Signal Sequence Conservation and Mature Peptide Divergence Within Subgroups of the Murine ß-Defensin Gene Family

Gillian M. Morrison, Colin A. M. Semple, Fiona M. Kilanowski, Robert E. Hill and Julia R. Dorin

MRC Human Genetics Unit, Western General Hospital, Edinburgh, Scotland


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
ß-defensins are two exon genes which encode broad spectrum antimicrobial cationic peptides. We have analyzed the largest murine cluster of these genes which localizes to chromosome 8. Using hidden Markov models, we identified six ß-defensin exon 2–like sequences and subsequently found full-length expressed transcripts for these novel genes. Expression was high in brain and reproductive tissues. Eleven ß-defensins could be grouped into two clear subgroups by virtue of their position and high signal sequence (exon 1 encoded) identity. In contrast, however, there was a very low level of sequence conservation in the exon 2 region encoding the mature antimicrobial peptide. Examination of the gene sequences of orthologs in other rodents also revealed an excess of nucleotide changes that altered amino acids in the mature peptide region. Evolutionary analysis revealed strong evidence that following gene duplication, exon 1 and surrounding noncoding DNA show little divergence within subgroups. The focus for rapid sequence divergence is localized in the DNA encoding the mature peptide and this is driven by accelerated positive selection. This mechanism of evolution is consistent with the role of this gene family as defense against bacterial pathogens and the sequence changes have implications for novel antibiotic design.

Key Words: adaptive evolution • ß-defensin • positive selection • Mus musculus


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
Defensins are antimicrobial cationic peptides of which three groups have been described in vertebrates. The {alpha} and ß forms are distinguishable by the spacing and connectivity of the conserved cysteine residues within the mature peptide (Ganz and Lehrer 1994). The ß-defensins are two exon genes which code for a prepropeptide that includes a signal sequence, a short propiece, and a mature carboxy-terminal peptide, which is liberated by proteolytic cleavage (Lehrer and Ganz 2002). The signal sequence is exon 1 encoded and the pro and mature peptides are coded for by the second exon. Recent studies have shown ß-defensins to be present in the airway epithelia of several species, including humans, rodents, and cattle where they contribute to the host defense system by the eradication of pathogens at the mucosal surface.

Four human ß-defensins have been functionally analyzed, and all of them have displayed antimicrobial activity in vitro (Lehrer and Ganz 2002). Interestingly, several human ß-defensins also display chemoattractant activity for immature dendritic cells and memory T-cells, suggesting that these peptides may also function as an important link between the innate and adaptive immune system (Yang et al. 1999; Garcia et al. 2001). Until recently, only eight murine ß-defensin genes had been identified, all mapping to a region of conserved synteny with the human locus on chromosome 8 (Huttner, Kozak, and Bevins 1997; Bals, Goldman, and Wilson 1998; Morrison et al. 1998; Morrison, Davidson, and Dorin 1999; Jia et al. 2000; Bauer et al. 2001; Yamaguchi et al. 2001). Synthetic murine ß-defensin peptides also possess antimicrobial activity (Bals, Goldman, and Wilson 1998; Morrison et al. 1998; Bals et al. 1999; Bauer et al. 2001; Yamaguchi et al. 2001; Morrison et al. 2002) demonstrating the functional conservation of these peptides between mouse and man. However, dramatic differences are evident within the murine ß-defensin gene family in terms of their tissue distribution and responsiveness to inflammatory stimuli (Lehrer and Ganz 2002). We have also reported functional differences within members of the murine ß-defensin gene family in terms of their ability to kill specific pathogens (Morrison et al. 1998, 2002). These results suggest that the murine ß-defensins may have evolved under positive selection to have different profiles of antimicrobial activity and thus maximize the host's ability to fight opportunistic infections.

Here we report the further characterization of the murine ß-defensin gene cluster and investigate the molecular mechanisms responsible for the divergence between these molecules.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
Isolation of BACs Containing ß-Defensins
A murine C57BL/6J BAC library (RPCI-23) was screened using radiolabeled double-stranded 40mers comprising two 24mers with a complementary 8-bp overlap (overgos) specifically designed to the exon 1 regions of the previously identified murine ß-defensins (http://genome.wusl.edu/gsc/overgo). The positive BACs were then screened for the presence of the individual defensin genes by PCR and hybridization with radiolabeled oligonucleotides. The sequences of the PCR primers and hybridization oligonucleotides are detailed in supplementary information.

BAC Sequencing
BACs were sequenced as part of the UK mouse sequencing program (http://mrcseq.har.mrc.ac.uk), and the sequence is freely available.

