Martin Malmsten and Artur SchmidtchenAndersson Nordahl, Matthias Mörgelin, Mukesh Pasupuleti, Björn Walse, Emma Invertebrates to HumansComplement Peptide C3a, from Preservation of Antimicrobial Properties ofDevelopmental Biology:Molecular Basis of Cell and

doi: 10.1074/jbc.M607848200 originally published online November 27, 20062007, 282:2520-2528.J. Biol. Chem.

10.1074/jbc.M607848200Access the most updated version of this article at doi:

.JBC Affinity SitesFind articles, minireviews, Reflections and Classics on similar topics on the

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

http://www.jbc.org/content/suppl/2006/11/28/M607848200.DC1.html

http://www.jbc.org/content/282/4/2520.full.html#ref-list-1

This article cites 31 references, 10 of which can be accessed free at

by guest on March 1, 2014

http://ww

w.jbc.org/

Dow

nloaded from

by guest on March 1, 2014

http://ww

w.jbc.org/

Dow

nloaded from

Preservation of Antimicrobial Properties of ComplementPeptide C3a, from Invertebrates to Humans*!S

Received for publication, August 16, 2006, and in revised form, November 8, 2006 Published, JBC Papers in Press, November 27, 2006, DOI 10.1074/jbc.M607848200

Mukesh Pasupuleti‡, Bjorn Walse§, Emma Andersson Nordahl‡, Matthias Morgelin¶, Martin Malmsten!,and Artur Schmidtchen‡1

From the ‡Section of Dermatology and Venereology, ¶Section of Clinical and Experimental Infectious Medicine, Department ofClinical Sciences, Biomedical Center, Lund University, Tornavagen 10, SE-221 84 Lund, §SARomics AB, P.O. Box 724,SE-220 07 Lund, !Department of Pharmacy, Uppsala University, SE-751 23 Uppsala, Sweden

The human anaphylatoxin peptide C3a, generated duringcomplement activation, exerts antimicrobial effects. Phyloge-netic analysis, sequence analyses, and structural modeling stud-ies pairedwith antimicrobial assays of peptides fromknownC3asequences showed that, in particular in vertebrate C3a, crucialstructural determinants governing antimicrobial activity havebeen conservedduring the evolution ofC3a. Thus, regions of theancient C3a from Carcinoscorpius rotundicauda as well as cor-responding parts of human C3a exhibited helical structuresupon binding to bacterial lipopolysaccharide permeabilizedliposomes and were antimicrobial against Gram-negative andGram-positive bacteria. HumanC3a andC4a (but not C5a) wereantimicrobial, in concert with the separate evolutionary devel-opment of the chemotactic C5a. Thus, the results demonstratethat, notwithstanding a significant sequence variation, func-tional and structural constraints imposed on C3a during evolu-tion have preserved critical properties governing antimicrobialactivity.

In vertebrates, the complement system is activated by theclassical, alternative, and lectin pathways, each converging atthe step of complement factor 3 (C3)2 with release of multipleproteolytic fragments, including the anaphylatoxin C3a (1). Inaddition to its multiple proinflammatory functions involvinghistamine release from mast cells, smooth muscle contraction,and increased vascular permeability (1), C3a exerts a direct andpotent antimicrobial effect against Gram-negative and Gram-positive bacteria (2). C3 deficiency is connected with increasedsusceptibility to bacterial infections in humans (3) as well as in

animal models (4, 5), findings compatible with this direct anti-bacterial effect of C3a (5). Thus, C3a, in concert with otherantimicrobial peptides (AMPs), executes pivotal roles in theinnate immune system, providing a rapid and nonspecificresponse against potentially invasive pathogenic microorgan-isms (6). It has been demonstrated that cathelicidins anddefensins (for review, see Refs. 6–8) representing two impor-tant AMP families display a wide sequence heterogeneity, thusreflecting positive selection and an adaptation of organisms tovarious bacterial environments (9–11). The complement factorC3 represents an evolutionarily old molecule (12) identified inthe deuterostomeCiona intestinalis (13) as well as in the horse-shoe crabCarcinoscorpius rotundicauda, a protostome consid-ered a “living fossil” originating over 500 million years ago (14).These animals, which lack adaptive immunity, mount an effec-tive antimicrobial defense in response to pathogens. In thiswork utilizing a combination of phylogenetic studies and struc-tural and sequence alignments paired with biophysical andfunctional analyses, we have shown that structural prerequi-sites governing antimicrobial activity can be traced from thehuman C3a molecule back to the C3a molecules of inverte-brates, such as those found in C. rotundicauda.

EXPERIMENTAL PROCEDURES

Microorganisms—Escherichia coli 37.4, Enterococcus faecalis2374, Pseudomonas aeruginosa 27.1, E. coli American TypeCulture Collection (ATCC) 25922, Bacillus subtilisATCC 6633,and Candida albicans ATCC 90028 were obtained from theDepartment ofMicrobiology, Lund University, Lund, Sweden.Peptides and Proteins—C3a andC4awere obtained fromThe

Binding Site, Inc. (San Diego, CA), whereas C5a-desArg wasfrom Calbiochem. The peptides GKE31, LGE33, CNY21,CQF20, CVV20 (for sequences, see Fig. 2), and tetramethylrho-damine (TAMRA)-conjugated CNY21 and CQF20 were syn-thesized by Innovagen (Lund, Sweden). The purity (!95%) andmolecular weight of these peptides were confirmed by massspectral analysis (matrix-assisted laser desorption ionizationtime-of-flight, Voyager). 20-mer peptides corresponding to vari-ous regions of C3a (Fig. 2 and supplemental Table 2) were fromSigma (PEPscreen", Custom Peptide Libraries, SigmaGenosys).Phylogenetic Analyses—C3a, C4a, and C5a amino acid

sequences were retrieved from the NCBI site. Each sequencewas analyzed with Psi-Blast (NCBI) (15) to find the orthologand paralog sequences. Sequences that showed structuralhomology !70% were selected. These sequences were aligned

