Rapid Evolution by Positive Darwinian Selection in the Extracellular Domain of the Abundant Lymphocyte Protein CD45 in Primates
http://www.100md.com
分子生物学进展 2004年第8期
Department of Biological Anthropology, University of Oxford, United Kingdom
E-mail: nim21@cam.ac.uk.
Abstract
CD45, encoded by PTPRC in humans, is the most abundantly expressed protein on the surface of many lymphocytes. We investigated whether the extracellular region of CD45 was under positive selection in Old World primates, and whether there was differential selection across this region, particularly on exons that were involved in alternative splicing and those that were not alternatively spliced. The results show extraordinarily strong and consistent positive Darwinian selection on the extracellular part of CD45 throughout the evolution of Old World monkeys, apes and humans. Positive selection is concentrated in exons 9 and 14, which code for the previously neglected linker and fibronectin III domains. These exons have a high rate of evolution at nonsynonymous sites that is roughly twice as high as that of the intronic rate in this gene. In contrast, alternatively spliced exons 4–6, which code for the variable domains, are under weaker positive selection and are evolving more slowly than the intronic rate. These data provide a striking example of positive selection in a well-known gene that should provide an impetus for further functional studies to elucidate its species-specific function.
Key Words: positive selection ? lymphocyte ? immune system ? PTPRC
Introduction
The immune system of vertebrates has provided some of the most compelling examples of positive Darwinian evolution at the molecular level. This can be attributed to the strong selective pressures created by the need to respond to rapidly evolving pathogens with short generation times. The best known cases involve major histocompatibility complex (MHC) Class I (Hughes and Nei 1988), MHC Class II (Hughes and Yeager 1998), and immunoglobulin heavy chain (Tanaka and Nei 1989) genes that code for proteins that have a clear role in direct binding of pathogen antigens as part of the specific acquired immune response. Although there is some evidence for rapid evolution of immune system genes in general (Hughes 1997), there are relatively few other genes expressed in the immune system which have been shown to be under positive Darwinian evolution. These include defensins (Hughes and Yeager 1997), eosinophil ribonucleases (Zhang et al. 1998), and CD59 (Osada et al. 2002).
CD45, also known as B220, PTPRC, or the leukocyte common antigen, is a transmembrane protein tyrosine phosphatase that comprises up to 10% of the total surface protein of nucleated hematopoietic cells. It has important functions in B- and T-cell maturation and activation (reviewed in Trowbridge and Thomas 1994). For example, in CD45 null mice, the number of single positive T cells in the periphery is greatly reduced, and the few single positive T cells that reach the periphery show increased affinity for self-MHC complexes, leading to an increased incidence of autoimmune disorders (Trop et al. 2000). Mutations in CD45 have been linked to cases of severe combined immunodeficiency (SCID) in humans (Tchilian et al. 2001).
The extracellular region of CD45 comprises a variable domain, a linker region, and three type III fibronectin domains. The variable domain defines isoforms of CD45 that differ in size and degree of glycosylation. The isoforms are generated by alternative splicing of exons 4–6 and are expressed in a tightly regulated cell-type and stage-specific manner that is highly conserved among vertebrates (Okumura et al. 1996). The extracellular region of CD45 associates with the component of the T-cell receptor (Leitenberg et al. 1996) and is necessary for controlling lck activity (Irles et al. 2003). Signaling properties may vary according to which CD45 isoform is expressed (McKenney et al. 1995). There is still much ignorance about the molecular interactions and function of the extracellular portion of CD45, and, in particular, no function has been ascribed to the linker region and type III fibronectin domains. In contrast, the function of the intracellular region of CD45 is well understood: it has two phosphatase domains that are responsible for dephosphorylating two of the Src family kinases critical to T-cell activation and proliferation, p56lck and p59fyn, and promoting the secretion of cytokines such as interleukin-2 (McKenney et al. 1995). The human gene for CD45 (PTPRC) is on chromosome 1 and comprises 33 exons, of which the first 15 code for the extracellular portion.
A recent study in cattle provided evidence for positive Darwinian evolution in the extracellular portion of CD45 (Ballingall et al. 2000). The question arises whether this positive selection is unique to cattle or whether it occurs in other mammals, and over longer periods of evolutionary time (across species). In addition, it is interesting to ask two questions that were not addressed in the cattle study. First, are there similar patterns of selection on the alternatively spliced domains and the other extracellular exons? Second, how does the rate of molecular evolution of CD45 exons, particularly those under positive selection, compare with that of introns? In order to address these questions we sequenced six extracellular exons and one intron (intron 6) from eight species of catarrhine primate representative of the full extant radiation of apes, humans, and Old World monkeys. The exons examined were the alternatively spliced exons 4–6, adjacent exon 7, exon 9, which codes for the linker domain, and exon 14, which is the largest exon coding for part of the fibronectin type III domain. The results provide evidence for consistently strong positive selection and rapid evolution on the extracellular portion of CD45 throughout catarrhine evolution that is concentrated in exons 9 and 14.