BAC Sequence Analysis
Sequence generated from the BAC 353A15 and 357B4 was analyzed using NIX, a Web tool designed to allow the viewing of multiple DNA analysis programs (http://www.hgmp.mrc.ac.uk). The sequence was also analyzed for the presence of novel ß-defensins using a calibrated HMMER model (version 2.1.1). The HMMER model was constructed from a ClustalW (version 1.82 with default settings) (Higgins, Thompson, and Gibson 1996) multiple sequence alignment of ß-defensin sequences retrieved from GenBank.

Tissue Expression of Novel Murine ß-Defensin Genes
The RACE protocol was adapted from (Townley et al. 1997), as previously described (Morrison, Davidson, and Dorin 1999), and RT-PCR was carried out as described (Morrison, Davidson, and Dorin 1999). Primer sequences are available in supplementary information.

Phylogenetic Inference and Evolutionary Analysis
Sequences were aligned using ClustalW (version 1.82) (Higgins, Thompson, and Gibson 1996), and gapped positions were omitted from subsequent analyses. The phylogenetic tree was constructed by neighbor-joining (Saitou and Nei 1987) based on the proportion of different amino acid sites and was rooted with chicken Gallinacin 1; its reliability was assessed with 1,000 bootstrap replications using MEGA (version 2.1) (Kumar et al. 2001). In pairwise comparisons between nucleotide sequences within subgroups, the number of synonymous substitutions per synonymous site (dS) and the number of nonsynonymous substitutions per nonsynonymous site (dN) were estimated using the Nei-Gojobori test. Standard errors for dS and dN were calculated using 1,000 bootstrap replicates. In addition, the Jukes-Cantor correction (Jukes and Cantor 1969) was applied to account for multiple substitutions at the same site. A one-tailed Fisher's exact test was used to test the null hypothesis that the proportions of synonymous and nonsynonymous differences are the same (Zhang, Kumar, and Nei 1997). All distance calculations and codon-based tests of selection were carried out using MEGA (version 2.1). Estimates of the proportions of radical and conservative nonsynonymous substitutions were made according to the methods of Zhang (2000) using the HON-NEW program. The radical or conservative nature of nonsynonymous substitutions was assessed with respect to charge and to the polarity and volume of the amino acids (the Miyata-Yasunaga amino acid classification) as defined by Miyata, Miyazawa, and Yasunaga (1979). Intron sequences for Defb11, Defb10, and Defb2 were aligned using DIALIGN (version 2.1) (Morgenstern 1999). The numbers of substitutions between the introns were estimated using Kimura's two-parameter method (Kimura 1980).

Sequence Analysis of Exon 2 Within the Rodentia Genus
The exon 2 sequences of nine murine ß-defensin genes, representing members of both subgroups, were amplified from genomic DNA from the inbred Mus musculus mouse lab stains C57Bl/6N, 129/Ola, CBA, and BALB/cJ; from the Mus musculus species Mus musculus domesticus, Mus musculus musculus, Mus musculus castaneous, and Mus musculus hortulanus; from the Mus subspecies Mus spretus, Mus caroli, and Mus pahari; and from the non-Mus species Apodemus sylvaticus and Rattus norvegicus. DNA was purchased from the Jackson Laboratories. The primer sequences are detailed in supplementary information. Genomic samples that failed to generate a PCR product were amplified with at least one set of alternative primers designed specifically towards the murine gene exon 2 sequence. PCR products were sequenced as previously described (Morrison, Davidson, and Dorin 1999) and the resulting sequence compared with that obtained from C57Bl/6 DNA. Isoelectric points (pI) were calculated from predicted mature peptide protein primary sequence using the ExPasy compute pI tool (http://us.expasy.org/tools/pi_tool.html).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
Construction of Two BAC Based Contigs Containing Six Novel and Eight Known ß-Defensin Genes
We have isolated a series of BACs containing the murine chromosome 8 ß-defensin gene family from a C57BL/6J library using overlapping oligonucleotides designed on the previously identified murine ß-defensins. Data from a BAC end sequence database (www.tigr.org), together with the results of the hybridization experiments and PCR, allowed the BACs to be ordered into two contigs separated by approximately 850 kb (fig. 1A). The first contig contained five previously described genes (Defb7 [Bauer et al. 2001] Defb3 [Bals et al. 1999], Defb5 [GenBank accession number AF318068], Defb6 [Yamaguchi et al. 2001], and Defb 4 [Jia et al. 2000]). An additional gene, Defr1, was identified from this contig. It had antimicrobial activity and clear ß-defensin homology, although it was lacking one of the canonical cysteines in the mature peptide (GenBank accession number AJ344114). This gene is 98% identical to Defb8 (Bauer et al. 2001) but has significant changes in the exon 2 sequence, resulting in a tyrosine residue rather than a cysteine residue (Morrison et al. 2002).



