Department of Ecology and Evolutionary Biology, University of Michigan
Correspondence: E-mail: jianzhi{at}umich.edu.
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Abstract |
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Key Words: RNase ruminant colobine adaptive evolution digestion
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Introduction |
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Ruminant artiodactyls and colobine monkeys mainly eat leaves. A foregut-fermenting alimentary system independently originated in these organisms to facilitate the digestion of the cellulose-rich food (Kay and Davies 1994). That is, symbiotic bacteria in the foregut ferment the leaves, and the mammals then recover nutrients by lysing and digesting the bacteria with various enzymes. One important digestive enzyme is pancreatic RNase, which is secreted from the pancreas and transported into the small intestine to degrade RNAs. Earlier studies revealed a substantially greater amount of RNase in the pancreas of ruminants and colobines than in other mammals (Barnard 1969; Beintema 1990). This is related to the fact that rapidly growing bacteria have the highest ratio of RNA-nitrogen to total nitrogen of all cells, and high concentrations of RNase are needed to break down bacterial RNAs so that nitrogen can be recycled efficiently (Barnard 1969). Pancreatic RNases of many mammals are also expressed outside the pancreas (Futami et al. 1997) and have an activity (EAdsRNA) in degrading double-stranded (ds) RNA (Libonati and Floridi 1969). This activity is unrelated to digestion and is thought to be involved in the host-defense against pathogenic viruses (Sorrentino and Libonati 1997; Libonati and Sorrentino 2001).
While most mammals have only one copy of the pancreatic RNase gene that is also known as RNase1, all ruminants examined have three gene copies that are results of two consecutive gene duplications in an ancestral ruminant (Beintema and Kleineidam 1998; Breukelman et al. 2001). The three genes have different expression patterns and they are known as the pancreatic, seminal, and brain RNases, respectively (Beintema and Kleineidam 1998). They also differ in RNase activity and protein structure. For example, seminal RNase forms homodimers, whereas the other two RNases are monomers. Pancreatic RNase has a decreased activity in degrading dsRNA, suggesting that its function is specialized in digesting RNAs released from foregut bacteria (Jermann et al. 1995; Libonati and Sorrentino 2001). On the other hand, seminal RNase has an increased activity against dsRNA (Libonati and Sorrentino 2001). Recently, an independent duplication of the RNase1 gene was found in the douc langur (Pygathrix nemaeus), an Asian colobine monkey (Zhang, Zhang, and Rosenberg 2002). Interestingly, while the sequence and function of one daughter gene (RNase1A) has remained unchanged, the other gene (RNase1B) has a dramatically reduced activity against dsRNA (Zhang, Zhang, and Rosenberg 2002). However, the digestive function of RNase1B has been enhanced by a reduction in its optimal catalytic pH from 7.4 to 6.3, an adaptive response to the lower pH in the colobine small intestine (6 to 7), in comparison to those in other primates (7.4 to 8) (Zhang, Zhang, and Rosenberg 2002). RNases purified from the pancreas of a related colobine species appear to be RNase1B (Beintema 1990; Zhang, Zhang, and Rosenberg 2002), suggesting that RNase1B has taken the digestive role and has left the putative antiviral function (by EAdsRNA) to its paralog, RNase1A. Therefore, specialization in digestion and reduction in the activity of degrading dsRNA occurred independently in ruminant pancreatic RNase and colobine RNase1B after gene duplication, which constitutes functional parallelism. Molecular mechanisms behind the reductions in EAdsRNA have been investigated in ruminants and colobines separately and the molecular mechanism underlying the shift of optimal pH in colobines is also known to some extent. Here, I perform a comparative evolutionary analysis and conduct experiments derived from this analysis to test the hypothesis of sequence parallelism underlying the functional parallelism of the digestive RNases.
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Materials and Methods |
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Evolutionary Analyses
Previously reported DNA sequences of RNase1 genes of artiodactyls and primates were obtained from GenBank. The protein and DNA sequences were aligned using ClustalX (Thompson et al. 1997). Gene trees were reconstructed using the neighbor-joining method (Saitou and Nei 1987) implemented in MEGA2 (Kumar et al. 2001), as well as the likelihood method (Felsenstein 1981) implemented in PAUP* (Swofford 1998). Several different distance measures or substitution models (Jukes-Cantor, Kimura's two-parameter, Tajima-Nei, Tamura-Nei, and general reversible models, with or without the gamma distribution for rate variation among sites [Nei and Kumar 2000]) were used. The interior branch test (Rzhetsky and Nei 1992; Nei and Kumar 2000) and the bootstrap test (Felsenstein 1985) with 1,000 replications were used to examine the reliability of the reconstructed trees.