Materials and Methods
Samples
The following species were included in the study (lab ID in parentheses): human (Hsa228); common chimpanzee: Pan troglodytes(Ptr2), Western lowland gorilla: Gorilla g. gorilla(Ggo), Orangutan: Pongo pygmaeus(Ppy3), Mueller's gibbon: Hylobates muelleri(Hmu1), pig-tailed macaque: Macaca nemestrina(Mne1), De Brazza's monkey (a guenon): Cercopithecus neglectus(Cne1), and black-and-white colobus: Colobus guereza(Cgu2). Genomic DNA had previously been extracted from blood or tissue samples using Qiagen kits (Qiagen, Valencia, Calif.).
Laboratory Methods
PCR reactions were performed in a Techne Genius thermal cycler. For polymerase chain reaction (PCR) product sizes less than 1 kb (exons 4, 5, and 14), PCR reactions were performed in a total volume of 25 μl containing 0.1-μl thermostable polymerase (Thermoprime Plus DNA Polymerase, Abgene, Rochester, N.Y.), 1 μl each of forward and reverse primers (10 mM), 0.05 μl each dNTP (25 mM), 1.5 mM MgCl2, 1X reaction buffer and 10–100 ng DNA with the following cycling parameters: 94°C, 120 s; 35X (94°C, 30 s; annealing temperature, 45 s; 72°C, 90 s or 150 s); 72°C, 5'. For PCR product sizes greater than 1 kb (exons 6, 7, and 9), PCR reactions were performed in a total volume of 10 μl containing 5 μl extensor polymerase (ReddyMix Hi-Fidelity Master Mix, 2x concentration, Abgene), 1 μl each of forward and reverse primers (10 mM), and 10–100 ng DNA, with the following cycling parameters: 94°C, 120 s; 30 or 35X (94°C, 30 s; annealing temperature, 45 s; 68°C, 3' or 6'); 72°C, 5'. PCR products were purified using Qiaquick PCR Purification Kits (Qiagen), and sequenced on both strands using internal primers (table 1) with Big Dye terminator (Applied Biosystems, Foster City, Calif.). Products were run on an ABI Prism 377 DNA Sequencer. Sequences were edited and aligned using Editseq and Seqman (DNAStar, GATC, Madison, Wisc.).
Table 1 Oligonucleotide Primers Used in This Study.
Primers and annealing temperatures are shown in table 1. PCR primers were also used for sequencing reactions.
Sequence Analysis
Most analyses were performed on a 993-bp dataset of concatenated exons 4–7, 9, and 14. Comparisons of genetic distances between exons and introns were were performed on a 555-bp dataset of concatenated exons 4–7, a 438-bp dataset of exons 9 and 14, and a 795-bp datset of intron 6, using HKY85 distances.
Phylogenetic analyses were performed in PAUP* (Swofford 1999). Neighbor-joining trees were obtained using HKY85 genetic distances, and maximum likelihood phylogenies were obtained using the HKY85 model.
Estimation of dN/dS ratios was primarily carried out by maximum likelihood using a codon-based substitution model in PAML version 3.13 (Yang 1997). We implemented several site-specific models in which selective pressure varies among different sites, but the site-specific pattern is identical across all lineages (Yang et al. 2000): model M0 (null model with no variation among sites), M1 ("neutral" model, with two categories of site with fixed dN/dS ratios of 0 and 1), M2 ("selection" model—three categories of site, two with fixed dN/dS ratios of 0 and 1, and a third estimated dN/dS ratio), M3 ("discrete" model—three categories of site, with the dN/dS ratio free to vary for each site), M7 ("beta model"—eight categories of site, with eight dN/dS ratios in the range 0–1 taken from a discrete approximation of the beta distribution), M8 ("beta plus omega" model—eight categories of site from a beta distribution as in model M7 plus an additional category of site with a dN/dS ratio that is free to vary from 0 to greater than 1), M8a (as M8, but the ninth category of the site is constrained to have dN/dS = 1, Swanson et al. 2002). Analyses were run with all parameters estimated in these models. Likelihood ratio tests to determine whether particular models provided a significantly better fit to the data than other nested models were performed by comparing the likelihood ratio test statistic (–2[LogLikelihood1–LogLikelihood2]) to critical values of the chi square distribution with the appropriate degrees of freedom (Yang 1998, Yang et al. 2000). The posterior probability that a site was under positive selection was obtained using the Bayesian approach implemented in PAML. Posterior probabilities were considered in models M3 and M8 to reduce the chance of false positives (Anisimova et al. 2001).
Pairwise dN and dS were estimated using the Pamilo-Bianchi-Li method (Li 1993; Pamilo and Bianchi, 1993) in Mega2 (Kumar et al. 2000). Tests of dN > dS in pairwise comparisons were conducted using z-tests in Mega2. Pairwise genetic distances of intron 6 were estimated using the Kimura-2-parameter method in PAUP*, in order to facilitate comparisons with dN and dS estimates.
Prediction of O-linked glycosylation sites was performed using NetOGlyc v. 2.0 (Hansen et al. 1998).
