Published as doi: 10.1096/fj.08-114579.
(The FASEB Journal. 2009;23:13-20.)
© 2009 FASEB
A fossil antibacterial peptide gives clues to structural diversity of cathelicidin-derived host defense peptides
Shunyi Zhu1 and
Bin Gao
Group of Animal Innate Immunity, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
1 Correspondence: Datun Rd., Chaoyang District, Beijing 100101, China. E-mail: zhusy{at}ioz.ac.cn
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ABSTRACT
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Cathelicidins, a group of vertebrate- specific immune effector molecules, developed into a multigene family through gene duplication in the Cetartiodactyla lineage, in which structural changes of the antimicrobial domains (AMDs) among paralogs during evolution are clearly associated with functional diversification of cathelicidins. However, the evolutionary mechanism for such structural diversity remains largely unsolved. Although at the genomic level, the 3'-exon alone encoding the variable AMD repertoire would favor a role of exon shuffling, the observation that two structurally unrelated subfamilies of AMDs (protegrins and prophenins) display high similarity in some nucleotide regions of the 3'-exons of their genes provides evidence for postduplication sequence remodeling. This opinion is further strengthened by finding a proline-arginine-rich antibacterial peptide of 38 residues (named PR-38) encoded by an open reading frame located in the 3' untranslated region of protegrins, which is in frame with prophenins. The latter appears to have undergone internal motif repeats in the region corresponding to PR-38. PR-38 exhibits similar amino acid sequence and carboxyl-terminal amidation feature to PR-39s, a subfamily of cathelicidin peptides conserved across Cetartiodactyla. Functional assays of the chemically synthesized PR-38 confirmed its antibacterial activity with a similar action mode to PR-39 against both gram-positive and gram-negative bacteria at micromolar concentrations, supporting an ancient functional link among cathelicidin members belonging to different subfamilies. Resurrecting the fossil peptide highlights the evolutionary position of PR-39 in generating structurally variable subfamilies of porcine cathelicidins through postduplication sequence remodeling.—Zhu, S., Gao, B. A fossil antibacterial peptide gives clues to structural diversity of cathelicidin-derived host defense peptides.
Key Words: functional divergence • gene duplication • innate immunity • exon shuffling • Cetartiodactyla • sequence remodeling
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STRUCTURAL DIVERSITY OF CATHELICIDIN-DERIVED PEPTIDES
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HOST DEFENSE PEPTIDES (HDPs) of the cathelicidin family are essential components of vertebrate immune response. These immune molecules were initially identified as natural peptide antibiotics with a broad spectrum of microbicidal activities against various bacteria, fungi, and viruses (1
2
3)
. Recently, some of them have been found to possess other functions beyond direct antimicrobial effects, such as immunomodulatory activities through inducing proinflammatory cytokine production and modulating the responses of adaptive immune cells (4)
. Their protective roles have also been evidenced by the cathelicidin-knockout mice showing increased susceptibility to pathogenic bacteria (5
, 6)
and the expression of a porcine cathelicidin peptide (PR-39) in mice providing protection against bacterial skin infection (7)
.
A notable feature of the cathelicidin family is that the number of different genes varies greatly among species (8)
. For example, primates have only one cathelicidin gene, whereas in other mammals belonging to the clade Euungulata (Cetartiodactyla and Perissodactyla), gene duplications frequently occurred to generate cathelicidin multigene families (3)
that often exhibit unique bipartite features, as identified by their structurally diverse carboxyl-terminal antimicrobial domain (AMD) (i.e., HDP) of 12–100 residues linked to a conserved amino-terminal cathelin-like domain (CLD) of 99–114 residues (8
, 9)
. Despite the high degree of sequence conservation, several sites of CLD have been found to undergo adaptive evolution to produce activating effects on cathepsin L (8
, 9)
. In contrast, AMD varied largely in size and fold type. For example, pigs belonging to Cetartiodactyla have 11 paralogous cathelicidins that have diversified their AMDs into 3 distinct structural subfamilies, i.e., linear 
-helical peptides such as PMAP-37, PMAP-36, and PMAP-23; peptides with disulfide bridges and β-sheet structures, such as protegrin-1 to protegrin-5 (PG-1 to PG-5); and peptides enriched for specific amino acids, such as prophenin-1 (PF-1), prophenin-2 (PF-2), and PR-39 (Fig. 1
). These three structural classes are also typical representatives of all known antimicrobial peptides isolated from multicellular organisms (10
11
12)
. Interestingly, the -helical cathelicidins are the only one structural class conserved throughout vertebrate evolution, supporting their early emergence during evolution (2
, 8)
.