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FIG. 1. Genomic organization of the murine defensin genes. (A) Diagrammatic representation of mouse chromosome 8 region A3 showing the relative positions of murine ß-defensin genes. The ß-defensin gene names are abbreviated as follows: 7 = Defb7, R1 = Defr1, 3 = Defb3, 5 = Defb5, 6 = Defb6, and 4 = Defb4. Arrowheads indicate gene orientation. The two BACs selected for sequence analysis, 353A15 and 357B4, are shown. (B) Relative positions of the new ß-defensin genes identified within BAC 357B4. The gene Atp7b, which is not a ß-defensin gene, is also shown

 
The second contig consisted of two overlapping BACs, 353A15 and 357B4, which contained Defb1 and Defb2, respectively (Huttner, Kozak, and Bevins 1997; Bals, Goldman, and Wilson 1998; Morrison et al. 1998; Morrison, Davidson, and Dorin 1999). These BACs were chosen for sequencing because of reduced stringency hybridization experiments that had indicated they contained multiple ß-defensin related sequences (data not shown). Complete sequence was generated from BACs 353A15 and 357B4 by the UK MRC mouse genome sequencing program, funded by the UK Medical Research Council and can be obtained from ftp://ftp.sanger.ac.uk/pub/mouse/ and analyzed using NIX (http://www.hgmp.mrc.ac.uk). This analysis identified the known ß-defensins Defb1 and Defb2 and multiple {alpha}-defensin genes (cryptdins) as expected, in addition to the Cu2+ transporting, b-polypeptide gene, Atp7b, which is not related to the murine defensins. Analysis using the HMMER model described in the methods section resulted in the identification of six novel ß-defensin exon 2–like sequences. Following translation, all of these sequences were found to contain the six canonical cysteine residues within their predicted mature peptide sequence. A recent study described the identification of multiple putative murine ß-defensin sequences also using a computational search strategy (Schutte et al. 2002). The six ß-defensin genes described here were also identified as gene fragments in their publication, and the names given to them were Defb7, Defb11, Defb9, Defb15, Defb13, and Defb35. We name them here following the recently approved HUGO nomenclature for the ß-defensin gene family, respectively, Defb10, Defb15, Defb9, Defb7, Defb13, and Defb35 (fig. 1B). 5' RACE sequence analysis and examination of EST libraries allowed us to identify full-length transcripts for all of these sequences and verify that they are expressed transcripts indicative of functional genes.

Expression Profiles of the Novel Murine ß-Defensin Genes Defb10, Defb11, Defb9, Defb15, Defb13, and Defb35
Full-length transcripts for two of the six sequences were present in the murine EST database. Defb11 was present in a neonate cerebellum and an adult testis expression library and Defb9 was present in an adult hippocampus library. 5' RACE analysis from trachea, testis, and brain tissue resulted in the identification of full-length transcripts for four of these sequences, Defb10, Defb11, Defb15, and Defb13.

Defb10 and Defb11 were found to be expressed in both adult and neonate brain (fig. 2) with weak expression being detected from kidneys, epididymis, and testis after increased numbers of cycles of PCR (data not shown). Defb15 and Defb13 are expressed in the testis and to a lesser extent in the epididymis (fig. 2). Weak expression of Defb15 and Defb13 was detected in the kidneys, and Defb15 was also found to be expressed at low levels in the colon (data not shown). Expression of Defb9 was only detected from brain cDNA after 35 cycles of RT-PCR followed by hybridization of the PCR product with a radiolabeled internal oligonucleotide probe. RT-PCR analysis of Defb35 using primers specific to the putative exon 2 sequence indicated Defb35 to be expressed in the testis and epididymis (fig. 2). RT-PCR using primers from exon 1 to 2 indicated expression also in kidney and neonatal and adult brain (data not shown). The predicted peptide sequences of the genes contained in the two separate contigs are shown in figure 3.