Results
CD45 Dataset
Sequences were obtained from six exons spread across the major extracellular domains—exons 4–7 (variable domain), exon 9 (linker region) and exon 14 (fibronectin type III domain)—plus one intron (intron 6) from eight species of catarrhine primates. Two indels were present in exons: a five amino acid deletion in exon 9 was present in humans and apes, and a single amino acid deletion in exon 14 occurred in cercopithecine Old World monkeys (macaque and guenon) (fig. 3). The human sequences were identical to those of the human CD45 genbank reference sequence.
FIG. 3. Alignment of CD45 amino acid sequences. Asterisks show sites identified as having a 95% posterior probability of being under positive selection. Gaps show breaks between noncontiguous exons
Phylogenetic reconstructions obtained using neighbor joining and maximum likelihood were identical to the well-established primate species phylogeny, except that there was poor resolution of the human-chimpanzee-gorilla trichotomy (not shown).
As evolution of O-linked glycosylation has been suggested to be important in positive selection of glycophorins (Baum et al. 2002), this was investigated in CD45. There were a few changes in predicted O-linked glycosylation in exons 4–7, but no predicted glycosylation sites in any of the exon 9 or exon 14 sequences obtained (not shown).
Patterns of Selection on CD45
Estimation of synonymous and nonsynonymous substitution rates over the six exons was performed by maximum likelihood using a codon-based substitution model in PAML (Yang 1997), using the known species phylogeny. Almost all lineages showed more reconstructed nonsynonymous substitutions than synonymous substitutions, and in many cases there was a large excess of nonsynonymous substitutions, e.g., in orangutan, guenon, and colobus terminal lineages (fig. 2), suggesting a strong signal of positive Darwinian evolution.
FIG. 2. Reconstruction of CD45 evolution over primate phylogeny. Estimated numbers of non-synonymous and synonymous substitutions are given for each lineage over the known, unrooted, species phylogeny. Upper branches: estimates for exons 4–7, 9, and 14 together. Lower branches: estimates for exons 9 + 14 (upper) and exons 4–7 (lower) separately. Branch lengths are arbitrary
The estimated dN/dS ratio over all exons and lineages was 2.53 (table 2), and this value was significantly greater than the neutral expectation of 1 in a likelihood ratio test (model M0 vs. model M0', test A, table 3). This indicates that the extracellular part of CD45 has been under positive Darwinian selection throughout the evolution of catarrhine primates.
Table 2 Maximum Likelihood Estimation of dN/dS Ratios over Six Exons of CD45.
Table 3 Likelihood Ratio Tests.
In order to determine whether positive selection was evenly distributed across extracellular domains, we investigated models in which the dN/dS ratio is allowed to vary among different classes of sites (models M1–M3, M7, M8, M8a; table 2; Yang et al. 2000). There was again significant positive selection across all lineages (table 3, tests B–E), and estimated dN/dS ratios at sites under positive selection were in the range 12.80–13.16. These values are extremely high and indicate the presence of "hotspots" of positive selection. Using a Bayesian approach, the same 30 sites were predicted to have a greater than 95% probability in models M3 and M8. Of these 30 sites, 27 were in exons 9 and 14, and 3 were in exons 4 and 6 (fig. 3).
Results using the more conventional pairwise dN/dS ratios were consistent with those using PAML in demonstrating the concentration of positive selection in exons 9 and 14. For exons 9 and 14, 25 out of 28 pairwise comparisons had dN/dS > 1, and in 17 of these cases dN was significantly greater than dS (not shown). For exons 4–7, 11 out of 28 pairwise comparisons had dN/dS > 1, and dN > dS was significant in 5 cases (not shown).
Rates of Evolution in Exons and Intron 6
In sequences under strong positive selection, the rate of molecular evolution may exceed the neutral rate. In order to examine this possibility we compared rates of evolution of CD45 exons with that of intron 6, which is expected to evolve neutrally. As positive selection appears to be concentrated in exons 9 and 14, these exons were compared separately to exons 4–7.
The comparison of pairwise genetic distances for intron 6 versus these groups of exons is shown in figure 4. In a large majority of pairwise comparisons (24/28) the rate of evolution of nonsynonymous sites in exons 9 + 14 is greater than that of intron 6, and in most cases there is at least a twofold greater rate for these exons compared to the intron. In contrast, the pairwise dN for exons 4–7 is greater than that of intron 6 in only 7 of 28 pairwise comparisons. The pairwise dS values for exons 9 + 14, and exons 4–7 are approximately equal to those of intron 6, with some evidence for a slightly higher rate for exons 9 + 14, and a lower rate for exons 4–7.
FIG. 4. Comparison of pairwise genetic distances for CD45 exons and intron 6. Diagonal lines represent equal evolutionary rates. (A) Intron 6 vs. exons 9 + 14 dS. (B) Intron 6 vs. exons 9 + 14 dN. (C) Intron 6 vs. exons 4–7 dS. (D) Intron 6 vs. exons 4–7 dN.
