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: 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 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. References 1. Magor, B.G. & Magor, K.E. (2001) Evolution of effectors and receptors of innate im munity. Dev. Comp. Immunol. 25,651– 682. 2. Fernandes,J.M.,Kemp,G.D.,Molle,G.&Smith,V.J.(2002) Antimicrobial properties of h istone H2A from skin s ecretions of rainbow trout, Oncorhynchus mykiss. Biochem. J. 368, 611–620. 3. Fernandes, J.M. & Smith, V.J. (2002) A novel antimicrobial function for a ribosomal peptid e from rainbow trout sk in. Bio- chem. Biophys. Res. Commun. 296, 167–171. 4. 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. 5. 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