* Department of Organismic and Evolutionary Biology, Harvard University
Department of Pharmacy, Faculty of Medicine, University of Tromsø, and Norwegian Institute of Gene Ecology, Tromsø, Norway
Department of Immunology and Infectious Diseases, Harvard School of Public Health
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Abstract |
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Key Words: falcipain gene conversion paralogs Plasmodium falciparum polymorphisms
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Introduction |
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Analysis of genetic variability among isolates of P. falciparum reveals anomalously low levels of polymorphisms of synonymous sites and introns (Rich et al. 1998; Volkman et al. 2001). This finding has been interpreted to imply that the parasite underwent a severe bottleneck in population size in the human population during the last 10,000 to 40,000 years, resulting in a relatively recent common ancestor of all extant P. falciparum. Nevertheless, many genes in P. falciparum have high levels of nonsynonymous single-nucleotide polymorphisms (SNPs) (Verra and Hughes 2000; Hughes and Verra 2001), which is apparently inconsistent with the recent common ancestor hypothesis. Other analyses of SNPs have suggested an age for the most recent common ancestor of 100,000 to 200,000 years (Mu et al. 2002), 300,000 to 400,000 years (Hughes and Verra 2001), or even older (Hughes 1999).
Critical assumptions in the use of SNP polymorphisms for estimating the age of the most recent common ancestor are (1) that the SNPs undergo spontaneous mutation independently and at the same rate and (2) that the SNPs undergo mutation and random genetic drift as selectively neutral genetic markers. In this paper, we demonstrate that some polymorphisms in P. falciparum are in gross violation of the first assumption. In particular, we have determined the distribution of polymorphisms within two of the genes encoding the major cysteine proteases responsible for hemoglobin degradation in infected red blood cells (Rosenthal et al. 1988; Francis, Sullivan, and Goldberg 1997). Close examination of 12 kb of chromosome 11 in a region encoding falcipain 2 and falcipain 3 unexpectedly revealed a previously unrecognized paralog closely related to falcipain 2. Here, we designate the products of these paralogs as falcipain 2A and falcipain 2B. Analysis of synonymous differences between the paralogs suggests that the duplication occurred a minimum of 10 to 20 MYA, considerably prior to the divergence between P. falciparum and the chimpanzee parasite P. reichenowi.
Although both the falcipain 2A and falcipain 2B genes are highly polymorphic among natural isolates, we find that virtually all of the polymorphisms result from gene conversion between the paralogs. Among 36 synonymous and 46 nonsynonymous nucleotide sites that are polymorphic in the gene for falcipain A, falcipain B, or both, all except possibly one are consistent with an origin by gene conversion. This result is supported not only by the identity of the polymorphic nucleotides but also by the significant clustering of polymorphic nucleotides within each gene. Positioning of the polymorphisms within derived three-dimensional models of the mature protease indicated that three of the five amino acid differences present in the mature protein between falcipain 2A and falcipain 2B are located near the predicted active site. All three of these are polymorphic in one or the other of the paralogs, and hence these polymorphisms may have some functional significance. These data imply that gene conversion between paralogs can be a powerful mechanism generating genetic variability among lineages of P. falciparum.
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Materials and Methods |
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Primer Design, PCR Amplification, and Sequencing
Primers were designed based on the published 3D7 sequences as in PlasmoDB (The Plasmodium Genome Database Collaborative 2001) using Primer 3 software written by S. Rozen and H. J. Skaletsky and available at http://www-genome.wi.mit.edu/ftp/distribution/software. Primers used for amplification and sequencing correspond to the PlasmoDB coordinates in chromosome 11 indicated below. The italicized primers were used to specifically amplify each gene, and they and the others were used for DNA sequencing. The amplifications are specific because the amplification primers match the flanking sequences, which have little similarity between the paralogs. For the gene for falcipain 2A, the primers were the sequences between coordinates (590813, 590838), (578933, 578959) and (589616, 589642), and complements of the sequences between coordinates (591210, 591185), (580063, 580038) and (590567, 590541). For the gene for falcipain 2B, the primers were the sequences between coordinates (579655, 579681), (579339, 579365) and (578826, 578852), and complements of the sequences between coordinates (580301, 580279), (579754, 579729), and (579440, 579414).