* This work was supported by grants from the Swedish Research Council(Projects 13471 and 621-2003-4022), the Royal Physiographic Society inLund, the Welander-Finsen, Soderberg, Schyberg, Groschinsky, Crafoord,Åhlen, Alfred Osterlund, Lundgrens, Lions, and Kock Foundations, and theSwedish Government Funds for Clinical Research. The costs of publicationof this article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

!S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. 1 and Tables 1 and 2.

1 To whom correspondence should be addressed: Section of Dermatologyand Venereology, Dept. of Clinical Sciences, Lund University, BiomedicalCtr., Tornavagen 10, SE-22184 Lund, Sweden. Tel.: 46-46-222-4522; Fax:46-46-157-756; E-mail: artur.schmidtchen@med.lu.se.

2 The abbreviations used are: C3, complement factor 3; AMP, antimicrobialpeptide; CF, carboxyfluorescein; TSB, trypticase soy broth; TAMRA, tetra-methylrhodamine; CD, circular dichroism; LPS, lipopolysaccharide.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 4, pp. 2520 –2528, January 26, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

2520 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 4 • JANUARY 26, 2007

by guest on March 1, 2014

http://ww

w.jbc.org/

Dow

nloaded from

using ClustalW (16) using Blosum 69 protein weight matrixsettings (17). Internal adjustmentsweremade taking the structuralalignment into account utilizing the ClustalW interface. The levelof consistency of each position within the alignment was esti-mated by using the alignment-evaluating software Tcoffee (18).C3a, C4a, and C5a sequences were used for phylogenetic treeconstruction by using the neighbor-joiningmethod (19). The gen-erated tree was rootedwith humanC3a, and the reliability of eachbranch was assessed using 1000 bootstrap replications. For deter-mination of non-synonymous (amino acid-altering) and synony-mous (silent) nucleotide substitutions (dN and dS) (20) we usedthe codeml PAML software. cDNA sequences corresponding tothe variousC3apeptides of vertebratesweredownloaded fromtheNCBI site (supplemental Table 1).Molecular Modeling—A comparative homology model of

C. rotundicauda C3a was created based on the structure of

human C3a (Protein Data Bank(PDB) code 2A73 (21)). The sequenceof human C3a (residues 651–719 inC3, corresponding to 2–70 in C3a)was aligned against the sequence ofC. rotundicauda C3a using thealignment in Fig. 2. For clarity,amino acid numbering in the textbelow is based on the position in therespective anaphylatoxin peptide,defining the N terminus after theRKKR processing site. Structuralalignment of C3a and C5a struc-tures revealed that the regions cor-responding to Arg8–Tyr15, Leu19–Met27, and Lys50–Leu63 in humanC3a (Fig. 2) correspond to struc-ture-conserved regions. ResiduesIle2–Asn67 of C. rotundicauda C3awere built using the Prime module(22) from the Schrodinger compu-tational chemistry suite of programs(Schrodinger, L.L.C., Portland, OR).The sequence identity was 26%(similarity 38%), and rotamers fromthe conserved residues wereretained. Terminal tails beyond sec-ondary structure elements were notbuilt. Loops 1 (Glu11–Arg13) and 2(Asp27–Arg29) were refined one at atime using default sampling in theloop refinement protocol built intoPrime, except the long inserted loop3 (Glu39–Glu47) where extendedmedium sampling was used. Disul-fur bridges Cys17–Cys49, Cys18–Cys56, and Cys31–Cys57 were builtand minimized prior to refinement.A stepwise refinement protocolinvolving minimization of (a) sidechains from non-structure-con-served regions, (b) both backbone

and side chains from non-structure-conserved regions, and (c)all side chains were employed. The refined comparative modelof C. rotundicaudaC3a was extended in the C terminus by res-idues Ile68–Arg75. These residues were added in an !-helicalconformation andminimized. Some adjustment of the "/# val-ues of Leu64, Leu65, Lys66, and Asn67 to helical values were nec-essary prior to minimization. All atoms of residues Leu64–Arg75 were minimized of this extended model. Finally, all sidechains were minimized resulting in the described model. Min-imization was performed using the MacroModel module fromthe Schrodinger computational chemistry suite of programsusing a dielectric constant of 1 and the OPLS2005 force field.Comparative homology models of human C4a (residues 1–77)and C. intestinalis C3a (residues 2–76) were constructed usingthe same template, refinement protocol, and minimization asabove and the alignment in Fig. 2. The last seven and five resi-

FIGURE 1. Evolution of anaphylatoxins. Phylogenetic tree presenting C3a, C4a, and C5a sequences using theneighbor-joining method. Numbers on the branches indicate the reliability of each branch using 1000 boot-strap replications.