Site-Directed Mutagenesis, Isolation of Recombinant Protein, and RNase Assay
The douc langur RNase1A gene was subcloned into the pFLAG CTS bacterial expression vector (Kodak, New Haven, Conn.) with a bacterial signal peptide sequence at the N-terminus and an octapeptide DYKDDDDK (FLAG) sequence at the C-terminus (Zhang, Zhang, and Rosenberg 2002). Previous studies showed that the FLAG octapeptide does not interfere with the folding or the catalytic activity of recombinant RNases (Rosenberg and Dyer 1995). The QuikChange site-directed mutagenesis kit of Stratagene (La Jolla, Calif.) was used to generate designed mutations in the gene construct following manufacturer's instructions. The mutations were confirmed by DNA sequencing. Recombinant proteins were isolated from 6 L of bacterial cultures after 4-h induction with isopropyl-1-thio-ß-galactoside (IPTG, 10 µM). After harvest and cell lysis by freeze-thaw and sonication, recombinant proteins were concentrated and isolated by M2 anti-FLAG monoclonal antibody affinity chromatography (Sigma). The concentration of the recombinant protein was determined by quantitative Western analysis with a FLAG-conjugated BAP protein at known concentration (Sigma). The enzyme activity (EAdsRNA) of the recombinant RNase against dsRNA [poly(U)poly(A) combined from poly(U) and poly(A) of Pharmacia (Piscataway, NJ)] was measured by the initial reaction rate at 25°C in 1 ml buffer of 0.15 M sodium chloride and 0.015 M sodium citrate (pH 7.0) with 40 µg substrate and 10 to 100 pmol RNase and was determined from ultraviolet absorbance at 260 nm (Libonati and Floridi 1969). At least three replications of experiments were conducted, with the means and their standard errors computed. This experimental procedure was identical to that used in Zhang, Zhang, and Rosenberg (2002) and the obtained EAdsRNA values are directly comparable. We further measured the EAdsRNA of the recombinant human pancreatic RNase prepared in our lab and that of the cow pancreatic RNase (Sigma). We found that our results are virtually identical to those reported in the literature (Opitz et al. 1998; Libonati and Sorrentino 2001), suggesting direct comparability between our measures of EAdsRNA and those in the literature. Our previous study showed that EAdsRNA is highest at pH 7.0 (Zhang, Zhang, and Rosenberg 2002). Since EAdsRNA is unrelated to the digestion of bacterial RNA and is probably biologically relevant in tissues other than the small intestine, we here measured and reported EAdsRNA at pH 7.0 for all subjects.
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Results |
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I aligned the primate and artiodactyl RNase1 sequences to find whether the five important sites in ruminant proteins that affect EAdsRNA and the nine important sites in colobine proteins overlap. The alignment (fig. 3) showed that there is only one overlapping site between the two sets of sites. However, this overlapping site showed a different substitution in monkeys (R32L) than that (R32C) in ruminants. If I consider only those amino acid changes that reduced EAdsRNA, G38D that occurred in bovine pancreatic RNase did not occur in langur RNase1B; G38 is invariant in primates (Zhang, Zhang, and Rosenberg 2002). The nine amino acid changes in langur RNase1B did not occur in bovine pancreatic RNase either. Furthermore, even when I consider all the amino acid substitutions that occurred in the exterior branches leading to bovine pancreatic RNase and langur RNase1B, none are parallel. Therefore, the parallel functional changes in EAdsRNA observed from colobine and ruminant RNases was due to divergent, rather than parallel, amino acid substitutions. At this time, it is unknown which amino acid substitutions are responsible for the change in catalytic activity against bacterial RNAs in ruminants, and a direct comparison with the langur RNase1B cannot be made. Nevertheless, the fact that there were no parallel amino acid substitutions in bovine pancreatic RNase and langur RNase1B indicates that this functional change likewise was not due to parallel amino acid substitutions.