PCR amplifications using the HotStarTaq DNA Master mix (Qiagen, Calif.) with 30 cycles at 2 min at 94°C, 1 min at 57°C to 62°C (usually 58°C), and 1 min 20 s at 73°C. Single PCR bands were purified for direct sequencing using shrimp alkaline phosphatase and exonuclease I (USB Biochemicals). To guard against polymerase incorporation error, products from two separate PCR amplifications were sequenced in both directions on a 3100 DNA capillary sequencer (Applied Biosystems) using Big Dye chemistry. Editing of raw chromatograms and multiple sequence alignments were carried out using the Sequencher v.3.1.1 software (GeneCodes).
Molecular Modeling
Computations
Energy minimizations and molecular dynamics simulations were performed using the all-atomic force field of the AMBER (Assisted Model Building with Energy Refinement) 6.0 programs (Pearlman et al. 1995). Models of falcipains 2A and 2B were refined with 500 steps of steepest-descent minimization, followed by conjugate gradient energy minimization until convergence with a 0.005 kcal mol-1 -1 root mean square energy gradient difference between successive minimization steps. Molecular dynamics simulations were performed for 100 ps at 300 K. The bond lengths involving hydrogens were constrained by using the SHAKE algorithm, allowing an integration step of 0.001 ps. A distance-dependent dielectric function (e = rij) and a 10
cut-off radius for nonbonded interactions were used.
The substrate benzyloxycarbonyl-Phe-Arg-7-amino-4-methyl coumarin (Z-FR-AMC) was geometry-optimized using AM1. HF/6-31G* single-point calculation with the program Gaussian 98 (Gaussian, Carnegie, Penn.) was performed in order to obtain electrostatic potentials for the AM1 geometry-optimized structure. The atomic point charges used for the molecular mechanics and dynamics calculations were derived from electrostatic potentials using the RESP (restrained electrostatic potential) program implemented in AMBER 6.
The ICM (Internal Coordinate Mechanics) 2.8 program (Molsoft, La Jolla, Calif.) was utilized for comparative modeling, structure alignments, computer graphics visualizations, substrate docking, and calculations of molecular surfaces and electrostatics potentials.
Falcipain 2A and 2B Structures
The mature form of P. falciparum falcipain 2A includes only residues Q244 to E484 (Shenai et al. 2000; Sijwali, Shenai, and Rosenthal 2002). Compared with other cysteine proteases, the mature form of falcipain 2A includes 17 additional amino-terminal residues (Q244 to F260) that are important for proper folding (Sijwali, Shenai, and Rosenthal 2002). An initial model of the mature form of falcipain 2A, including residues A263 to E484, was built by comparative modeling using the homology module of ICM (Abagyan, Totrov, Kuznetsov 1994). The crystal structure of human cathepsin V associated with the irreversible vinyl sulfone inhibitor APC-3316 (pdb code: 1FH0) (Somoza et al. 2000) was selected as a template due to its high sequence similarity with falcipain 2A (36% identity over 230 residues).
Disulphide bridges were introduced between Cys282 and Cys323, Cys316 and Cys357, Cys342 and Cys362, and Cys411and Cys472. The Cys342 to Cys362 disulphide bridge was conserved among P. falciparum falcipains 1, 2A, 2B, and 3, whereas the three other disulphide bridges were conserved among cysteine proteases. Due to the acid environment in the P. falciparum food vacuole, all histidine residues in the model (H270, H417, and H440) were modeled using the AMBER HIP residue with protonated delta and epsilon nitrogen atoms. The catalytic H417 and C285 residues were modeled as an imidazolium-thiolate ion pair. Iterative energy refinement was performed using the refineProtein macro in ICM, and the resultant model was further energy minimized using the AMBER force field.
Models of polymorphisms in the mature forms of falcipain 2A and 2B were constructed by mutating residues in the energy-minimized models of the consensus sequences of falcipains 2A and 2B. The models of the mutant proteins were energy minimized using the AMBER force field. By using ICM, substrate Z-FR-AMC was placed at its putative binding site in the falcipain 2A model utilizing knowledge from X-ray structures of inhibitor-cysteine protease complexes. The substrate-falcipain 2A complex was energy minimized and the energy-minimized structure was used as a starting structure for a 100 ps molecular dynamics simulations in which the protein backbone atoms were kept in fixed positions. The conformation observed at 100 ps was energy minimized. Near the active site, falcipain 2B differs from falcipain 2A by three amino acid replacements (V393I, A400P, and Q414E). A model of falcipain 2B was constructed by introducing these three amino acid replacements in the energy-minimized model of the falcipain-2A-Z-FR-AMC complex, and the resultant model was energy minimized.