Structural Conservation of Antimicrobial C3a

JANUARY 26, 2007 • VOLUME 282 • NUMBER 4 JOURNAL OF BIOLOGICAL CHEMISTRY 2521

by guest on March 1, 2014

http://ww

w.jbc.org/

Dow

nloaded from

dues, respectively, were added in !-helical conformations andminimized. Sequence identities were 29% (51% similarity) forhuman C4a and 17% (37% similarity) for C. intestinalis C3awhen compared with human C3a.Radial Diffusion Assay—Essentially as described earlier (23),

bacteria were grown to mid-log phase in 10 ml of full-strength(3% w/v) trypticase soy broth (TSB) (BD Biosciences). Themicroorganisms were washed once with 10 mM Tris, pH 7.4.4 " 106 bacterial colony-forming units were added to 5 ml ofthe underlay-agarose gel (0.03% TSB, 1% low electroendosmo-sis-type agarose (Sigma) and 0.02% Tween 20 (Sigma)). Theunderlay was poured into an 85-mm-diameter Petri dish (or144 mm diameter for the experiment with PEPscreen pep-tides). After agarose solidification, 4-mm-diameter wells werepunched, and 6 $l of test peptide was added to each well. Plateswere incubated at 37 °C for 3 h to allow diffusion of the pep-tides. The underlay gel was then covered with 5 ml of moltenoverlay (6%TSB and 1% low electroendosmosis-type agarose indistilled H2O). The antimicrobial activity of a peptide was visu-alized as a zone of clearing around each well after incubating18–24 h at 37 °C. The activity of the human complement-de-rived peptides LGE33, CNY21, CQF21, and CVV20 as well asGKE31 of C. rotundicauda (at 50 and/or 100 $M) were com-pared with the activity of the peptide LL-37. In all cases, tripli-cate samples were used.

ViableCountAnalysis—P. aeruginosa 27.1 orE. faecalis 2374bacteriawere grown tomid-log phase inTodd-Hewittmedium.Bacteria were washed and diluted in 10 mM Tris, pH 7.4, con-taining 5 mM glucose. For dose-response experiments,P. aeruginosa (50 $l; 2 " 106 colony-forming units/ml) wereincubated at 37 °C for 2 h with the peptides GKE31 and LGE33(P. aeruginosa) at concentrations ranging from 0.03 to 60 $M.For analysis of the effects of C3a, C4a, and C5a-desArg (see Fig.7A), P. aeruginosa and E. faecalis 2374 were used, and the pep-tide concentration was 3$M. To quantify the bactericidal activ-ity, serial dilutions of the incubation mixture were plated onTodd-Hewitt agar followed by incubation at 37 °C overnight,and the number of colony-forming units was determined.Electron Microscopy—P. aeruginosa 27.1 (1–2 " 107/sam-

ple) were incubated for 2 h at 37 °C with the peptides GKE31or LGE33 at #90% of their required bactericidal concentra-tion (6 $M), as judged by dose-response experiments usingviable count assays (not shown). LL-37 (6 $M) was includedas a control. Samples of P. aeruginosa bacteria suspensionswere adsorbed onto carbon-coated copper grids for 2 min,washed briefly on two drops of water, and negatively stainedon two drops of 0.75% uranyl formate. The grids were ren-dered hydrophilic by glow discharge at low pressure in ambi-ent air. Specimens were observed in a Jeol JEM 1230 electronmicroscope operated at 60 kV of accelerating voltage. Images

FIGURE 2. Alignment of C3a, C4a, and C5a sequences showing identical and similar amino acids. The shading represents the degree of conservation ateach position in the alignment taking into account similar physicochemical properties of the residues. The peptide sequences corresponding to regions 1– 6in the human C3a sequence are indicated. Note that this only applies to the C3a sequences (full information on these peptides is given in supplemental Table2). The amino termini of peptides LGE33 (C3a Homo), CNY21 (C3a Homo), CQF21 (C4a Homo), CVV20 (C5a Homo), and GKE31 (C3a, C. rotundicauda) are indicatedby an asterisk in the alignments. All peptides terminate at the final arginine residue. Disulfide bonds are indicated (bottom).

Structural Conservation of Antimicrobial C3a

2522 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 4 • JANUARY 26, 2007

by guest on March 1, 2014

http://ww

w.jbc.org/

Dow

nloaded from

were recorded with a Gatan Multiscan 791 charge-coupleddevice camera.Circular Dichroism (CD) Spectroscopy—The CD spectra of

the peptides in solution were measured on a Jasco J-810 spec-tropolarimeter (Jasco). The measurements were performed at37 °C in a 10-mm quartz cuvette under stirring, and the effecton peptide secondary structure was monitored in the range of200–250 nm. The background value (detected at 250 nm,where no peptide signal is present) was subtracted, and signalsfrom the bulk solution were corrected. The peptide secondarystructure was monitored at a peptide concentration of 10 $Mboth inTris buffer and in the presence ofE. coli lipopolysaccha-ride (0.02%w/w) (E. coli 0111:B4, highly purified,$1% protein/RNA; Sigma).

Liposome Preparation and Leak-age Assay—Dry lipid filmswere pre-pared by dissolving either dio-leoylphosphatidylcholine (AvantiPolar Lipids, Alabaster, AL) (60 mol%) and cholesterol (Sigma, St Louis,MO) (40 mol %) or dioleoylphos-phatidylcholine (30 mol %), dio-leoylphosphatidic acid (AvantiPolar Lipids, Alabaster, AL) (30 mol%), and cholesterol (40 mol %) inchloroform and then removing thesolvent by evaporationunder vacuumovernight. Subsequently, buffer (10mM Tris, pH 7.4) was added togetherwith 0.1 M carboxyfluorescein (CF)(Sigma). After hydration, the lipidmixture was subjected to eightfreeze-thaw cycles consisting offreezing in liquid nitrogen and heat-ing to 60 °C. Unilamellar liposomesof #100 nm diameter were gener-ated by multiple extrusions throughpolycarbonate filters (pore size 100nm) mounted in a LipoFast miniex-truder (Avestin, Ottawa, Canada) at22 °C. Untrapped CF was thenremoved by two gel filtrations(Sephadex G-50) at 22 °C with theTris buffer as eluent. The CF releasewas determined by monitoring theemitted fluorescence at 520 nmfrom a liposome dispersion (10 mMlipid in 10 mM Tris, pH 7.4). Anabsolute leakage scale was obtainedby disrupting the liposomes at theend of the experiment through theaddition of 0.8 mM Triton X-100(Sigma), therebycausing100%releaseand dequenching of CF. A SPEX-flu-orolog 1650 0.22 monochromaticdouble spectrometer (SPEX Indus-tries, Edison, NJ) was used for theliposome leakage assay.