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Figure 1. Structural diversity of AMDs encoded by the 3'-exons of paralogous cathelicidins arose from gene duplication in the pig lineage. A) Precursor organization and gene structure of 3 subfamilies of the porcine cathelicidins. B) Structural diversity of AMDs. The structure of PMAP-37 was predicted based on the solution structure of the rabbit cathelicidin peptide CAP18 (pdb entry 1LYP) through homology modeling generated with programs TITO and MODELLER. Models were evaluated by Verify3D and PROSA (http://bioserv.cbs.cnrs.fr/). The ab initio structure of PF-2 was predicted using Robetta (http://robetta.bakerlab.org/). The 3-dimensional models of PMAP-37 and PF-2 have been submitted to the Protein Model database (http://mi.caspur.it/PMDB/) under identification numbers PM0075252 and PM0075253, respectively. The pdb entry of PG-1 is 1PG1 (http://www.ncbi.nlm.nih.gov/structure/index. shtml).
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Although structural changes in the AMDs of duplicated cathelicidins can provide the host a multidrug and synergistic defense system by acting as pore formers (e.g., protegrins) and metabolic inhibitors (e.g., PR-39) (13
, 14)
, how the structurally diverse AMDs originated following gene duplication remains obscure. In this work, we present evidence based on a combined analyses of sequences and functional data in support of postduplication protein remodeling rather than exon shuffling as a major mechanism shaping the AMD repertoire of a multigene family of cathelicidins.
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SEQUENCE SIMILARITY AND GENE STRUCTURE DISFAVORING THE INVOLVEMENT OF EXON SHUFFLING
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As a major evolutionary mechanism involved in the creation of new genes with dramatic structural and functional changes, intronic recombination-mediated exon shuffling accounts for the formation of 
19% of exons in eukaryotic genes (15)
. Such a mechanism has been proposed to explain the evolution of AMD structural diversity due to the existence of a correspondence between AMDs and their coding exons (3
, 11
, 16)
. As shown in Fig. 1
, the AMD lies in a single exon (3'-exon) and in this case, a preexisting and autonomous antimicrobial module is presumably excised and reintegrated downstream CLD of a duplicated cathelicidin gene through 3'-intron-mediated nonhomologous recombination. Obviously, this type of recombination of exons cannot be considered as classical exon shuffling in that in this case the so-called shuffled AMDs are not flanked by two introns with identical phases (Fig. 1)
(17
18
19)
. If one assumes that this process occurs, it should be expected that genetically unrelated 3'-exons will be generated among different paralogous genes. However, as observed previously (20)
, two distinct cathelicidin subfamilies (protegrins and prophenins) with completely different AMD structural types display relatively high sequence similarity at the nucleotide level in their 3'-exons (Fig. 2
, PF-2, and PG-3 used here as representatives considering high sequence similarity within subfamilies). With the exception of the protegrin-coding region that bears no resemblance to the corresponding region of prophenins, other alignable parts of their 3'-exons share 95% sequence identity. Such similarity cannot be accounted for in terms of this nonclassical exon shuffling of autonomous module. Besides containing all of the AMD region of the cathelicidin precursor, the 3'-exon also encodes the conserved residues involved in post-translational processing (Fig. 2)
downstream of the hypothetical shuffling site, again not in agreement with this hypothesis. Instead, the protegrin-coding region could be considered as an independent shuffled module to insert into a gene originally specifying prophenin by a classical exon shuffling event (20)
. However, this mechanism appears to be less likely due to the lack of symmetric introns in the flanking region of the protegrin-coding sequence. Moreover, this proposed shuffled domain has only 19 residues that are too small in size when compared with a typical domain of 100 to 250 residues (21)
. Further evidence comes from BLAST searches of the GenBank database (http://www.ncbi.nlm.nih.gov), which found no similar nucleotide sequences in this database when using protegrins as queries, implying that protegrin-coding region could generate de novo in the pig lineage.
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Figure 2. A) Sequence comparison of PG-3 and PF-2 with their 3'-exons emphasized. Internal repeat motifs are lined once in gray. Residues for C-terminal amidation (RCA) are boxed. Identical nucleotides and amino acids are shadowed in yellow, and italicized and underlined once, respectively. Asterisk (*) indicates motifs used for chemical synthesis. Note that the PG-3-coding region and the corresponding region in PF-2 cannot be aligned due to the absence of detectable sequence similarity. E, exon; I, intron. B) Schematic representation of 3'-exons of PG-3 and PF-2. PS, processing signal; TRR, 13 residues region; 3'UTR, 3' untranslated region. Colors in A and B are identical.