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FIG. 2. Tissue expression of Defb10, Defb11, Defb15, Defb13, and Defb35. RT-PCR of the new murine ß-defensin genes and Hprt (control) from various mouse tissues. All PCR products were verified by sequence analysis. Minus reverse transcriptase control reactions were performed in parallel for all samples and produced no PCR products (data not shown)

 


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FIG. 3. Peptide alignments phylogenetic tree mouse ß-defensins. (A) Alignment of Mus musculus ß-defensin genes from chromosome 8 A3. Subgroups 1 and 2 are indicated. The splice between first and second exons are indicated with an arrowhead, a new arrowhead indicating a change for the sequences below. The signal region and mature peptide regions of Defb1 according to the Swissprot annotation are indicated. Residues conserved in all but one of the ß-defensins are shaded in black. Residues conserved in 80% of subfamily members are shaded in grey. (B) Relationship between mouse ß-defensins only showing clades supported by bootstrap values of 70% and over. The numbers 1 and 2 refer to the subgroups to which the genes have been assigned in the text

 
Evolutionary Analysis of the Murine ß-Defensin Gene Family
A phylogenetic tree was constructed with the 14 validated and expressed murine ß-defensin genes (fig. 3B). Two clusters of genes are evident with Defb6, Defb4, Defb5, Defb3, Defb7, and r1 in one subgroup supported by a 99% bootstrap value and Defb9, Defb2, Defb10, and Defb11 in another subgroup with a statistically significant bootstrap value of 99%. Defb1 probably also falls into this group with a bootstrap value of 94%. We refer to these groups as subgroup 1 and 2, respectively. Defb35, Defb15, and Defb13 are not so strongly supported by bootstrap values as being subgroup members. The separation of the genes into subgroups 1 and 2 also reflects the spatial arrangement of the genes along chromosome 8 (fig. 4), that is, closely related genes being adjacent to each other. In addition the genomic organization (exon 1 length) is the same within subgroup members (fig. 3A).



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FIG. 4. Comparison of the mean identity of distinct regions of the ß-defensin genes and peptide sequences within subgroups 1 and 2. Standard deviations are shown for all values

 
Evidence of Positive Selection Within the Murine ß-Defensin Gene Family
The mean identities of the exon 2 sequences of subgroup 1 and subgroup 2 were significantly lower than the mean identities of the first exons at 65 % (standard deviation [SD] = 10.54) and 62 % (SD = 6.08), respectively (fig. 4). This difference between identities in the different regions of these genes was even more pronounced when the predicted peptide sequences were analyzed. The prepro part of the peptide has mean sequence identities of 86 % (SD = 6.44) and 92 % (SD = 6.6) in subgroup 1 and subgroup 2, respectively, but the predicted mature peptide had mean identities of only 45% (SD = 7.94) and 42 % (SD = 3.6) in subgroup 1 and subgroup 2, respectively. The identity between family members appears to be lost very rapidly after the first cysteine in the mature peptide. In order to characterize the driving force underlying the functional diversification of the murine ß-defensin genes, we tested the role of positive Darwinian selection. Figure 5 shows that when the synonymous (dS) and nonsynonymous (dN) nucleotide substitutions per site are calculated for the exon 2 sequences of paralogous genes within the subfamilies of Mus musculus C57Bl/6, it is clear that for subfamily 1 all except one pairwise comparison (Defr1 and Defb3) gives a dN value greater than the dS value. This implies positive selection, and using the Nei and Gojobori (1986) method, we can show that the excess of nonsynonymous changes is significant (using the Fisher's exact test) between Defb5 versus Defb7 and Defr1 versus Defb7. In subfamily 2, pairwise comparisons of family members with both Defb1 and Defb 9 do not show an excess of nonsynonymous changes. However, in diverging from Defb10 and Defb11, the evolution of the sequence encoding the Defb2 mature peptide has involved an excess of dN relative to dS. We also subjected this observation to rigorous analysis using the Nei and Gojobori (1986) method. The excess of nonsynonymous changes in both subfamilies are significant by the conservative Fisher's exact test of the null hypothesis that the proportions of synonymous and nonsynonymous differences are the same (Zhang, Rosenberg, and Nei 1998) (table 1).



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FIG. 5. Synonymous versus nonsynonymous substitutions within the mouse subgroups 1 and 2. The number of synonymous substitutions per nonsynonymous site (dN) plotted against number of synonymous substitutions per synonymous site (dS) for pairwise comparisons of genes within subgroups 1 and 2. A 45-degree line is drawn so a point above the line indicates dN > dS

 

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Table 1 Estimated Distances Between the Second Exons of Paralogous Genes from Subfamilies 1 and 2.