Discussion
The results presented here demonstrate that the extracellular domain of CD45 has been under strong and consistent positive selection through the evolution of catarrhine primates, representing some 25 to 35 Myr of primate evolution (Martin 1990). The presence of positive selection on CD45, which was first reported from intraspecific variation in cattle (Ballingall et al. 2000), is thus confirmed in another mammalian order and over a much greater time period. This consistent adaptive evolution over long time periods is in striking contrast to the large majority of reports of positive selection in primates (table 4), in which positive selection generally occurs as bursts in particular lineages. Such bursts of positive selection are generally thought to be associated with changes in function, often following duplication events (e.g., pancreatic ribonuclease and morpheus).
Table 4 Positive Selection in Primate Genes Identified by dN/dS or KA/KS > 1.
Two comparable examples in which genes are under consistent positive selection in primates appear to be the MHC genes (Hughes and Nei 1988) and glycophorins A, B, and E (Baum et al. 2002; Wang et al. 2003). In these cases, direct interaction with parasite-encoded molecules has been implicated in the selective pressure. In MHC genes, positive selection is concentrated in the peptide-binding region or antigen recognition site, which binds peptide antigens derived from pathogens for presentation to T cells. Positive selection on glycophorins, which are expressed on red blood cells, has been suggested to be a defensive mechanism against infection by malaria parasites (Plasmodium spp.), although direct evidence is lacking (Baum et al. 2002; Wang et al. 2003).
Positive selection is not evenly distributed among the CD45 exons examined. Results from estimates of dN/dS ratios show that positive selection is concentrated in exons 9 and 14 compared to exons 4–7. In addition to frequent changes in amino acid sequence resulting from point substitutions, exons 9 and 14 were also the only exons in the dataset to show deletions. These are some of the first data we are aware of in which strength of selection has been compared between alternatively spliced exons and non–alternatively spliced exons in the same gene. The results are perhaps counterintuitive at first sight since alternative splicing permits functional diversification of a gene, and alternatively spliced exons might therefore be more free to evolve under directional selection. However, directional selection would be expected to occur shortly after the acquisition of a novel pattern of alternative splicing, and data from sharks indicates that the cell and development-specific pattern of alternative splicing of CD45 is conserved through hundreds of millions of years of vertebrate evolution (Okumura et al. 1996). In addition, it has been shown that four regulatory elements for alternative splicing are present in exon 4, providing a constraint on the evolution of that exon (Lynch and Weiss 2001). In their study of molecular evolution on the extracellular part of CD45 (exons 7–15) among breeds of cattle, Ballingall et al. (2000) found that positive selection was unevenly distributed. However, unlike in primates, positively selected sites were concentrated in exon 9 and were not present in exon 14.
Further confirmation of the rapid rate of molecular evolution of exons 9 and 14 of CD45 in primates comes from comparison of rates of evolution of exons and introns. In particular, nonsynonymous sites in exons 9 + 14 have evolved at roughly twice as fast as intron 6. In contrast, rates among intron 6, synonymous sites in exons 9 + 14 and exons 4–7, and nonsynonymous sites in exons 4–7 are approximately equal. Some caution in assuming neutral evolution of CD45 introns is necessary since certain regulatory elements are present in the introns. However, there is no evidence that intron 6 participates in this regulation.
The intracellular part of CD45 contains two phosphatase domains and is strongly conserved during vertebrate evolution, including primates (Thomas 1989, Okumura et al. 1996, Montoya et al. 2002). Overall, therefore, the molecular evolution of CD45 combines regions of strong conservation of function with regions rapidly evolving under directional selection. These results imply a previously unsuspected species-specificity to CD45 function involving the spacer region and fibronectin III domains encoded by exons 9 and 14, respectively. These domains have attracted limited attention previously and there is little information available on the their function or molecular interactions, but they must have a critical role in CD45 function.
We propose that the extracellular spacer and fibronectin domains of CD45 determine binding (either directly or indirectly) to rapidly evolving host or parasite-encoded molecules in primates. However, the nature of the molecules/parasites involved is elusive. Evidence that CD45 in primates binds to parasites has not been described. The only host extracellular ligand for CD45 that is currently known, galectin-1, appears to be a poor candidate for such an interaction (Nguyen et al. 2001). Galectin-1 is strongly conserved in mammalian evolution, and has a broad binding specificity to O-linked galactosylamines, whereas the CD45 linker and fibronectin III domains encoded by exons 9 and 14 are not predicted to be glycosylated in any of the species we examined. In cattle, Ballingall et al. (2000) suggested more specifically that blood-borne trypanosomes (Babesia spp.) might be responsible for exerting selective pressure on CD45. Trypanosomes are not thought to have high prevalence in catarrhines so this is an unlikely explanation in primates.
FIG. 1. CD45 extracellular domains, and relationship to exons. Line segments above exons represent exons sequenced in this study
Acknowledgements
We thank Andrew Kitchener and Mike Bruford for samples, Joanne Kelly for technical assistance, and Eddie Holmes and the late Ryk Ward for helpful discussion and comments on an earlier version of the manuscript. Sequences have been deposited in GenBank (accession numbers AY539659–AY539714).
