted 25 May 2004)

800–1200 g were acclimatized at 20 ± 2C with a

photo-period of 14-h light : 10-h dark, for at least 3 weeks before

they were used Animals were anesthetized in a bath

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 isolates of the salmonid pathogens Yersinia ruckeri

and Pseudomonas sp were kindly provided by M

Fernan-dez (Fundacio´n Chile, Puerto Montt, Chile) and 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 (20C for fish

pathogens and P citreus and 37C for E coli)

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-Gel Blue-Gel (Bio-Rad), essentially as described by

Amthauer and coworkers [16] ApoA-I and A-II were

isolated from HDL particles according to Amthauer and

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) was loaded on a

Sephacryl S-200 (Pharmacia) column (100· 1.5 cm)

equil-ibrated with 10 mM Tris/HCl pH 8.6/8M urea/1 mM

EDTA and eluted with the same buffer at a flow rate of

0.3 mLÆmin)1 Fractions corresponding to the three peaks

were pooled, exhaustively dialyzed in 5 mMTris/HCl

pH 8.0/0.1 mM EDTA and concentrated 10-fold in a

Speed-Vac centrifuge Prior to its use in antimicrobial

assays, protein concentration of apolipoprotein samples was

determined by the bicinconinic acid method [17] and its

purity and integrity was checked by SDS/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 and 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/Pi and mixed with 1 mL of 8 mgÆmL)1

hemocyanin Twenty-five aliquots of 100 mM

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 days 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 (EC50) of the purified protein/peptide against each of the test bacteria used was performed using the microtiter broth dilution assay [19] One hundred microliters of each bacterial suspension containing 105 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, Cambridge, UK) The positive control contained bacteria and diluent only P citreus, Y ruckeri and Pseudo-monassp were incubated at 20C and E coli at 37 C and the attenuance (D) was read at 570 nm using a MRX II microtiter plate reader (Dynex, West Sussex, UK) against a blank comprising diluent only Values for experimental wells were recorded when the attenuance reached 0.2 in the positive control well The EC50 was considered to be the lowest concentration of protein that reduces the growth by 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 residual colony 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 diffusion assay of Lehrer et al

as described by Smith et al using P citreus Gram-positive

as a test bacteria [20,21] Briefly, bacteria grown 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

4C, 0.3-mm diameter wells were bored into 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 allowed to diffuse for at least 3 h at 4C 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

20C 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)1in 100 mMammonium bicar-bonate buffer) was incubated with bovine pancreas chymo-trypsin at 37C using a molar ratio of protease to lipoprotein (1 : 100) and taking aliquots each 30 min over

4 h The reaction was stopped by heating the samples at

100C for 5 min in sample buffer [62.5 mM Tris/HCl;

2% w/v SDS; 10% v/v glycerol; 5% v/v 2-mercaptoethanol

and bromophenol blue) The products of proteolysis 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 were blocked for 1 h with 1% (w/v) bovine

serum albumin in NaCl/Pi buffer and then alternatively

incubated a further hour with rabbit anti-carp apoA-I serum

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/Pi, the membranes were incubated for 1 h

with a 1 : 2000 dilution of alkaline phosphatase-conjugated

goat anti-rabbit IgG (Gibco BRL) The blot was developed

by incubating the membranes for 10 min in phosphatase

buffer (0.1MTris/HCl, pH 9.5; 0.1MNaCl; 5 mMMgCl2)

containing of 0.16 mgÆmL)1 5-bromo-4-chloroindolyl

phosphate and 0.33 mgÆmL)1nitroblue 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 bands 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 The first peak was shown to contain aggregates 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 demonstrated 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 EC50and a MBC

of approximately 0.4 lMagainst P citreus (Gram-positive) and at micromolar concentrations (2.6–4.0 lM) against two Gram (–) fish pathogens Pseudomonas sp and Yersinia ruckeri (Table 1) In addition, purified apoA-II also dis-played bacteriostatic activity against the Gram-positive and -negative bacteria at micromolar concentrations (Table 1) These results clearly show 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 on 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 proteases (e.g tryptase, chymase and several matrix metalloproteases) [23,24], we hypothes-ized that during acute inflammation one or more peptides could be released from the HDL particle by proteolysis either from the N- or C-terminal region of apoA-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 of amphipathic a-helix (Fig 2B) In particular, 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 and

concen-trated fractions of each peak were separated by SDS/PAGE (Insert)

Carp plasma (lane P), lanes 1–3 correspond to peaks 1–3, respectively

(50 lg protein per lane) Arrows indicate the migration of carp apoA-I

(27.5 kDa) and A-II (12.5), respectively.

Table 1 Bacteriostatic and bactericidal activities of carp apoA-I and A-II Each value in the table represents the mean ± SE of experiments performed in triplicate Similar results were obtained with different preparations of apolipoproteins ND, not determined.