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DISCOVERY AND RESURRECTION OF A FOSSIL ANTIBACTERIAL PEPTIDE HIGHLIGHTING THE ROLE OF PR-39 IN THE ORIGINATION OF PROTEGRINS AND PROPHENINS
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To investigate the mechanism that diversified the different subfamilies of cathelicidins after gene duplication, a detailed analysis of the 3'-exons of protegrins and prophenins was performed, which allowed us to identify an open reading frame (ORF) immediately following the stop codon of protegrins (Fig. 2)
. This ORF codes for a proline-arginine-rich peptide of 38 residues (RLRRQAFPPPIFPGPGFRPPIFPPPPFRPAPFGPPRFP-NH2, named PR-38) with the carboxyl terminus amidated and in frame with prophenins. The region of prophenins corresponding to PR-38 appears to have undergone internal motif repeats of a proline-rich decamer with a consensus of FPPPN/IF/VPGGR/P/W. Relative to PR-38 that contains only 1.5 repeat motifs (R1 and R2N), prophenins have six such motifs (R1–R6).
Interestingly, PR-38 exhibits marked amino acid sequence similarity to PR-39s (Fig. 3A
), a subfamily of proline-arginine-rich cathelicidin peptides conserved across Cetartiodactyla, and they both have an amidated carboxyl terminus. To further evaluate the evolutionary relationship of PR-38 and PR-39, we examined the 3'-exon sequences of protegrins (PG-3 as a representative) and PR-39 (Fig. 3B
). We found that except for the absence of a sequence corresponding to the protegrin-coding region in PR-39, their nucleotide and predicted amino acid sequences can be well aligned, with detectable similarity extending to their 3' untranslated regions (UTRs) (Fig. 3B
). These analyses suggest a clear paralogous relationship among the 3'-exons of protegrins, prophenins, and PR-39. This relationship is consistent with their genomic location in which the genes for protegrins and prophenins are closely linked to the gene for PR-39, all mapped on chromosome 13 (22)
.
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Figure 3. A) Multiple-sequence alignment of PR-38 and PR-39, all derived from mammals belonging to Cetartiodactyla. Identical amino acids between PR-38(PG-3) (PR-38 from PG-3) and other related peptides are shadowed in yellow. Ch, Capra hircus (goat); Oa, Ovis aries (sheep); Bt, Bos taurus (cow). B) Sequence comparison of 3'-exons of PG-3 and PR-39. Identical nucleotides or amino acids are shadowed in yellow or italicized and underlined once. 38/39, PR-38/PR-39; PS, processing signal; RCA, residues for C-terminal amidation.
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The recognition of PR-38, encoded by an ORF embedded in the 3'UTR of protegrins, as a paralog of the PR-39 subfamily offers a chance to evaluate an evolutionary link at the ancient functional level among different cathelicidin subfamilies. Sequence analysis of PR-38 revealed that it could still remain ancient antimicrobial activity owing to strong positively charged feature (pI=12.70) (23)
and a high proline content (42%) (24)
. In particular, it has two conserved positively charged residues in its amino terminus that has been found to be important for the function of PR-39 (25)
. We chemically synthesized PR-38 (Supplemental Fig. S1) and tested its antibacterial and antifungal activities by classical inhibition zone assays (26)
. Micrococcus luteus, Bacillus megaterium, Bacillus sp. DM-1, Agrobacterium tumefaciens, Escherichia coli, Shewanella oneidensis, Stenotrophomonus sp. YC-1, Salmonella typhimurium, Geotrichum candidum, Neurospora crassa, and Saccharomyces cerevisiae were used as target microorganisms. As described in Fig. 4A
, PR-38 displayed clear antibacterial activity against two gram-positive strains such as B. megaterium and Bacillus sp. DM-1 and two gram-negative strains, such as S. oneidensis and Stenotrophomonus sp. YC-1 at micromolar concentrations. The control 0.1% trifluoroacetic acid (TFA), which was used to dissolve the peptide, had no effect. PR-38 and PR-39 both are highly active on B. megaterium with a compatible lethal concentration (CL) (0.3 vs. 1.04 µM) (27)
. The absence of activity against E. coli and S. typhimurium, two bacterial species susceptible to PR-39, might reflect the ancient feature of PR-38, which might have been lost during evolution after becoming a molecular fossil. In addition, we evaluated the antimicrobial activity of two synthesized repeat motifs (R1 of PG-3 and R1 of PF2) (Supplemental Fig. S1), which displayed no effect on all of the microbial organisms used in this study, suggesting that these motifs represent nonfunctional elements, in contrast to some short repetitive motifs in several intrinsically unstructured proteins, in which they carry fundamental function (28)
.