 
In order to investigate what type of amino acid changes are favored in these genes, we calculated the proportions of radical versus conservative amino acid substitution. The averages for pR (radical nonsynonymous substitution)/pC (conservative nonsynonymous substitution) calculated over 47 mammalian genes were 0.81 for charge and 0.49 for polarity and volume (Miyata-Yasunaga amino acid classification) (Zhang 2000). The equivalent values (shown in table 2) for comparisons between the second exons Defb7 versus Defb5 and Defr1 are greater than 1 with respect to charge but not with respect to polarity and volume. That is, of the nonsynonymous changes observed, most have tended to change the charges of the residues encoded but have tended to conserve the polarities and volumes of those residues. For the Defb2, Defb11, and Defb10 second exons, comparisons the effect is weaker but pR/ pC is still greater for charge than for polarity and volume (table 2).


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Table 2 Estimated Rates of Radical and Conservative Changes for Nonsynonymous Substitutions Between the Second Exons of Genes Displaying Positive Selection.

 
The pI (isoelectric point) of Defb2, Defb10, and Defb11 mature peptides are calculated as 8.66, 9.13, and 7.8, respectively. Thus the change in amino acid content changes the charge quite significantly. This change in charge presumably reflects change in function of these cationic peptides. Only Defb2 peptide has been subjected to functional analysis and it has clear antibacterial function (data not shown). The pI of Defb7 mature peptide is calculated to be 9.59. For the mature Defr1 the pI is 9.26, and for the mature Defb5 it is 9.0. So the charge change is again evident between genes showing positive selection. The antimicrobial activity of Defr1 peptide, despite having a tyrosine rather than the canonical first cysteine, is still potent and is the only murine defensin described to date to be active against Burkholderia cepacia (Morrison et al. 2002).

We aligned the introns of the genes, which showed the evidence for positive selection (see Supplementary Material). The rates of substitution between the introns from Defb2 and Defb11 and between those from Defb2 and Defb10 were estimated as 0.111 ± 0.013 and 0.090 ± 0.012, respectively. The rates of substitution between the introns from Defb7 and Defb5, introns from Defb7 and Defr1, and introns from Defb5 and Defr1 were estimated as 0.104 ± 0.009, 0.240 ± 0.015, and 0.237 ± 0.014, respectively. These values (except for Defr1 and Defb7 comparison) are not significantly different from the rates of synonymous substitution estimated between the second exons of the same pairs of genes given in table 1. Thus the high dN/dS ratio observed between these pairs of exons is not attributable to artificially low dS estimates as a result of sampling error, but is caused by a real increase in dN relative to dS, which is the pattern expected to be generated by positive selection.

Gene Alignments Reveal That the Region of Adaptive Change Is Confined to a Small Portion of Exon 2
ClustalW lineups of Defb2, Defb10, and Defb11 reveal that exon 1 only has one nucleotide change between the three sequences, and the first 200 bp of the intron sequence 3' to exon 1 has 95% identity where all three sequences are present (see Supplementary Material). There are many examples of small deletions from the sequences, and Defb11 and Defb2 have independent deletions relative to Defb10. All three gene have small regions of dinucleotide expansions. Defb10 and Defb11 are recent duplications as shown by the peptide sequence identity and cladogram in figure 3. Within the intron, Defb10 and Defb11 have 98% identity where Defb10 has sequence to compare. In the coding sequence, there are only two amino acid changes in the first 44 residues of the peptide. This is up to the second cysteine of the mature peptide. From that point, however, to the end of the peptide, the DNA identity drops from 90% to only 30%. The amino acid identity between the two genes drops to only 50% in the region that encodes the 15 amino acids between cysteine 2 and 4 in the mature peptides. The dN/dS ratio of the second exon of Defb10 and Defb11 is 1.76 but does not reach significance for positive selection in Fisher's exact test. The intron sequence just 5' to exon 2, where all three sequences align, has 85% identity. Within the first 50 nucleotides of exon 2, the identity across the three genes falls to 66%, (although Defb10 and Defb11 are still 98% identical at the nucleotide level). Thus, the high similarity of these genes in both exon 1, the intron, and 3'and 5' of the first and second exons shows that these genes have recently duplicated.

The ClustalW lineups of Defr1, Defb5, and Defb7 are given in supplementary information and again it is clear that these genes have arisen by recent duplication. The DNA surrounding the exons and exon 1 are 85% identical between the three genes, whereas the exon 2 DNA are only 54% identical.

