Department of Biological Anthropology, University of Oxford, United Kingdom
Correspondence: E-mail: nim21{at}cam.ac.uk.
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Abstract |
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Key Words: positive selection lymphocyte immune system PTPRC
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Introduction |
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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 46 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 46, 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.
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Materials and Methods |
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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 10100 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 10100 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.).
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Sequence Analysis
Most analyses were performed on a 993-bp dataset of concatenated exons 47, 9, and 14. Comparisons of genetic distances between exons and introns were were performed on a 555-bp dataset of concatenated exons 47, 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" modelthree categories of site, two with fixed dN/dS ratios of 0 and 1, and a third estimated dN/dS ratio), M3 ("discrete" modelthree 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 01 taken from a discrete approximation of the beta distribution), M8 ("beta plus omega" modeleight 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[LogLikelihood1LogLikelihood2]) 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).
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Results |
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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 47, 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.
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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 47, 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 47.
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 47 is greater than that of intron 6 in only 7 of 28 pairwise comparisons. The pairwise dS values for exons 9 + 14, and exons 47 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 47.
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Discussion |
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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 47. 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 nonalternatively 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 715) 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 47, and nonsynonymous sites in exons 47 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.
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Acknowledgements |
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Footnotes |
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