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Figure 4. Functional characterization of PR-38. A) Antimicrobial activity. The inhibition zone assay and calculation of lethal concentration (CL) were performed according to the previously described methods by Hultmark’s method (26)
. LB medium and MEA medium, both containing 0.8% agarose, were used for bacterial or fungal growth, respectively. For sources of microorganisms used, see Supplemental Table S1. B) Permeabilization assays, performed according to the method of Amino et al. (40)
with some modifications. B. megaterium was incubated in LB medium to OD600 = 0.5. Then 100 µl of the culture was taken, and PR-38 or meucin-13 was added to a final concentration of 10 µM. After incubation for 30 min at 30°C, propidium iodide was added to a final concentration of 10 µg/ml, and cells were immediately photographed in a phase contrast/fluorescence microscope after washing 3 times with PBS. C) Hemolysis assay. The hemolytic activity of PR-38 and meucin-13 against fresh rabbit blood was assayed according to standard method (41)
. The percentage of hemolysis was determined as (Apep–Ablank)/(Atot–Ablank) x 100, where Apep is absorbance in the presence of the peptide, measured at 570 nm; Ablank is absorbance evaluated in the absence of the peptide; and Atot is absorbance at 100% hemolysis in the presence of 1% Triton X-100). Meucin-13, a scorpion venom pore-forming peptide, was used as a control (this peptide will be published elsewhere). Peptides, including PR-38, R1(PR-38), and R1/R2 (PF-2), were synthesized by Xi’an Huachen Bio-Technology Co., Ltd (Xi An, China) with more than 95% purity confirmed by HPLC and MALDI-TOF mass spectrometry (Supplemental Fig. S1). All peptides were dissolved in 0.1% trifluoroacetic acid (TFA).
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To determine whether PR-38 has similar action mode to PR-39, we studied its effect of membrane permeabilization on the highly susceptible bacterium B. megaterium by observing the uptake of propidium iodide, a classical DNA-binding dye that is excluded from cells with normal membrane integrity and can enter cells with increased membrane permeabilization (29)
. The results showed that the propidium iodide was not able to enter the PR-38-treated B. megaterium cells, suggesting its nonmembranolytic action mode (Fig. 4B
), in agreement with that of PR-39 that inhibits the growth of cells by interrupting both DNA and protein synthesis (13
, 30)
. Our observation thus supports the metabolic inhibition rather than pore-forming effect of PR-38 on the susceptible bacteria. In addition, PR-38 exhibits a nonhemolytic effect on fresh rabbit blood (Fig. 4C
), a typical feature of nonmembranolytic HDPs.
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DYNAMIC REMODELING
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Although a paralogous relationship among the 3'-exons of protegrins, prophenins, and PR-39 has been established, how did sequence remodeling change their structural types during evolution? To answer this question, the evolutionary history of these paralogs needs to be elucidated. In a previous study, it was proposed that the prophenin gene being the ancestor molecule and protegrin was evolved after inserting a hypothesized peptide-coding exon into prophenin by exon shuffling (20)
. However, as discussed above, this proposal is not supported by the gene structure of the protegrin domain (17
18
19
, 31)
. Moreover, the model requires a complicated process to accurately delete the 4.5 tandem repeat motifs (R2C, R3-R6) of prophenin and to replace its 13-residue region (TRR) by the protegrin-coding region (Fig. 2)
. Importantly, when only two sequences were compared, it is difficult to define which is the more ancient molecule due to the absence of a suitable outgroup.
Resurrecting the fossil peptide establishes an ancient functional, hence evolutionary, link among these structurally diverse subfamilies of cathelicidins. The distribution of the PR-39 subfamily throughout Cetartiodactyla, relative to the restricted distribution of the subfamilies of protegrin and prophenin to the pig lineage, suggests that PR39 is a more ancient progenitor molecule and can thus be taken as an outgroup to elucidate the evolutionary process of protegrins and prophenins (Fig. 5
). In this case, we can predict that a PR-39-like gene generated from an early gene duplication of PR-39 after the separation of the pig lineage from other Cetartiodactyla at 65 million years ago underwent a late duplication and formed two daughter genes, one of which later became protegrin after gaining an insertion sequence close upstream of the coding region of PR-39-like peptide, which finally became the fossil molecule (PR-38). In another daughter gene, the original peptide constitutes a part of prophenins through sequence insertion together with internal motif repeat. Thus, in these two cases, the duplicated daughter genes could independently obtain the short unrelated sequences immediately following the processing signal (Fig. 2)
. Obviously, this process was selected to ensure the release of mature peptides.