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Results |
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Evidence for Gene Conversion
To capture geographical polymorphisms, the falcipain 2A and 2B genes were sequenced from P. falciparum strains originating in The Netherlands (3D7), Sierra Leone (D6), Laos (W2), Honduras (HB3), Brazil (7G8), and Papua New Guinea (Muz12). Among these strains we found 10 synonymous polymorphisms in falcipain A and 25 synonymous polymorphisms in falcipain B, and we found nine nonsynonymous polymorphisms in falcipain A and 37 nonsynonymous polymorphisms in falcipain B. The locations of the polymorphisms across the codons 1 to 484 in the gene are shown in figure 1. The falcipain A sequences are shown as black lines and those of falcipain B are shown as gray lines. Underneath the lines are designations n for nonsynonymous differences between the paralogous sequences, s for synonymous differences, and d for deletion in falcipain B (equivalently insertion in falcipain A). The vertical arrays of up to three symbols indicate multiple differences in a single codon.
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The possibility that PCR artifacts account for the sequence polymorphisms in figure 1 can be ruled out for three reasons. First, the primers are specific to the divergent flanking sequences and hence specific to each of the paralogs. Second, the sequence of each paralog from each isolate is reproducible from one PCR reaction to the next. Third, note that the polymorphisms do not include the differences in the consensus sequences at each end of the paralogs. This spatial distribution rules out PCR artifacts as the cause of the differences between the sequences, since attributing them to recombinant PCR due to a change of template would require the occurrence of two template-switching events at specific sites in each independent PCR reaction.
Two lines of evidence support gene conversion as the source of most or all the polymorphisms in falcipain 2A and 2B, except that in codon 239. The first is based on sequence. With the exception of codon 239, all of the polymorphisms in each paralog are exact matches to the consensus sequence of the other paralog. The second line of evidence is that the polymorphisms are significantly clustered. For example, the 18 polymorphic codons in falcipain A include two runs, each six codons in length, that contain all the sites at which the paralogous consensus sequences differ. These runs comprise codons 15 to 59 in Muz12 and codons 255 to 266 in 3D7 and 1D6. Elementary combinatorial considerations indicate that the probability of a run of six such sites by chance alone is 0.012. Similarly, the falcipain B of Muz12 has a run of 52 codons from 105 to 434 in which all of the nucleotide sites match the consensus sequence of the gene for falcipain 2A. The probability of a run of this length by chance alone is 1.03 x 10-11. The isolates with polymorphisms that are subsets of significant runs are most easily explained as resulting from recombination within each paralog after each gene-conversion event, rather than as resulting from multiple, independent gene-conversion events.
Estimates of the Rate of Gene Conversion
We used the theory of Innan (2002) to estimate the rates of mutation, gene conversion, and recombination affecting the paralogous falcipain 2A and 2B genes. The parameters in the theory are = 4Nµ, where N is the effective population number and µ is the single-nucleotide mutation rate; C = 4Nc, where c is the rate of gene conversion between the paralogs; and R = 4Nr, where r is the rate of recombination between the paralogs. The data for the model fitting consist of
w, the average number of pairwise differences within paralogs;
b, the average number of pairwise differences between the paralogs; and d, the linkage disequilibrium between the paralogs (Innan 2002).
Innan's (2002) theory is an equilibrium theory, and the estimates assuming equilibrium are given in the top part of table 1, along with the standard deviations of the estimates calculated by means of jackknifing (Efron and Stein 1981). The rate of gene conversion, relative to that of mutation, is c/µ and estimated as C/. These estimates range from 19.4 to 74.2, depending on whether only synonymous or only nonsynonymous sites are analyzed. Over all sites, the ratio c/µ = 43.2.
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To address the likelihood of nonequilibrium, we also estimated the value of c/µ without assuming equilibrium by making use of Innan's (2002) iterative equations. For this estimation, we hoped to bracket the true value of c/µ by using the smallest plausible estimate of the most recent common ancestor. Taking this time as 7,500 years with six generations per year yields 45,000 generations for the iterations. We also assumed µ = 3.24 x 10-9, which is the average of the estimated mutation rate for fourfold degenerate synonymous sites assuming either a 5-Myr or a 7-Myr time for the divergence between P. falciparum and P. reichenowi (Rich et al. 1998). Finally, we assumed r = 0.0044 based on 1 cM per 15 to 30 kb (Su et al. 1999) across the approximately 10.8 kb between the paralogs. With these parameters, the iterative equations of Innan (2002) yield estimates of c/µ in the range 1,389 to 1,698 (table 1). These are approximately 20 to 70 times greater than the equilibrium estimates. If the most recent common ancestor of P. falciparum is older than the 7,500 years assumed here as the minimum plausible value, then the true value of c/µ may be smaller than 1,000. In any case, it is clear that across the 10.8 kb separating the falcipain 2A and 2B paralogs, gene conversion has been a much more potent process for generating variation than has been mutation.