The ß-Defensin Genes Have Evolved At an Accelerated Rate Since Mouse/Rat Divergence
Southern blot hybridization experiments with mouse and rat genomic DNA indicate the existence of an excess of mouse ß-defensin–like sequences over rat sequences (data not shown), indicative of a high rate of species specific divergence. In order to examine ß-defensin evolution within Rodentia in more detail, we used PCR primers designed to intron sequence and specific for the murine ß-defensin genes Defb1 to Defb6, Defb10, Defb11, and r1 to amplify the corresponding genomic regions from animals of the Mus genus and the non-Mus species (table 3). A negative signal implies the gene is either not present or too divergent to detect using this assay. Based on exon 2 sequence, it appears that Def b1 and Defb4 arose before rat/mouse divergence. The subgroup 1 and 2 genes then arose from these as a consequence of gene duplication in an ancestral species. The genes in subgroup 2, Defb10 and Defb11 arose before Mus divergence from an ancestral species around 8 to10 MYA and Defb2 from a duplication of one of these genes in the common ancestor of M. hortulanus and M. musculus. In subfamily 1, Defb3, Defb4, Defb5, and Defb6 arose by dramatic duplication of an ancestral Defb4 gene before the divergence of M. caroli and other Mus species 4 to 6 MYA. The inability to detect genes in more ancient species—for example, Defb10 being present in M. pahari but not M. caroli—is either due to their divergence rendering them undetectable by PCR or due to the loss of a functional gene in a particular species.


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Table 3 Genomic PCR Analysis of ß-Defensin Exon 2 Sequences in Rodentia.

 
Table 3 shows there are virtually no nucleotide variation between orthologs of the Mus musculus species. The other species tested displayed the highest frequency of coding changes within the mature peptide region. In particular, the Defb10 and Defb11 sequence identified in M. pahari had diverged from the Defb10 and Defb11 sequence present in Mus musculus by 17 and 13 nucleotides, respectively, leading to 13 and nine amino acid substitutions. Table 4 shows that the majority of amino acid changes are located in the mature peptide regions. Full nucleotide sequences are given in supplementary information.


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Table 4 Amino Acid Changes in the Mature Peptide Sequence of ß-Defensin Rodentia Orthologs and the Consequent Change in the pI.

 
Table 5 shows that using the same codon-based test for selection as before, further evidence for the role of positive selection in the evolution of ß-defensins was found. In this case for subfamily 2, positive selection was observed between the second exons of Defb2 sequence found in the lab mouse, M. musculus and M. domesticus versus Defb11 in M. spretus, M. m. hortulanus, and M. caroli. Positive selection was also seen to be acting between Defb2 versus Defb10 in Mus musculus sp., M. spretus, and M. pahari. Defb2 hortulanus showed positive selection against the same genes as Defb2 from the lab mouse. Defb10 M. pahari demonstrated positive selection against Defb11 Mus musculus sp. and M. spretus, in addition to Defb10 Mus musculus sp. and Mus spretus. In subfamily 1, positive selection was found for Defb7 Mus musculus C57Bl/6 versus Defb5 M. spretus and M. caroli. The pI values of the mature peptides are given in table 4. The changes in amino acids results in charge changes in the mature peptides, and Defb10 M. pahari (with a calculated pI of 9.10) shows positive selection between Defb11 (pI of 7.8), Defb11M. spretus (pI of 8.32), and M. m. hortulanus (pI of 7.8). These charge changes are dramatic, but Defb10 M. pahari also shows positive selection versus Defb10 Mus musculus with a very similar pI of 9.13. Conversely, Defb11 has a pI of 7.8 but does not show evidence of positive selection versus Defb11 spretus, although the pI change is to 8.92. It is thus unclear whether charge is the only important feature in changing function in these peptides.


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Table 5 Positive Selection in Exon 2 Between Paralogues and Orthologs Within Rodentia.

 
The only two known rat ß-defensins (Rbd-1 and Rbd-2) were amplified by the primers specific for the mouse genes Defb1 and Defb4, respectively, supporting our belief that Defb1 is orthologous to Rbd-1, and Defb4 is orthologous to Rbd-2. No other rat sequences were amplified in the PCR reactions specific for the other genes.

We recently identified and characterized a murine ß-defensin–like gene named Defensin-related 1 (Defr1) because it does not contain the first of the canonical six cysteine residues characteristic of the ß-defensin family (GenBank accession number AJ344114), however it does display very potent antimicrobial activity in vitro (Morrison et al. 2002). PCR analysis amplified Defr1 exon 2 sequence from C57BL/6 genomic DNA, but no signal was obtained from any of the other genomic samples. The lack of amplification in any other of the DNAs except C57B/6 was also obtained using two other sets of primers designed towards the Defr1 sequence. A product was also produced from C57BL/6 DNA using primers that had 100% identity to the very similar (98% identity) Defb8 gene described by Bauer et al. (2001) but contained mismatches to the Defr1 sequence. However, when this product was sequenced, it had 100% identity to Defr1 and not Defb8. We are forced to conclude that this novel gene arose by gene duplication very recently within the C57Bl/6 inbred strain, after duplication from an ancestral Defb7 gene.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
We report here the characterization of six new murine ß-defensin genes identified from a BAC containing the previously identified ß-defensin Defb2. The expression profile of these novel genes was surprising in that the highest expression of two of the genes, Defb10 and Defb11, was found in adult and neonatal brain. The Defb9 sequence was found in an adult hippocampal EST library, and we found Defb9 to be expressed in adult and neonatal brain tissue but only after an increased number of cycles of PCR, indicating its expression to be weak. It is unclear what functions antimicrobial peptides might have in the brain. The human peptides DEFB1 and DEFB2 have been detected in brain tissue and DEFB2 can be induced by lipopolysaccharide in astrocytes (Hao et al. 2001), which suggests the possibility that ß-defensins may play a role in host defense against bacterial central nervous system pathogenesis where infection would be catastrophic.