Fluorescence Microscopy—P. aeruginosa 27.1 bacteria weregrown to mid-logarithmic phase in Todd-Hewitt medium. Thebacteria were washed twice in 10 mM Tris, pH 7.4. The pelletwas dissolved to yield a suspension of 5 " 106 colony-formingunits/ml in the same buffer. Two hundred microliters of thebacterial suspension were incubated with either 1 $l ofTAMRA-CNY21 or TAMRA-CQF21 (2 mg/ml) on ice for 5min and washed twice in 10mMTris, pH 7.4. The bacteria werethen fixed by incubation on ice for 15min and in room temper-ature for 45 min in 4% paraformaldehyde. The suspension wasapplied onto poly-L-lysine-coated cover glass, and bacteriawere left to attach for 30 min. The liquid was poured away, andthe cover glass was mounted on a slide by Dako mountingmedium (Carpinteria, CA). This was performed using a Nikon

FIGURE 3. Molecular models of human C3a, C4a, and C5a and C. rotundicauda C3a and illustration ofamphipathicity of C-terminal peptides of C3a. A, molecular models. Human C3a is represented by thecorresponding part in the crystal structure of human C3 (PDB code 2A73; (21)). The last seven residues weremissing in this structure because of inherent flexibility and were added in an !-helical conformation for com-parable purposes. Human C4a and C. rotundicauda C3a are represented by homology models based on thecrystal structure of human C3a. Human C5a is represented by the NMR structure described in Zhang et al. (27),1997 (PDB code 1KJS). From top to bottom, ribbon, front and back surface representations, respectively, ofC. rotundicauda C3a, human C3a, C4a, and C5a. Color coding shows blue (positively charged residues (H, K, andR)), red (negatively charged residues (D and E), cyan (polar residues CSH, S, N, Q, T, Y, W), green (hydrophobicresidues A, CSS, F, G, I, L, M, P, V). B, helical wheel representation of the C3a-derived C-terminal peptides GKE31(from C. rotundicauda) and LGE33 (Homo). The amino acids are indicated.

Structural Conservation of Antimicrobial C3a

JANUARY 26, 2007 • VOLUME 282 • NUMBER 4 JOURNAL OF BIOLOGICAL CHEMISTRY 2523

by guest on March 1, 2014

http://ww

w.jbc.org/

Dow

nloaded from

EclipseTE300 inverted fluorescencemicroscope equippedwitha Hamamatsu C4742-95 cooled charge-coupled device camera,a Plan Apochromat 60" objective, and a high numerical aper-ture oil condenser.

RESULTS

Sequence Analyses of Anaphylatoxin Homologs—Phyloge-netic analysis of representativeC3a peptides from invertebratesaswell as vertebrates (Fig. 1) yielded valuable information. Con-sistent with a common ancestor, the C3a molecules all colocal-ized to a single group, suggesting that C3a has evolved via mul-tiple changes in the protogene, a finding consistent withprevious analyses of the evolution of the complement system(14, 24). On the other hand, C4a and C5a formed separateclades, C5a being most distant from the C3a. This pattern sug-gests that C5a and C4a likely evolved from C3a and that C5a isthe paralog to C4a and C3a (14). Comparisons of synonymousand non-synonymous substitution rates are useful for studyingthe mechanisms of gene evolution. Analysis of evolutionarilydistant sequences, however, is not useful because of the satura-tion of amino acid changes. Nevertheless, to get useful informa-tion on possible positive selection of C3a, relevant and homol-ogous vertebrate sequences were compared. The dS values(indicating synonymous nucleotide substitutions) ranged from0.7 to 1.0, except forMacropus eugenii, which showed a higher

substitution rate #2.7, whereas thedN (indicating non-synonymoussubstitutions) ranged from 0.1 to0.2 (supplemental Table 1). Thus,although there was the existenceof an extensive variability of cer-tain amino acids in C3a (Fig. 2),the results from the analysis ofvertebrate sequences indicated thatthe selection pressure imposed onthe C3a molecules results in a highdegree of conservation.Given this phylogenetic relation-

ship, it was of interest to examinethis molecular family closer, bothfrom a structural as well as a func-tional perspective. Several commonstructural features exist for the cor-responding C3a, C4a, and C5asequences of various organisms cru-cial for the integrity and stability ofthe molecules (Fig. 2). A most nota-ble feature is the existence of six dis-ulfide-bonded cysteines, which areconserved not only in C3a but alsoin C4a and C5a. The three disulfidebonds stabilize the conformation ofthe internal “core” portion of themolecules represented by residues22–57 in the human C3a sequence.The C3a of C. intestinalis lacks onedisulfide pair (Fig. 2) and will be dis-cussed separately below. Focusing