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E-mail: nim21@cam.ac.uk.
Abstract
CD45, encoded by PTPRC in humans, is the most abundantly expressed protein on the surface of many lymphocytes. We investigated whether the extracellular region of CD45 was under positive selection in Old World primates, and whether there was differential selection across this region, particularly on exons that were involved in alternative splicing and those that were not alternatively spliced. The results show extraordinarily strong and consistent positive Darwinian selection on the extracellular part of CD45 throughout the evolution of Old World monkeys, apes and humans. Positive selection is concentrated in exons 9 and 14, which code for the previously neglected linker and fibronectin III domains. These exons have a high rate of evolution at nonsynonymous sites that is roughly twice as high as that of the intronic rate in this gene. In contrast, alternatively spliced exons 4–6, which code for the variable domains, are under weaker positive selection and are evolving more slowly than the intronic rate. These data provide a striking example of positive selection in a well-known gene that should provide an impetus for further functional studies to elucidate its species-specific function.
Key Words: positive selection ? lymphocyte ? immune system ? PTPRC
Introduction
The immune system of vertebrates has provided some of the most compelling examples of positive Darwinian evolution at the molecular level. This can be attributed to the strong selective pressures created by the need to respond to rapidly evolving pathogens with short generation times. The best known cases involve major histocompatibility complex (MHC) Class I (Hughes and Nei 1988), MHC Class II (Hughes and Yeager 1998), and immunoglobulin heavy chain (Tanaka and Nei 1989) genes that code for proteins that have a clear role in direct binding of pathogen antigens as part of the specific acquired immune response. Although there is some evidence for rapid evolution of immune system genes in general (Hughes 1997), there are relatively few other genes expressed in the immune system which have been shown to be under positive Darwinian evolution. These include defensins (Hughes and Yeager 1997), eosinophil ribonucleases (Zhang et al. 1998), and CD59 (Osada et al. 2002).
CD45, also known as B220, PTPRC, or the leukocyte common antigen, is a transmembrane protein tyrosine phosphatase that comprises up to 10% of the total surface protein of nucleated hematopoietic cells. It has important functions in B- and T-cell maturation and activation (reviewed in Trowbridge and Thomas 1994). For example, in CD45 null mice, the number of single positive T cells in the periphery is greatly reduced, and the few single positive T cells that reach the periphery show increased affinity for self-MHC complexes, leading to an increased incidence of autoimmune disorders (Trop et al. 2000). Mutations in CD45 have been linked to cases of severe combined immunodeficiency (SCID) in humans (Tchilian et al. 2001).
The extracellular region of CD45 comprises a variable domain, a linker region, and three type III fibronectin domains. The variable domain defines isoforms of CD45 that differ in size and degree of glycosylation. The isoforms are generated by alternative splicing of exons 4–6 and are expressed in a tightly regulated cell-type and stage-specific manner that is highly conserved among vertebrates (Okumura et al. 1996). The extracellular region of CD45 associates with the component of the T-cell receptor (Leitenberg et al. 1996) and is necessary for controlling lck activity (Irles et al. 2003). Signaling properties may vary according to which CD45 isoform is expressed (McKenney et al. 1995). There is still much ignorance about the molecular interactions and function of the extracellular portion of CD45, and, in particular, no function has been ascribed to the linker region and type III fibronectin domains. In contrast, the function of the intracellular region of CD45 is well understood: it has two phosphatase domains that are responsible for dephosphorylating two of the Src family kinases critical to T-cell activation and proliferation, p56lck and p59fyn, and promoting the secretion of cytokines such as interleukin-2 (McKenney et al. 1995). The human gene for CD45 (PTPRC) is on chromosome 1 and comprises 33 exons, of which the first 15 code for the extracellular portion.
A recent study in cattle provided evidence for positive Darwinian evolution in the extracellular portion of CD45 (Ballingall et al. 2000). The question arises whether this positive selection is unique to cattle or whether it occurs in other mammals, and over longer periods of evolutionary time (across species). In addition, it is interesting to ask two questions that were not addressed in the cattle study. First, are there similar patterns of selection on the alternatively spliced domains and the other extracellular exons? Second, how does the rate of molecular evolution of CD45 exons, particularly those under positive selection, compare with that of introns? In order to address these questions we sequenced six extracellular exons and one intron (intron 6) from eight species of catarrhine primate representative of the full extant radiation of apes, humans, and Old World monkeys. The exons examined were the alternatively spliced exons 4–6, adjacent exon 7, exon 9, which codes for the linker domain, and exon 14, which is the largest exon coding for part of the fibronectin type III domain. The results provide evidence for consistently strong positive selection and rapid evolution on the extracellular portion of CD45 throughout catarrhine evolution that is concentrated in exons 9 and 14.