The high similarity these peptides share over their first exons (particularly within subgroups 1 and 2), and dissimilarity in their second exons, prompted us to investigate the sequence similarities and disparities of these peptides in more detail.

A phylogenetic tree constructed using all of the known mammalian ß-defensin genes showed species specific clustering (Hughes 1999). Many second exon fragments from known and novel mouse ß-defensins were recently reported by Schutte et al. (2002), but from these data it is impossible to tell which fragments are pseudogenes and which are functioning genes. Although we acknowledge the value of examining pseudogene sequence in determining "birth and death" mechanisms of evolution (Nei, Gu, and Sitnikova 1997), in the present study (because we are primarily interested in function), we restricted our attention to complete gene sequences. These are either previously known or determined from the analysis of the BAC clone based map of the region and verified as encoding real transcripts by RNA analysis.

The existence of independent, species-specific gene clusters suggests that multiple duplications and diversification events have occurred at these loci after mammalian radiation. The increased number of mouse hybridizing fragments compared with rat under reduced stringency conditions implies that gene duplications took place after mouse and rat divergence approximately 40 MYA (Hedges et al. 1996). Our cross-species PCRs indicates that only Defb4 and Defb1 can be found in the rat. The construction of a phylogenetic tree with only the Mus musculus C57Bl/6J sequences separated the genes into subgroups, and it became apparent that within subgroups 1 and 2, there were distinct regional constraints upon the divergence of these sequences. Within subgroups 1 and 2, the genes appear to have risen by recent duplication and the extremely high level of sequence identity in the first exons may be due to this and possibly some level of gene conversion after duplication. However, in marked contrast to exon 1, the exon 2 sequences show rapid divergence as a result of positive selection. Although statistical significance is not achieved for all of the genes showing dN > dS, only Defb1 and Defb9 show dS > dN. Intriguingly, primate ß-defensin 1 orthologs (Del Pero et al. 2002), in common with the mouse Defb1 gene, also demonstrate neutral rather than adaptive evolution.

In addition to the clear evidence we have for positive Darwinian selection in the M. musculus ß-defensin genes, there is also evidence that the subgroup evolved relatively recently by a "birth and death" mechanism (Nei, Gu, and Sitnikova 1997). Rapid "birth and death" and "gene sorting" has recently been described for the rodent eosinophil-associated RNase family. "Gene sorting" is defined as the process leading to differential retention of ancestral genes or gene lineages in different species, where "birth" implies gene duplication and "death" refers to gene deactivation (Zhang, Dyer, and Rosenberg 2000). We postulate that the inability to detect particular genes in certain species is as a result of this process. For example, Defb10 and Defb11 arose by duplication of an ancestral gene in an ancestor of M. pahari. Defb10 was then lost from M. caroli, but in other Mus species, the gene was functionally important and was retained. In addition, it appears that once a gene arises by duplication, rapid change occurs until optimal function arises and is selected so the gene sequence is stabilized and adaptive change is relaxed.

Hill and Hastie (1987) first demonstrated that certain regions of genes can demonstrate an excess of nonsynonymous nucleotide change. Subsequent research has shown that gene duplication and specialization by positive Darwinian selection is a hallmark of vertebrate host-defense, although a few other genes, including those involved in reproduction (Swanson and Vacquier 2002), are also known to be under strong positive selection. Interestingly, the subgroup 1 genes Defb15, Defb13, and Defb35 are strongly expressed in the testis and epididymis, and indeed expression in male testis/epididymis is seen for all the novel genes described here. Recently, a rat gene Bin1b that is exclusively expressed in the caput region of the rat epididymis, and which is responsible for sperm maturation, storage, and protection, was described. Bin1b exhibits structural characteristics and antimicrobial activity similar to that of ß-defensins. Bin1b appears to be a natural epididymis-specific antimicrobial peptide that plays a role in reproductive tract host defense and male fertility, and the mouse defensins we describe may also be involved in these processes (Li et al. 2001).