Estimated Age of the Paralogs
The synonymous differences between the consensus sequences of the paralogous genes can be used to estimate the most recent common ancestor (MRCA in table 2) of the genes. This is not necessarily the time at which the original gene duplication occurred. It is rather the time since the paralogs were last identical in sequence. Identity could be created if a large gene conversion in a lineage rendered the paralogs identical, and then this chromosome went to fixation in the population. In other words, comparison of the consensus sequences cannot distinguish between the most recent complete homogenization due to gene conversion and the time of the original gene duplication.
The estimated age of the most recent common ancestor of the falcipain 2A and 2B paralogs ranges from 8.8 to 20.6 MYA, with an average across all estimates of 15.2 MYA (table 2). The estimation procedure used in table 2 follows that of Rich et al. (1998) and makes use of estimates of the mutation rates of twofold (µ2) and fourfold (µ4) degenerate sites. The latter estimates are obtained by comparisons of coding sequences between P. falciparum and either P. reichenowi (a chimpanzee parasite) or P. berghei (a rodent parasite), taking into account the uncertainty in the time of divergence of these species (5 to 7 MYA for the former species pair and 55 to 129 MYA for the latter). In table 2, the numbers 688 and 250 refer to the number of twofold and fourfold degenerate sites in both paralogs together, and S is the number of twofold or fourfold degenerate sites at which the paralogs differ.
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The mature form of falcipain 2A consists of residues Q244 to E484 (Shenai et al. 2000; Sijwali, Shenai, and Rosenthal 2002). Across this region of the protein, the consensus sequences of falcipain 2A and 2B differ in 10 residues. Five of the residues (M245I, E248D, E249A, R255L, and E257N) are in the region Q244 to F260 that is important for proper folding (Sijwali, Shenai, and Rosenthal 2002).
The localization of the remaining five differences are shown in figure 3. The two differences on the left (P343T and G345D) are localized in a loop region far away from the catalytic site. The remaining three differences between falcipain 2A and 2B (V393I, A400P, and Q414E) are localized in close proximity to residues that are predicted to interact with the substrate Z-FR-AMC (D413, D397, D398, L415, and S392). As noted in the Discussion, the difference Q414E is a candidate for affecting substrate affinity and specificity.
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Discussion |
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That gene conversion may play a potentially important role in generating polymorphisms among related sequences is indicated by the results in table 1. Although the falcipain paralogs are separated by a distance of 10.8 kb, the rate of gene conversion between them is estimated from nonequilibrium theory to be approximately 1,400 to 1,700 times greater than the rate of mutation (table 1). This may be somewhat of an overestimate because it is based on the assumption that P. falciparum shared a most recent common ancestor 7,500 years ago, but even the equilibrium theory implies that the rate of gene conversion relative to mutation is 20 or more (table 1). Gene conversion as a source of genetic variation in P. falciparum may therefore be more important than previously realized. Especially among the AT-rich sequences in 5' untranslated regions, 3' untranslated regions, and introns, there may be regions close enough together and with sufficient similarity to serve as templates for gene conversion. If these regions are short, they may be difficult to detect, but one indication of possible gene conversion would be significant clustering of polymorphisms within a sequence.
Implications for Selective Constraints Due to High-AT Content
The divergence at synonymous sites between the falcipain paralogs strongly suggests that there are no exceptionally strong constraints on the nucleotides at synonymous sites as a result of high AT content or T skew (Jongwutiwes et al. 2002; Mu et al. 2002). This is also true of the 5' and 3' flanking sequences (fig. 4). The flanking sequences are very AT rich, but they are not conserved. Although the falcipain paralogs have been evolving for a very long time to accumulate so many differences, the large number of differences suggests that the lack of polymorphisms observed in the introns and flanking sequences of other genes cannot easily be attributed to their skewed AT content alone (Jongwutiwes et al. 2002).