The occurrence of functional parallelism by different sets of substitutions in two groups of organisms suggests the possibility that the number of sites (N) that can potentially affect the function (i.e., EAdsRNA) is large. With the observation of one overlapping site (although with different substitutions) from two sets of independent substitutions, one may estimate N. The likelihood of observing n overlapping sites is
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Discussion |
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Because it is the parallel reduction in EAdsRNA that is studied here, one may think that the case simply represents functional deterioration, which of course can be due to many different amino acid substitutions. This view is too simplistic. While having reduced EAdsRNA, the digestive RNases of ruminants and colobines have become more specialized in digesting bacterial RNAs. The digestive activity is shown to have increased in colobines (Zhang, Zhang, and Rosenberg 2002), which is probably true in ruminants as well (Libonati and Sorrentino 2001). Therefore, although the reduction in EAdsRNA itself is unlikely an adaptation, the evolution of the RNases is under specific functional constraints and natural selection. It is interesting to see that under parallel pressures for enhanced digestive function, ruminant and colobine RNases independently lost EAdsRNA by different means. More surprisingly, EAdsRNA was enhanced in seminal RNase by substitutions at a different set of sites.
The molecular basis of the RNase activity against dsRNA has been studied quite extensively in bovine seminal and pancreatic RNases, which show 25-fold difference in this activity. Libonati and Sorrentino (2001) proposed that positively charged amino acids (arginines and lysines) are critical to the activity, whereas Opitz et al. (1998) contested their hypothesis. It is worth noting that the difference in EAdsRNA between RNase1A and RNase1B of the douc langur is much greater than that between bovine pancreatic and seminal RNases (fig. 2). Of the nine functionally important substitutions in langur RNase1B, seven involved charge changes (fig. 2B). This observation appears in support of Libonati's hypothesis. However, there are two substitutions (P42S and D83E) that do not alter the net charge of the protein, yet are among the most influential ones in changing EAdsRNA (Zhang, Zhang, and Rosenberg 2002). Therefore, Libonati's theory on charged amino acids is valuable, but additional factors certainly exist.
It is interesting to note here that another protein, lysozyme, has been suggested to undergo parallel evolution in ruminants and colobines, from a bactericidal defense protein to a digestive stomach protein that lyses symbiotic bacteria (Stewart, Schilling, and Wilson 1987). Parallel amino acid substitutions have been identified (Stewart, Schilling, and Wilson 1987; Zhang and Kumar 1997), although they have not been shown to be important to the proposed new role of lysozyme in digestion. Furthermore, as pointed out by Kay and Davies (1994), stomach lysozyme has virtually no catalytic activity in the acidic stomach environment (pH 2 to pH 3) (Dobson, Prager, and Wilson 1984), and its presence in stomach is enigmatic.
Parallel evolution at the protein sequence level has been an interesting subject to many molecular evolutionists because it is often presumed that observed parallel substitutions are functionally important and are under adaptive selection. Rigorously speaking, four requirements appear necessary to establish a case of sequence parallelism that would indicate functional importance of and adaptive selection on the parallel amino acid changes. First, parallel amino acid substitutions are observed in independent lineages. Second, proteins under investigation have independently evolved similar functions. Third, the parallel substitutions are indeed responsible for the parallel functional changes. Fourth, the number of observed parallel substitutions is greater than expected by chance alone. Most claims of sequence parallelism satisfy the first requirement or the first and second requirement. To fulfill the third requirement, experiments involving site-directed mutagenesis and recombinant protein techniques are usually needed. The final necessity is a statistical test demonstrating that the parallel substitutions are not attributable to chance alone. Such a test has been developed by Zhang and Kumar (1997). To my knowledge, there is only one case of sequence parallelism so far discovered that satisfies all four requirements, and it is the red and green opsins of vertebrates. Opsin is the protein component of visual pigments that determine the wavelength of light that turns on photoreceptors. Three amino acid sites experienced parallel substitutions in fish and primate opsins, which have similar changes in maximum wavelength absorption (Yokoyama and Yokoyama 1990). Experimental studies confirmed the functional roles of these three substitutions (Asenjo, Rim, and Oprian 1994) and a statistical test by the method of Zhang and Kumar (1997) rejects the hypothesis that these parallel substitutions were due to chance alone (Zhang, unpublished data). In fact, these parallel substitutions were later found to have occurred on more than two occasions, all being correlated with changes in maximum wavelength absorptions (Boissinot et al. 1998; Yokoyama and Radlwimmer 2001), strengthening the claim that they were under adaptive selection. One can imagine that the number of sites (N in equation 1) that potentially affect the wavelength absorption in red/green opsins is small. Indeed, it has been found that functional variations of red/green opsins of most vertebrates can be explained by amino acid substitutions at five sites (Yokoyama and Radlwimmer 2001). In the future, it would be interesting to reexamine the previously reported cases of sequence parallelism using the above four rules and reevaluate the frequency of adaptive parallel evolution at the protein sequence level.
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Acknowledgements |
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Footnotes |
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Mark Springer, Associate Editor
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