on C3a, it is evident that, apart from cysteines, the four glycines(positions 13, 26, 46, and 74), phenylalanine (53), as well aslysines and arginines (21, 64, 77) constitute additional con-served features, suggesting their importance for the structuralstability as well as function of C3a.Structural Modeling of Anaphylatoxin Peptides—Computa-

tional modeling utilizing available structural data on humanC3a (21, 25) as well as C5a peptides (26, 27) was employed toprovide further structural information. Considering the recentidentification of C3 and a putative anaphylactic peptide in thearthropod C. rotundicauda (14), we decided to compare thesetwomolecules. As demonstrated in Fig. 3A, the similarity at thethree-dimensional level between human C3a and the predictedC. rotundicauda C3a peptide is apparent, although having anextensive overall sequence discrepancy (26% sequence iden-tity and 38% similarity). Both peptides share a striking simi-larity in the four helical regions and in the two first loopslocated before the cysteines at positions 22 and 36 and posi-tions 17 and 31 in the human and C. rotundicauda peptides,respectively. The C. rotundicauda C3a has a five-amino-acid-long insert in the third loop region between the cysteines atpositions 31 and 49, a feature only shared with C3a from C. in-testinalis. Analogous to human C3a, the C. rotundicauda pep-tide contains a prominent cationic and amphipathic helical Cterminus predicted to extend a few residues further than in

FIGURE 4. Antibacterial activities of C3a peptides. Overlapping peptides (regions 1– 6; see Fig. 2 and alsosupplemental Table 2) of C3a were analyzed for antimicrobial activities against P. aeruginosa. The regions,inhibitory zones, the respective organism, as well as net charge of respective peptides are indicated in thethree-dimensional graph. For determination of antibacterial activities, the P. aeruginosa isolate (4 " 106 colony-forming units) was inoculated in 0.1% TSB-agarose gel. Each 4-mm-diameter well was loaded with 6 $l ofpeptide (at 200 $M). The zones of clearance correspond to the inhibitory effect of each peptide after incubationat 37 °C for 18 –24 h (mean values are presented, n % 3). RDA, radial diffusion assay.

Structural Conservation of Antimicrobial C3a

2524 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 4 • JANUARY 26, 2007

by guest on March 1, 2014

http://ww

w.jbc.org/

Dow

nloaded from

human C3a. In human C3a, the C-terminal part of this region,being flexible in solution, strongly conforms to an !-helicalconformation in anisotropic environments (1). Helical wheeldiagrams (Edmundson projection) illustrate the amphipathicnature of the C-terminal helices (given a helical conformation)(Fig. 3B), as defined by the peptides GKE31 and LGE33, whichencompass this region of C. rotundicauda and human C3a,respectively (see Fig. 2 for sequence). The amphipathic organi-zation of these helices is also seen in the three-dimensionalmodels of C. rotundicauda and human C3a (Fig. 3A).Antimicrobial Activities of C3a Peptides—To explore the

structure-function relationships of C3a epitopes, overlappingpeptide sequences comprising 20-mers (Fig. 2 and supplemen-tal Table 2) were synthesized and screened for antimicrobialactivities against P. aeruginosa, a ubiquitous pathogen foundamong both vertebrates as well as invertebrates. The experi-ments showed that, particularly, peptides derived from theC-terminal regions of C. rotundicauda, Ciona, and Branchios-

toma as well as from the vertebrates Homo, Sus, Mus, Rattus,andGuinea, were antimicrobial (Fig. 4), whereas peptides orig-inating from Cobra, Paralicchtys, Onchorhyncus, Eptatretus,Xenopus, and Gallus did not show any activity against bacteria(Fig. 4). Properties common for most AMPs include minimumlevels of cationicity, amphipathicity, and hydrophobicity (6).Therefore, it was interesting to note thatC-terminal regions of thetwo formergroups comprisedcationicpeptides,whereas the lattergroup comprisednegatively charged peptides (illustrated by colorsin Fig. 4). These results corresponded well with the phylogeneticanalysis, which showed that these animals belong to differentsubclades, and with single nodes of divergence (Fig. 1). Theglobal analysis of biophysical parameters showed that, in gen-eral, peptides displaying antimicrobial activity had a net chargeof &2 to &4 and #20–40% hydrophobic amino acids. Thedegree of amphipathicity, as judged by the relative hydrophobicmoment ($Hrel), ranged from #0.2 to 0.4, values comparablewith those observed inmany helical AMPs (28). Although intu-

FIGURE 5. Activities of C3a-derived human and C. rotundicauda peptides. A, antibacterial effects. Peptides were tested in radial diffusion assay in low saltconditions. P. aeruginosa, E. coli, B. subtilis, and C. albicans (4 " 106 colony-forming units) were inoculated in 0.1% TSB-agarose gel. Each 4-mm-diameter wellwas loaded with 6 $l of the peptides GKE31 and LGE33 as well as LL-37 peptide at 100 $M. The zones of clearance (y-axis) correspond to the inhibitory effect ofeach peptide after incubation at 37 °C for 18 –24 h. Mean values and S.D. are presented (n % 3). The inset illustrates the antibacterial effects of the peptidesagainst P. aeruginosa. B, the C3a-derived peptides GKE31 and LGE33 generate breaks in bacterial plasma membranes. P. aeruginosa was incubated with GKE31and LGE33 peptides (at 6.0 $M) and analyzed with electron microscopy. The peptides are indicated in the figure. The scale bar corresponds to 0.5 $m. Control,buffer control. C, CD spectrum of GKE31 and LGE33 in Tris buffer and in the presence of LPS. For control, CD spectra for buffer and LPS alone are presented.D, effects of GKE33 and LGE31 on liposome leakage. The membrane-permeabilizing effect was recorded by measuring the fluorescence release of CF fromliposomes. Values represent mean of triplicate samples. E, kinetic analysis of liposome permeabilization using 1 $M GKE31 and LGE33 peptides, respectively.ODC, ellipticity (in millidegrees).