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Figure 5. A possible evolutionary route of different subfamilies of the cathelicidins. A) Two rounds of gene duplications of the PR-39 ancestral gene are discussed in the framework of the tree of the PR-39 subfamily. The neighbor-joining method implemented in MEGA 3.1 (http://www.megasoftware.net/) was selected to construct an unrooted tree based on the aligned amino acid sequences presented in Fig. 3A
, with >70% bootstrap values shown at the nodes. ED, early duplication; LD, late duplication; SP, signal peptide; CLD, cathelin-like domain; IS, insertion sequence; 38/39, PR-38/PR-39; IR, internal repeat. B) Phylogenetic distribution of PR-39 and other two evolutionarily related cathelicidin subfamilies, mapped onto the species tree with speciation time shown at the nodes (42)
. +, presence; –, absence. For a detailed description, see Dynamic Remodeling in the text.
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The nearly complete conservation in some regions of 3'-exons of protegrins and prophenins (Fig. 2)
indicates that they diverged very recently, and sequence degeneration did not occur in such a short evolutionary time. This explains the insertions of the protegin-coding region and TRR as a result of the independent event rather than rapid mutation accumulation. However, how did the inserted sequence in the protegrin evolve into a new AMD to replace the original PR-39-like HDP? Selection could have acted on the subsequent evolutionary process. As we know, protegrins and some invertebrate antimicrobial peptides [e.g., spider gomesin and scorpion androctonin (12
, 32)
] constitute an superfamily due to structural similarity, all members of which is composed of two-stranded antiparallel β-sheet connected by two internal disulfide bridges. Obviously, these peptides are evolutionarily unrelated in that they significantly differ in sequence and disulfide bridge position. In this superfamily, the amphipathic design other than sequence conservation as a key determinant of antibacterial activity, indicating that ab initio evolution of such a molecule with the amphipathic architecture, could be easily fulfilled. A similar evolutionary strategy has also been proposed to trace the origin of cathelicidins from the cystatin scaffold, in which an -helical HDP is presumably generated de novo from the noncoding genomic DNA (33)
.
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CONCLUSIONS
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Gene duplication represents a major mechanism for evolutionary innovation of a multigene family through providing organisms genetic raw materials on which selection can act to fix advantageous mutations (34
, 35)
. Examples of immune-related molecules include the highly diverse mammalian - or β-defensin family, which both were generated through gene duplications and subsequent rapid sequence divergence during evolution (36
, 37)
. Some recent studies also highlight the fundamental importance of species-specific gene duplication in species-specific adaptation, as exemplified in the contribution of the RNase1 duplication in a leaf-eating monkey toward its adaptation to changing digestive environments (34)
. In contrast to these defensins and RNase1 that evolved new functions through positive Darwinian selection-driven amino acid substitutions in a conserved structural scaffold, paralogous cathelicidins have diversified their AMDs into distinct structural subfamilies by sequence remodeling in the Cetartiodactyla lineage. This poses a challenge for a prevailing viewpoint, that is, dramatic changes in AMD structural types are a consequence of exon-shuffling (3
, 11
, 16)
.
In fact, sequence mutation-mediated structural and functional change of proteins has also been observed in other immune-related molecules. For instance, the rodent-specific C-terminal frameshift mutation in the duplicated NKG2 natural killer cell receptor clearly links to the functional switch of this family from inhibition to activation by altering the immunoreceptor tyrosine-based inhibitory motif (ITIM) (38)
. In addition, evolutionary emergence of a premature stop codon in duplicated -defensins of Old World Monkeys led to the generation of the 
-defensins with unusual structural and functional features (39)
. In these examples, gene duplication-mediated structural diversity of immune molecules appears to frequently occur in a lineage-specific manner. If we believe that selective pressure from pathogens may have prompted such lineage-specific gene duplication and subsequent structural divergence of these molecules, probing evolutionary mechanisms responsible for such diversity will be undoubtedly helpful in further understanding the role of gene duplication in species-specific immunological adaptation.
Finally, it is worth mentioning that although sequence remodeling has been highlighted as an important mechanism for structural alterations of cathelicidins between peptides with β-sheet structures and peptides enriched for specific amino acids, how -helical AMDs, the firstly emerged domain, develop into other structural types following gene duplication remains an open question.
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ACKNOWLEDGMENTS
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This work was supported by the Bairen Plan from the Chinese Academy of Sciences to S.Z.
Received for publication July 13, 2008.
Accepted for publication August 28, 2008.
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ABSTRACT
STRUCTURAL DIVERSITY OF...