Implications for Highly Divergent Genes
The result in figure 2 is potentially important in understanding how certain alleles in P. falciparum can be extremely divergent, even though the population may have gone through one or more recent bottlenecks in population size. In particular, two highly divergent alleles may originally have been highly divergent paralogs (Hartl et al. 2002). Both paralogs could have passed through a bottleneck, even one as extreme as a single haploid genome. Afterward, if one lineage lost one paralog and another lost the other paralog, and these deletions replaced the duplication in the population, the coding sequences in the deleted chromosomes would once again become alleles, but extremely divergent alleles.
A possible example of this process is found in the gene encoding merozoite surface protein 1 (Msp1). Based on the extreme divergence of the MAD and K1 types of Msp1 alleles, Hughes (1999) has estimated that they may have diverged from a common ancestor about 48 MYA. This would make the Msp1 polymorphism the oldest known polymorphism in any organism, and such an ancient divergence seems to be completely inconsistent with a recent common ancestor for P. falciparum. To account for such an apparently extreme age, we have suggested that the MAD and K1 alleles may originally have been highly divergent paralogs (Hartl et al. 2002). Both paralogs could have passed through a bottleneck, and then one lineage lost the MAD paralog and another lost the K1 paralog.
Implications for Protein Structure
The biological significance of the 26 amino acid replacements in the consensus sequences for the prodomains of falcipain 2A and 2B is unclear because of the unique N-terminal region for which structural models are not yet available. All of the falcipains have prosequences 2 to 3 times the length of other known papain family proteases. The prosequence contains a putative type II membrane-spanning domain. Similar proforms of the proteases have been identified in the aspartic proteases, which also are active in red blood cells during hemoglobin degradation (Francis, Sullivan, and Goldberg 1997; Ban-erjee et al. 2002). The prodomain has been reported to be a potent, reversible inhibitor of mature falcipain-2 (Sijwali, Shenai, and Rosenthal 2002), possibly involved in the controlled release of parasites from red blood cells through skeletal protein degradation (Dua et al. 2001; Sijwali et al. 2001). One might speculate that the differences in the prodomain might affect activation of the enzyme for hemoglobin degradation and control of parasite release, as well as confer resistance to drugs involved in the hemoglobin degradation pathway.
The two differences P343T and G345D affecting the loop in figure 3 may also affect protein conformation. Inspection of a structural alignment of cysteine proteases including human cathepsins L, K, and V, Zingiber officinale protease II, Trypanosoma cruzi cruzain, Carica papaya papain, porcine cathepsin H, and Actinidia chinesis actinidin revealed that the conformation of the first part of this loop (corresponding to D344 to S351 in falcipain 2A) was conserved among cysteine proteases. It is possible that the nonconservative substitutions in the P343-D344-G345-D346 motif in falcipain 2A may affect the local loop conformation as well as the folding of the whole protein.
The differences between falcipain 2A and 2B that are near the catalytic site (V393I, A400P, and Q414E in figure 3) are likely to be important. These residues are located in close proximity to residues that are predicted to interact with the substrate Z-FR-AMC (D413, D397, D398, L415, and S392). The position of Q414 in falcipain 2A is near residues D413 and L415 that are predicted to interact directly with the substrate. It is therefore possible that the Q414E difference influences substrate affinity and specificity. In falcipain 2A, Z-LR-ACE has been reported to be slightly more effective as a substrate than Z-FR-ACE and Z-VLR-ACE (Shenai et al. 2000). Compared with falcipain 2A, all tested Z-P2-P1-ACE substrates had lower second-order rate constants (kcat/Km) in falcipain 1 and 3 (Salas et al. 1995; Francis et al. 1996; Sijwali et al. 2001).
Implications for Malaria Therapy
Owing to the essential role of proteases in P. falciparum pathogenicity, protease inhibitors are heavily investigated as potential chemotherapeutic agents (Rosenthal et al. 1991; Semenov, Olsen, and Rosenthal 1998; Joachimiak et al. 2001; Singh and Rosenthal 2001). Studies administering cysteine protease inhibitors that block hemoglobin hydrolysis in mice infected with P. vinckei or Trypanosoma cruzi have shown promising results (Rosenthal, Lee, and Smith 1993; Engel et al. 1998; Olsen et al. 1999). Most of the studies performed have assumed a single protein target where the falcipain 2 enzyme has been reported to constitutes 93% of soluble trophozoite cysteine protease activity, as measured with the substrate Z-Phe-Arg-AMC (Shenai et al. 2000). The discovery of a second copy of the falcipain-2 in the malaria genome is therefore of importance for further development of protease inhibitors.
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Acknowledgements |
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Footnotes |
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Rose Crozier, Associate Editor
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