Interestingly, for functional studies of the nonsynonymous changes seen for subgroup 2, there has been a tendency to change the charge but not the polarity and volume of the amino acids. However, this is not so strong for subgroup 1, where positive selection is observed between Defb7 (pI = 9.59), Defr1 (pI = 9.26), and Defb5 (pI = 9.03). In this subgroup, the ß-defensins appear to remain very cationic, but in subgroup 2, the charge varies from 7.8 to 9.13. Again, the signal peptide remains virtually unchanged between the paralogs. Inefficient secretion may be too damaging to the organism to allow amino acid change. However, we see no evidence for a coordinated change of charge in the prepropeptide, such as is observed with the {alpha}-defensins (Hughes and Yeager 1997). The propeptide region is very small and does not balance the change in charge observed in the mature peptides. In the cross-species analysis, Defb11 shows very rapid accumulation of amino acid changes by positive selection. Independent changes occur within different species. Interestingly, one amino acid (leucine), which is nonpolar, is present in the Defb11 M. pahari sequence and in M. caroli and M. m. hortulanus but is phenylalanine (also nonpolar) in M. spretus and the other strains more closely related to the inbred lines. This amino acid is coded for by three different codons in the three species that have a leucine rather than a phenylalanine. The phenylalanine codon is TTT, and the leucine codons are TTA, TTG, and CTT, respectively, in M. m. hortulanus, M. caroli, and M. pahari. We conclude from these data that this amino acid change must be highly advantageous for functional change, as its change has been selected three independent times.

How have these genes evolved to rapidly accumulate nucleotide changes in the mature region of the peptide and to remain relatively unchanged in the signal peptide and intron? The rapid, accelerated evolution of the exon 2 domain is achieved by positive selection, and the striking sequence similarity between genes adjacent on the chromosome implies recent duplication. After duplication, both relaxation of purifying and the existence of strong positive selection may have had complementary roles in allowing the mature peptide region of duplicate genes to functionally diverge, as recently postulated for the evolution of the ribonuclease gene in a leaf-eating monkey (Zhang, Zhang, and Rosenberg 2002). Our cross-species analysis demonstrates that the duplications of all genes except Defb4 and Defb1 occurred after the divergence of Mus. Thus, the amino acid changes have occurred over a small time scale and imply a strong selective pressure. Bauer et al. (2001) recently showed that Defb7 and Defb8 (Defr1 variant) mature peptides are similar in tertiary structure, despite a high level of sequence change (58% identity). A high level of sequence change must be permissible without loosing antimicrobial function, and change is advantageous to confer pathogen specifity. The rapid evolution of these mouse-specific clades is reminiscent of the morpheus gene family recently described by Johnson et al. (2001), which arose after chromosomal duplication and positive selection during the emergence of humans and African apes.

Functional differences between murine ß-defensins identified to date have been observed (Lehrer and Ganz 2002). Our own data have demonstrated a species-specific antimicrobial action of Defb1 (Morrison et al. 1998; Morrison et al. 2002). We demonstrate here that positive selection acting on ß-defensins can account for the diversity observed between the mature peptide regions of paralogous and orthologous genes. Selection appears to promote changes in the charges of the residues encoded. This rapid adaptive evolution is in keeping with their proposed specialized role in innate immunity diversity and a necessity to respond to pathogen diversity.


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The novel genes in this paper have been submitted to the EMBL Nucleotide Sequence Database and assigned the following accession numbers: AJ437646 (Defb11 gene); AJ437647 (Defb9 gene); AJ437648 (Defb15 gene); AJ437649 (Defb13 gene); AJ437650 (Defb35 gene); and AJ437645 (Defb10 gene). These sequence data were produced by the UK MRC mouse genome sequencing program, funded by the UK Medical Research Council and can be obtained from ftp://ftp.sanger.ac.uk/pub/mouse/.

Intron alignments, gene alignments, primer sequences, and nucleotide sequences from the cross Rodentia gene analysis are included in supplementary information available online only at the journal Web site.


    Acknowledgements
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We would like to thank the UK mouse sequencing program. This work was supported by The Cystic Fibrosis Trust UK, and the Medical Research Council, UK. Thanks to Paul Sharp for initial advice and to Nick Hastie for his support and encouragement.


    Footnotes
 
Naruya Saitou, Associate Editor Back

E-mail: julia{at}hgu.mrc.ac.uk. Back


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Accepted for publication December 3, 2002.