Materials and Methods
Samples
The following species were included in the study (lab ID in parentheses): human (Hsa228); common chimpanzee: Pan troglodytes(Ptr2), Western lowland gorilla: Gorilla g. gorilla(Ggo), Orangutan: Pongo pygmaeus(Ppy3), Mueller's gibbon: Hylobates muelleri(Hmu1), pig-tailed macaque: Macaca nemestrina(Mne1), De Brazza's monkey (a guenon): Cercopithecus neglectus(Cne1), and black-and-white colobus: Colobus guereza(Cgu2). Genomic DNA had previously been extracted from blood or tissue samples using Qiagen kits (Qiagen, Valencia, Calif.).
Laboratory Methods
PCR reactions were performed in a Techne Genius thermal cycler. For polymerase chain reaction (PCR) product sizes less than 1 kb (exons 4, 5, and 14), PCR reactions were performed in a total volume of 25 μl containing 0.1-μl thermostable polymerase (Thermoprime Plus DNA Polymerase, Abgene, Rochester, N.Y.), 1 μl each of forward and reverse primers (10 mM), 0.05 μl each dNTP (25 mM), 1.5 mM MgCl2, 1X reaction buffer and 10–100 ng DNA with the following cycling parameters: 94°C, 120 s; 35X (94°C, 30 s; annealing temperature, 45 s; 72°C, 90 s or 150 s); 72°C, 5'. For PCR product sizes greater than 1 kb (exons 6, 7, and 9), PCR reactions were performed in a total volume of 10 μl containing 5 μl extensor polymerase (ReddyMix Hi-Fidelity Master Mix, 2x concentration, Abgene), 1 μl each of forward and reverse primers (10 mM), and 10–100 ng DNA, with the following cycling parameters: 94°C, 120 s; 30 or 35X (94°C, 30 s; annealing temperature, 45 s; 68°C, 3' or 6'); 72°C, 5'. PCR products were purified using Qiaquick PCR Purification Kits (Qiagen), and sequenced on both strands using internal primers (table 1) with Big Dye terminator (Applied Biosystems, Foster City, Calif.). Products were run on an ABI Prism 377 DNA Sequencer. Sequences were edited and aligned using Editseq and Seqman (DNAStar, GATC, Madison, Wisc.).
Table 1 Oligonucleotide Primers Used in This Study.
Primers and annealing temperatures are shown in table 1. PCR primers were also used for sequencing reactions.
Sequence Analysis
Most analyses were performed on a 993-bp dataset of concatenated exons 4–7, 9, and 14. Comparisons of genetic distances between exons and introns were were performed on a 555-bp dataset of concatenated exons 4–7, a 438-bp dataset of exons 9 and 14, and a 795-bp datset of intron 6, using HKY85 distances.
Phylogenetic analyses were performed in PAUP* (Swofford 1999). Neighbor-joining trees were obtained using HKY85 genetic distances, and maximum likelihood phylogenies were obtained using the HKY85 model.
Estimation of dN/dS ratios was primarily carried out by maximum likelihood using a codon-based substitution model in PAML version 3.13 (Yang 1997). We implemented several site-specific models in which selective pressure varies among different sites, but the site-specific pattern is identical across all lineages (Yang et al. 2000): model M0 (null model with no variation among sites), M1 ("neutral" model, with two categories of site with fixed dN/dS ratios of 0 and 1), M2 ("selection" model—three categories of site, two with fixed dN/dS ratios of 0 and 1, and a third estimated dN/dS ratio), M3 ("discrete" model—three categories of site, with the dN/dS ratio free to vary for each site), M7 ("beta model"—eight categories of site, with eight dN/dS ratios in the range 0–1 taken from a discrete approximation of the beta distribution), M8 ("beta plus omega" model—eight categories of site from a beta distribution as in model M7 plus an additional category of site with a dN/dS ratio that is free to vary from 0 to greater than 1), M8a (as M8, but the ninth category of the site is constrained to have dN/dS = 1, Swanson et al. 2002). Analyses were run with all parameters estimated in these models. Likelihood ratio tests to determine whether particular models provided a significantly better fit to the data than other nested models were performed by comparing the likelihood ratio test statistic (–2[LogLikelihood1–LogLikelihood2]) to critical values of the chi square distribution with the appropriate degrees of freedom (Yang 1998, Yang et al. 2000). The posterior probability that a site was under positive selection was obtained using the Bayesian approach implemented in PAML. Posterior probabilities were considered in models M3 and M8 to reduce the chance of false positives (Anisimova et al. 2001).
Pairwise dN and dS were estimated using the Pamilo-Bianchi-Li method (Li 1993; Pamilo and Bianchi, 1993) in Mega2 (Kumar et al. 2000). Tests of dN > dS in pairwise comparisons were conducted using z-tests in Mega2. Pairwise genetic distances of intron 6 were estimated using the Kimura-2-parameter method in PAUP*, in order to facilitate comparisons with dN and dS estimates.
Prediction of O-linked glycosylation sites was performed using NetOGlyc v. 2.0 (Hansen et al. 1998).