Structural Conservation of Antimicrobial C3a

JANUARY 26, 2007 • VOLUME 282 • NUMBER 4 JOURNAL OF BIOLOGICAL CHEMISTRY 2525

by guest on March 1, 2014

http://ww

w.jbc.org/

Dow

nloaded from

itively apparent (Fig. 4), the results confirmed that net chargecorrelates to antimicrobial activity (supplemental Fig. 1). Con-sidering these findings, it is notable that a disproportionatealteration of charge appears to characterize the evolution of%-defensins (9, 29). Analogous relationships were recentlyreported to apply to the evolution of primate cathelicidin,showing positive selection affecting charge while keeping hy-drophobicity and amphipathicity fairly constant (11).Structural and Functional Congruence of C Termini of C3a—In

humans, peptides derived from the well defined C-terminalregion of C3a exert antimicrobial effects. Furthermore, humanneutrophilic enzymes release similar C3a-derived peptidesexerting antimicrobial effects, proving the physiological impor-tance of this helical and antimicrobial region of C3a (2). Con-sidering this, the following experimental analyses focused onthe C-terminal region of human C3a as well as the related pep-tide “ancestor” from C. rotundicauda (see Figs. 2 and 3B).Indeed, the experiments showed that theC. rotundicauda pep-tide GKE31, spanning the whole C-terminal part of C. rotundi-cauda C3a, exerted similar antibacterial effects as the humanhomolog LGE33 against both the Gram-negative P. aeruginosaand E. coli and the Gram-positive B. subtilis (Fig. 5A). The“classical” human cathelicidin LL-37 yielded similar effects asthe two C3a-derived peptides. The C. rotundicauda peptidewas not active against C. albicans. To examine whether theGKE31 and LGE33 peptides interact with and permeabilizebacterial plasmamembranes, P. aeruginosawas incubated witheach of the two peptides at concentrations yielding #90% bac-terial killing (6 $M) and analyzed by electron microscopy (Fig.5B). Clear differences in the morphology of peptide-treatedbacteria in comparison with the control were demonstrated bythis approach. The peptides caused local perturbations andbreaks along P. aeruginosa plasma membranes, and occasion-ally intracellularmaterial was found extracellularly. These find-ings were similar to those seen after treatment with the antimi-crobial human cathelicidin LL-37 (Fig. 5B). Furthermore, CDspectroscopy was used to study the structure and the organiza-tion of the GKE31 and LGE33 peptides in solution and uponinteraction with E. coli lipopolysaccharide (LPS) (Fig. 5C). Nei-ther GKE31 nor LGE33 adopted an ordered conformation inaqueous solution; however, the CD spectra revealed that a sig-nificant and almost identical structural change, largely indicat-ing an induction of helicity, taking place in the presence ofE. coli LPS (Fig. 5C). The remarkably similar profile of bothpeptides indicates that, despite a marked difference in primarysequence, the peptides structure themselves in a similar way inthe presence of negatively charged LPS-rich bacterial mem-branes. Both of the peptides also induced leaking of liposomes,thus establishing their membrane-breaking activities (Fig. 5D).Kinetic analysis showed that #80% of the maximum fluores-cence was reached within #200 s for both peptides (at 1 $M)(Fig. 5E). The results therefore indicate that the GKE31 andLGE33 peptides indeed function similar to most helical AMPs,such as LL-37 (6, 28), by interactions with LPS and most likelypeptidoglycan at bacterial surfaces, leading to induction of an!-helical conformation, which in turn facilitates membraneinteractions, membrane destabilization, and, finally, bacterialkilling. The fact that the two C3a-derived peptides are sepa-

rated by as much as over a half-billion years of evolutionarydistance elegantly demonstrates the remarkable structural andfunctional conservation of this C-terminal peptide region.Comparison of C3a Molecules of C. rotundicauda and

C. intestinalis—The C3a molecule of Ciona is functionallyactive; it exerts chemotactic effects (13) and, as shown here, hasa C-terminal antibacterial part (Fig. 4). However, in contrast tothe other C3a molecules, C3a of Ciona lacks one disulfidebridge (Fig. 2). Despite this, and a sequence identity of only 17%(37% similarity) with humanC3a,molecularmodeling and con-formational analysis show that Ciona C3a adopts a predictedconformation similar to other C3a molecules (Fig. 6A). Inter-estingly, the first missing cysteine in CionaC3a is replaced by aglycine residue (Gly18), whereas the second cysteine is replacedby a glutamate residue (Glu57). Hypothetically, these changescould enable the second helix to approach and interact with theC-terminal helix by formation of a salt bridge between Glu57and Lys22, thus compensating for the loss of a disulfide bond.These interactions, together with main structural constraintsimposed by the remaining two disulfide pairs preserve the over-all topology of Ciona C3a (Fig. 6B). Furthermore, it is notablethat residues forming the inner core throughout the anaphyla-toxin family (Gly21, Ile/Val39, and Phe54) are all conserved inCiona C3a (Fig. 2). Taken together, these structural consider-ations, combined with the functional data, further underscorethe conservation of C3a.Structure and Activities of C4a and C5a—The phyloge-

netic analysis indicated that C5a evolved separately from thefamily of C3a as well as C4a molecules (Fig. 1). To address

FIGURE 6. Molecular model of C. intestinalis C3a and comparison withC. rotundicauda C3a. A, composite model incorporating Ciona C3a (gray)and C. rotundicauda C3a (magenta). The location of the missing disulfidebond in Ciona C3a is indicated by an arrow. B, front and back surface repre-sentations of Ciona C3a. The color coding is as in Fig. 3. Note the amphipathiccharacter of the C-terminal helix.