EC 50 (l M ) MBC (l M ) EC 50 (l M )

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-terminal peptide and evaluated its antimicrobial activity in vitro The synthetic peptide was active against P citreus displaying

an EC50of 3–6 lM Considering that other cationic proteins and peptides have been shown to exhibit synergism with hen egg white lysozyme [27–29], we attempted to ascertain if the carp apoA-I peptide would be also able to synergize with lysozyme As shown in 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 concentrations of 6 lgÆmL)1 and 0.8 mM of lysozyme and peptide, respectively (Fig 3)

Limited proteolysis of HDL-associated apoA-Iin vitro

To determine if one or more peptides could be liberated after limited proteolysis of apoA-I in vitro, we analyzed the kinetics of carp HDL 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 than 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 ofa-helicity of apoA-I peptide and three-dimensional

model of carp apoA-I (A) Helical wheel projection of 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 faces The preferential localization of the positively

charged residues on the upper face of the helix 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 partial

carp apoA-I sequence was generated by SWISS - MODEL (http://

www.expasy.org/swissmod/SWISS-MODEL.html) based on the

crystallographic data for human apoA-I The N-terminal residue (N)

corresponds to the first residue of the carp apoA-I partial sequence

(GenBank accession 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) Bacterial growth in the presence of different combinations of the 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) or 0.8 m M (r) of the synthetic peptide The experiments were performed in triplicate and the error bars cor-respond to the standard error around the mean (B) Depicts the increased inhibitory halo observed with increasing concentrations of peptide were used in combination with 6 lgÆmL)1of lysozyme Wells 1–5 correspond to the same peptide concentration as in (A), ranging from 0 to 0.8 m M , respectively.

Fig 4B, the intact apoA-I (band a), an intermediary

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 immunodetected while the third band, which was the

most abundant after 30 min of digestion was not detected

by this antiserum, indicating that it would correspond to a

fragment truncated both at the N- and C-terminal end 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) recognized by the antipeptide antibodies

Using the same antiserum we could not detect a band in the

range of molecular masses 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 the same experiment, no degradation was

observed for apoA-II neither by direct staining (Fig 4D)

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 studies of mammalian HDL and

its principal apolipoproteins A-I and 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 be relevant for teleost fish [8]

Here we clearly demonstrate the important 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 as 1 gÆdL)1irrespective, of the acclimatization condition of the fish [11] Although the relative abundance of HDL varies among different teleosts,

it is generally accepted that this lipoprotein is clearly more abundant in fish plasma than in higher 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 present 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 and HDL in the plasma In both, apoA-I and apoA-II are derived from HDL particles While the size

of skin nascent HDL is different from that of plasma HDL,

it contains both apoA-I and apoA-II, molecules shown by the present paper to have potent antimicrobial properties

in vitro Work is currently underway to investigate the precise mechanism by which HDL and the associated apolipoproteins act The results of these studies 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 several amphipatic a-helices in a horseshoe-shape structure [12] In fact, it has been demonstrated that various HDL functions (e.g activation of lecithin-cholesterol acyltransferase or lipid binding) are dependent on these structural features of apoA-I [31] In view of the fact that 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 to salt as no activity was detected at

150 mM 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 mMNaCl [19] In the particular case of carp apoA-I, it could be argued that if a C-terminal peptide would be released in vivo it would be expected to be more active in a low-salt environment like the mucus of this freshwater fish than in its blood stream Another interesting feature of this C-terminal peptide is its ability to synergize with lysozyme Synergy of several antimicrobial peptides and proteins with lysozyme has been previously described [27,28] In teleosts, the blood and skin mucosa are particularly rich in lysozymes [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 we rule out a possible synergism between intact apolipoproteins and lysozyme

Fig 4 Limited proteolysis of HDL-associated apoA-I (A)

Tricine-SDS/PAGE and Coomassie blue staining were used to analyze the

progress of HDL-associated apoA-I proteolysis with chymotrypsin.

(B,C) Western blot analyses of the gel in (A) immunodetected with a

specific anti-apoA-I and anti-peptide serum, respectively Arrows

indicate the different bands of apoA-I: (a) intact form; (b) intermediary

fragment and (c) stable truncated apoA-I Incubation time is shown

above each gel (D) Tricine-SDS/PAGE and Coomassie blue staining

of HDL-associated apoA-II incubated with chymotrypsin under the

same conditions as in (A) (E) Western blot analysis of the gel in

(D) using a specific anti-apoA-II serum.

Once the antimicrobial mechanism of 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 effectors

The above results raise the possibility that not only the

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, which has previously been shown to produce human

apoA-I truncated either at the N- or at the C-terminus [23]

In this same study it was also demonstrated that apoA-II is

resistant to degradation under the conditions used Our

results are in close agreement with these data as following the

digestion with chymotrypsin, a stable apoA-I fragment that

seems to lack 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 vitroconditions of protease digestion, the peptide could

be very short-lived and therefore extremely hard to detect

Based on these preliminary results we postulate that besides

the constitutive contribution of HDL and its apolipoproteins

in teleost fish innate immunity, an additional mechanism

might involve the release of one or more antimicrobial

peptides by limited proteolysis of HDL-associated apoA-I

possibly triggered by one or more insult-regulated proteases,

e.g elastase or chymase Such a mechanism has already been

described for another nonimmune protein, histone H2A, in

catfish skin, where a complex cascade of injury-induced

proteases is involved in the regulation of the AMP parasin I

production [5] Therefore further studies will attempt to

evaluate the presence of the peptide in the 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 the multifunctionality

of these proteins Moreover the synergism observed between

the apoA-I synthetic peptide and lysozyme suggests that a

mechanism involving the regulated release of peptides from

the HDL-associated apoA-I present in plasma and 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 Direccio´n

de Investigacio´n y Desarrollo, Universidad Austral de 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 Andrews, Scotland, UK and of V.J.S to the Institute

of Biochemistry, Faculty of Sciences, Universidad Austral de Chile,

Valdivia, Chile.

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