Results
CD45 Dataset
Sequences were obtained from six exons spread across the major extracellular domains—exons 4–7 (variable domain), exon 9 (linker region) and exon 14 (fibronectin type III domain)—plus one intron (intron 6) from eight species of catarrhine primates. Two indels were present in exons: a five amino acid deletion in exon 9 was present in humans and apes, and a single amino acid deletion in exon 14 occurred in cercopithecine Old World monkeys (macaque and guenon) (fig. 3). The human sequences were identical to those of the human CD45 genbank reference sequence.
FIG. 3. Alignment of CD45 amino acid sequences. Asterisks show sites identified as having a 95% posterior probability of being under positive selection. Gaps show breaks between noncontiguous exons
Phylogenetic reconstructions obtained using neighbor joining and maximum likelihood were identical to the well-established primate species phylogeny, except that there was poor resolution of the human-chimpanzee-gorilla trichotomy (not shown).
As evolution of O-linked glycosylation has been suggested to be important in positive selection of glycophorins (Baum et al. 2002), this was investigated in CD45. There were a few changes in predicted O-linked glycosylation in exons 4–7, but no predicted glycosylation sites in any of the exon 9 or exon 14 sequences obtained (not shown).
Patterns of Selection on CD45
Estimation of synonymous and nonsynonymous substitution rates over the six exons was performed by maximum likelihood using a codon-based substitution model in PAML (Yang 1997), using the known species phylogeny. Almost all lineages showed more reconstructed nonsynonymous substitutions than synonymous substitutions, and in many cases there was a large excess of nonsynonymous substitutions, e.g., in orangutan, guenon, and colobus terminal lineages (fig. 2), suggesting a strong signal of positive Darwinian evolution.
FIG. 2. Reconstruction of CD45 evolution over primate phylogeny. Estimated numbers of non-synonymous and synonymous substitutions are given for each lineage over the known, unrooted, species phylogeny. Upper branches: estimates for exons 4–7, 9, and 14 together. Lower branches: estimates for exons 9 + 14 (upper) and exons 4–7 (lower) separately. Branch lengths are arbitrary
The estimated dN/dS ratio over all exons and lineages was 2.53 (table 2), and this value was significantly greater than the neutral expectation of 1 in a likelihood ratio test (model M0 vs. model M0', test A, table 3). This indicates that the extracellular part of CD45 has been under positive Darwinian selection throughout the evolution of catarrhine primates.
Table 2 Maximum Likelihood Estimation of dN/dS Ratios over Six Exons of CD45.
Table 3 Likelihood Ratio Tests.
In order to determine whether positive selection was evenly distributed across extracellular domains, we investigated models in which the dN/dS ratio is allowed to vary among different classes of sites (models M1–M3, M7, M8, M8a; table 2; Yang et al. 2000). There was again significant positive selection across all lineages (table 3, tests B–E), and estimated dN/dS ratios at sites under positive selection were in the range 12.80–13.16. These values are extremely high and indicate the presence of "hotspots" of positive selection. Using a Bayesian approach, the same 30 sites were predicted to have a greater than 95% probability in models M3 and M8. Of these 30 sites, 27 were in exons 9 and 14, and 3 were in exons 4 and 6 (fig. 3).
Results using the more conventional pairwise dN/dS ratios were consistent with those using PAML in demonstrating the concentration of positive selection in exons 9 and 14. For exons 9 and 14, 25 out of 28 pairwise comparisons had dN/dS > 1, and in 17 of these cases dN was significantly greater than dS (not shown). For exons 4–7, 11 out of 28 pairwise comparisons had dN/dS > 1, and dN > dS was significant in 5 cases (not shown).
Rates of Evolution in Exons and Intron 6
In sequences under strong positive selection, the rate of molecular evolution may exceed the neutral rate. In order to examine this possibility we compared rates of evolution of CD45 exons with that of intron 6, which is expected to evolve neutrally. As positive selection appears to be concentrated in exons 9 and 14, these exons were compared separately to exons 4–7.
The comparison of pairwise genetic distances for intron 6 versus these groups of exons is shown in figure 4. In a large majority of pairwise comparisons (24/28) the rate of evolution of nonsynonymous sites in exons 9 + 14 is greater than that of intron 6, and in most cases there is at least a twofold greater rate for these exons compared to the intron. In contrast, the pairwise dN for exons 4–7 is greater than that of intron 6 in only 7 of 28 pairwise comparisons. The pairwise dS values for exons 9 + 14, and exons 4–7 are approximately equal to those of intron 6, with some evidence for a slightly higher rate for exons 9 + 14, and a lower rate for exons 4–7.
FIG. 4. Comparison of pairwise genetic distances for CD45 exons and intron 6. Diagonal lines represent equal evolutionary rates. (A) Intron 6 vs. exons 9 + 14 dS. (B) Intron 6 vs. exons 9 + 14 dN. (C) Intron 6 vs. exons 4–7 dS. (D) Intron 6 vs. exons 4–7 dN.