Structural Conservation of Antimicrobial C3a

2526 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 4 • JANUARY 26, 2007

by guest on March 1, 2014

http://ww

w.jbc.org/

Dow

nloaded from

whether this also reflected a functional difference, we com-pared the antibacterial activities of human C3a with C4a, andC5a (the latter only available in desArg form) and their cor-responding peptides from the C terminus. C3a and C4a bothexerted antibacterial effects, whereas the C5a peptide wasinactive (Fig. 7A). Corroborating results were obtained withthe corresponding C-terminal peptides (Fig. 7B; for sequences,see Fig. 2). Although the C3a-and C4a-derived peptides(CNY21 and CQF21, respectively) killed the Gram-negativeP. aeruginosa and E. coli, the Gram-positive B. subtilis, as wellas the fungusC. albicans, theC5a peptide (CVV20)was inactiveagainst these microbes. The fact that the C5a-derived peptideCVV20 contained a terminal arginine and that there is no dif-ference in antimicrobial activity between C3a and C3adesArgas well as the corresponding C3a-derived peptides CNY21 andCNY20 (2) demonstrated that the terminal arginine is dispen-sable for antimicrobial activity of these peptides. Finally, theinteraction between the antimicrobial peptides CNY21 (C3a)and CQF21 (C4a) and bacterial plasma membranes was exam-ined by fluorescence microscopy. The peptides were labeledwith the fluorescent dye TAMRA and incubatedwith P. aerugi-nosa. As demonstrated, the peptides bound to the bacterial sur-face, and the binding was completely blocked by the negativelycharged glycosaminoglycan heparin (Fig. 7C). As seen in thethree-dimensional models, the last ten residues in C5a form a

short !-helix that is bent backwards through a short loop posi-tioning the carboxyl terminal arginine (Arg74) in close proxim-ity to arginine 62 (Fig. 3A), a feature that is believed to be impor-tant for receptor binding (27). Thus, C5a display a significantlydifferent structure when compared with both C4a and C3a,lacking the typical C-terminal and antimicrobial protrudingpeptide (Fig. 3A), thus compatiblewith the experimental resultspresented herein. This observation, paired with the separateevolutionary development of C5a, indicate that this moleculehas evolved separately in higher organisms into a purely che-motactic and highly spasmogenic molecule.

DISCUSSION

In conclusion, the combination of phylogenetic, structural,biophysical, and biological analyses point at a preservation ofstructures crucial for antimicrobial activity of C3a in inverte-brates as well as vertebrate lineages. Although speculative, thedifference noted in C3a of jawless fishes, likely abolishing theantimicrobial activity of C3a, may not only reflect the multi-functional role of C3a but could also indicate a gain of otherfunctions during the separation from invertebrates in theordovician period (500million years ago). The observation thatanaphylatoxins have unforeseen biological effects in fish iscompatible with this hypothesis (30). Asmentioned previously,significant changes in cationicity are observed in defensins as

FIGURE 7. Activities of human C3a, C4a, and C5a and related C-terminal peptides. A, activities of C3a, C4a, and C5a (as desArg) on P. aeruginosa 27.1 andE. faecalis 2374. In viable count assays, C3 and C4a (but not C5a) displayed antibacterial activities against both bacterial isolates. 2 " 106 colony-formingunits/ml of bacteria were incubated in 50 $l of peptides at a concentration of 3 $M. B, antibacterial activities of synthetic peptides derived from the C-terminalregions of C3a, C4a, and C5a. P. aeruginosa, E. coli, B. subtilis and C. albicans (4 " 106 colony-forming units) were inoculated in 0.1% TSB-agarose gel. Each4-mm-diameter well was loaded with 6 $l of the peptides CNY21, CQF21, or CVV20 (100 $M) derived from the C terminus of C3a, C4a, and C5a, respectively. Thezones of clearance correspond to the inhibitory effect of each peptide after incubation at 37 °C for 18 –24 h. Antibacterial activity is indicated (y-axis). Meanvalues and S.D. are presented (n % 3). For comparison, LL-37 was used. The inset illustrates the antibacterial effects of the peptides against P. aeruginosa.C, binding of TAMRA-labeled peptides to P. aeruginosa 27.1 and inhibition of binding by excess of heparin. The lower panels show red fluorescence ofbacteria (1 " 107 ml'1) stained with the indicated TAMRA-conjugated peptides (10 $g ml'1) in the absence or presence of heparin. All images were recordedusing identical instrument settings. The corresponding Nomarski images are shown in the upper panel. Scale bar represents 10 $m.