Discussion
The results presented here demonstrate that the extracellular domain of CD45 has been under strong and consistent positive selection through the evolution of catarrhine primates, representing some 25 to 35 Myr of primate evolution (Martin 1990). The presence of positive selection on CD45, which was first reported from intraspecific variation in cattle (Ballingall et al. 2000), is thus confirmed in another mammalian order and over a much greater time period. This consistent adaptive evolution over long time periods is in striking contrast to the large majority of reports of positive selection in primates (table 4), in which positive selection generally occurs as bursts in particular lineages. Such bursts of positive selection are generally thought to be associated with changes in function, often following duplication events (e.g., pancreatic ribonuclease and morpheus).
Table 4 Positive Selection in Primate Genes Identified by dN/dS or KA/KS > 1.
Two comparable examples in which genes are under consistent positive selection in primates appear to be the MHC genes (Hughes and Nei 1988) and glycophorins A, B, and E (Baum et al. 2002; Wang et al. 2003). In these cases, direct interaction with parasite-encoded molecules has been implicated in the selective pressure. In MHC genes, positive selection is concentrated in the peptide-binding region or antigen recognition site, which binds peptide antigens derived from pathogens for presentation to T cells. Positive selection on glycophorins, which are expressed on red blood cells, has been suggested to be a defensive mechanism against infection by malaria parasites (Plasmodium spp.), although direct evidence is lacking (Baum et al. 2002; Wang et al. 2003).
Positive selection is not evenly distributed among the CD45 exons examined. Results from estimates of dN/dS ratios show that positive selection is concentrated in exons 9 and 14 compared to exons 4–7. In addition to frequent changes in amino acid sequence resulting from point substitutions, exons 9 and 14 were also the only exons in the dataset to show deletions. These are some of the first data we are aware of in which strength of selection has been compared between alternatively spliced exons and non–alternatively spliced exons in the same gene. The results are perhaps counterintuitive at first sight since alternative splicing permits functional diversification of a gene, and alternatively spliced exons might therefore be more free to evolve under directional selection. However, directional selection would be expected to occur shortly after the acquisition of a novel pattern of alternative splicing, and data from sharks indicates that the cell and development-specific pattern of alternative splicing of CD45 is conserved through hundreds of millions of years of vertebrate evolution (Okumura et al. 1996). In addition, it has been shown that four regulatory elements for alternative splicing are present in exon 4, providing a constraint on the evolution of that exon (Lynch and Weiss 2001). In their study of molecular evolution on the extracellular part of CD45 (exons 7–15) among breeds of cattle, Ballingall et al. (2000) found that positive selection was unevenly distributed. However, unlike in primates, positively selected sites were concentrated in exon 9 and were not present in exon 14.
Further confirmation of the rapid rate of molecular evolution of exons 9 and 14 of CD45 in primates comes from comparison of rates of evolution of exons and introns. In particular, nonsynonymous sites in exons 9 + 14 have evolved at roughly twice as fast as intron 6. In contrast, rates among intron 6, synonymous sites in exons 9 + 14 and exons 4–7, and nonsynonymous sites in exons 4–7 are approximately equal. Some caution in assuming neutral evolution of CD45 introns is necessary since certain regulatory elements are present in the introns. However, there is no evidence that intron 6 participates in this regulation.
The intracellular part of CD45 contains two phosphatase domains and is strongly conserved during vertebrate evolution, including primates (Thomas 1989, Okumura et al. 1996, Montoya et al. 2002). Overall, therefore, the molecular evolution of CD45 combines regions of strong conservation of function with regions rapidly evolving under directional selection. These results imply a previously unsuspected species-specificity to CD45 function involving the spacer region and fibronectin III domains encoded by exons 9 and 14, respectively. These domains have attracted limited attention previously and there is little information available on the their function or molecular interactions, but they must have a critical role in CD45 function.
We propose that the extracellular spacer and fibronectin domains of CD45 determine binding (either directly or indirectly) to rapidly evolving host or parasite-encoded molecules in primates. However, the nature of the molecules/parasites involved is elusive. Evidence that CD45 in primates binds to parasites has not been described. The only host extracellular ligand for CD45 that is currently known, galectin-1, appears to be a poor candidate for such an interaction (Nguyen et al. 2001). Galectin-1 is strongly conserved in mammalian evolution, and has a broad binding specificity to O-linked galactosylamines, whereas the CD45 linker and fibronectin III domains encoded by exons 9 and 14 are not predicted to be glycosylated in any of the species we examined. In cattle, Ballingall et al. (2000) suggested more specifically that blood-borne trypanosomes (Babesia spp.) might be responsible for exerting selective pressure on CD45. Trypanosomes are not thought to have high prevalence in catarrhines so this is an unlikely explanation in primates.
FIG. 1. CD45 extracellular domains, and relationship to exons. Line segments above exons represent exons sequenced in this study
Acknowledgements
We thank Andrew Kitchener and Mike Bruford for samples, Joanne Kelly for technical assistance, and Eddie Holmes and the late Ryk Ward for helpful discussion and comments on an earlier version of the manuscript. Sequences have been deposited in GenBank (accession numbers AY539659–AY539714).
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