Structural Conservation of Antimicrobial C3a

JANUARY 26, 2007 • VOLUME 282 • NUMBER 4 JOURNAL OF BIOLOGICAL CHEMISTRY 2527

by guest on March 1, 2014

http://ww

w.jbc.org/

Dow

nloaded from

well as cathelicidins, even in closely related species (such asprimates) (11, 31). Furthermore, indicative of a strong non-neutral evolution, gene duplications and subsequent variationshave led to further generation of different AMPs (29). Forexample, the C-terminal domains of cathelicidins may rangefrom a dozen to over eighty residues forming cysteine-bridgedhairpins (bovine bactenecin) and tryptophan-rich (bovineindolicidin), proline-rich (porcine PR-39), or !-helical mole-cules (human LL-37) (8). In this context, it is interesting to notethat a unifying structural motif (&-core) was recently revealedin many diverse AMPs, such as protegrins, defensins, and che-mokines, thus indicating the existence of previously unprece-dented higher level structural motifs that govern antimicrobialfunctions (6). Being multifunctional, also acting as immunemodulators, the generation of many structurally diverse AMPslikely reflects an evolutionary adaptation to the microbial envi-ronment as well as introduction of novel biological functions ina given organism.Contrasting to this, C3a hasmaintained strik-ingly similar features governing antimicrobial activity, despite asignificant primary sequence variation. Thus, in addition to the“innovative” generation of AMPs in nature, our findings onC3aillustrate an alternative concept where molecules are subjectedto strong and precise selection forces aiming at maintaining arobust and structurally intact innate defense system.

Acknowledgment—We thank Lotta Wahlberg for expert technicalassistance.

REFERENCES1. Hugli, T. E. (1990) Curr. Top. Microbiol. Immunol. 153, 181–2082. Nordahl, E. A., Rydengård, V., Nyberg, P., Nitsche, D. P., Morgelin, M.,

Malmsten, M., Bjorck, L., and Schmidtchen, A. (2004) Proc. Natl. Acad.Sci. U. S. A. 101, 16879–16884

3. Alper, C. A. (1998) Exp. Clin. Immunogenet. 15, 203–2124. Wessels, M. R., Butko, P., Ma, M., Warren, H. B., Lage, A. L., and Carroll,

M. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11490–114945. Kerr, A. R., Paterson, G. K., Riboldi-Tunnicliffe, A., and Mitchell, T. J.

(2005) Infect. Immun. 73, 4245–42526. Yount, N. Y., Bayer, A. S., Xiong, Y. Q., and Yeaman,M. R. (2006) Biopoly-

mers 84, 435–4587. Zasloff, M. (2002) Nature 415, 389–3958. Lehrer, R. I., and Ganz, T. (2002) Curr. Opin. Hematol. 9, 18–229. Maxwell, A. I., Morrison, G.M., and Dorin, J. R. (2003)Mol. Immunol. 40,

413–42110. Tomasinsig, L., and Zanetti, M. (2005) Curr. Protein Pept. Sci. 6, 23–3411. Zelezetsky, I., Pontillo, A., Puzzi, L., Antcheva, N., Segat, L., Pacor, S.,

Crovella, S., and Tossi, A. (2006) J. Biol. Chem. 281, 19861–1987112. Zarkadis, I. K., Mastellos, D., and Lambris, J. D. (2001)Dev. Comp. Immu-

nol. 25, 745–76213. Pinto, M. R., Chinnici, C. M., Kimura, Y., Melillo, D., Marino, R., Spruce,

L. A., De Santis, R., Parrinello, N., and Lambris, J. D. (2003) J. Immunol.171, 5521–5528

14. Zhu, Y., Thangamani, S., Ho, B., and Ding, J. L. (2005) EMBO J. 24,382–394

15. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller,W., and Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389–3402

16. Thompson, J. D., Higgins, D.G., andGibson, T. J. (1994)Nucleic Acids Res.22, 4673–4680

17. Henikoff, S., and Henikoff, J. G. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,10915–10919

18. Poirot, O., Suhre, K., Abergel, C., O’Toole, E., and Notredame, C. (2004)Nucleic Acids Res. 32, (suppl.) W37–W40

19. Saitou, N., and Nei, M. (1987)Mol. Biol. Evol. 4, 406–42520. Tennessen, J. A. (2005) J. Mol. Evol. 61, 445–45521. Janssen, B. J., Huizinga, E. G., Raaijmakers, H. C., Roos, A., Daha, M. R.,

Nilsson-Ekdahl, K., Nilsson, B., and Gros, P. (2005) Nature 437, 505–51122. Jacobson,M. P., Pincus, D. L., Rapp, C. S., Day, T. J., Honig, B., Shaw, D. E.,

and Friesner, R. A. (2004) Proteins 55, 351–36723. Lehrer, R. I., Rosenman, M., Harwig, S. S., Jackson, R., and Eisenhauer, P.

(1991) J. Immunol. Methods 137, 167–17324. Nonaka, M., and Yoshizaki, F. (2004)Mol. Immunol. 40, 897–90225. Huber, R., Scholze, H., Paques, E. P., and Deisenhofer, J. (1980) Hoppe-

Seyler’s Z. Physiol. Chem. 361, 1389–139926. Williamson,M. P., andMadison, V. S. (1990) Biochemistry 29, 2895–290527. Zhang,X., Boyar,W., Toth,M. J.,Wennogle, L., andGonnella,N.C. (1997)

Proteins 28, 261–26728. Zelezetsky, I., and Tossi, A. (2006) Biochim. Biophys. Acta 1758,

1436–144929. Tennessen, J. A. (2005) J. Evol. Biol. 18, 1387–139430. Sunyer, J. O., Boshra, H., and Li, J. (2005) Vet. Immunol. Immunopathol.

108, 77–8931. Semple, C. A., Rolfe, M., and Dorin, J. R. (2003) Genome Biol. 4, R31

Structural Conservation of Antimicrobial C3a

2528 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 4 • JANUARY 26, 2007

by guest on March 1, 2014

http://ww

w.jbc.org/

Dow

nloaded from