Institute of Molecular Evolutionary Genetics, Department of Biology, The Pennsylvania State University
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Abstract |
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Introduction |
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An interesting question regarding the evolution of Ig's is as to how and when the heavy-chain Ig emerged. A number of studies showed that Ruminantia (sheep and cattle) and Suidae (pigs), two groups of mammals closely related to Camelidae, possess only the conventional Ig (e.g., Sun et al.1994
; Dufour, Malinge, and Nau 1996
; Sinclair, Gilchrist, and Aitken 1997
). Therefore, the emergence of the heavy-chain Ig must have occurred only relatively recently. However, a detailed phylogenetic analysis is necessary to answer this question. Previous studies of phylogenetic relationships among Ig's from different species have concentrated primarily on the variable region genes (e.g., Ota and Nei 1994
; Andersson and Matsunaga 1996
; Sitnikova and Nei 1998
) because these genes encode antigen-binding regions and are largely responsible for the generation of diversity of antibody repertoires. We therefore conducted a phylogenetic analysis of the variable region (VHH) genes of the heavy-chain Ig from camels and llamas, together with the VH genes of the conventional Ig from seven mammalian species, namely humans, mice, rabbits, sheep, cattle, swine, and camels.
The loss of the L chain and the CH1 region in this novel type of Ig raises an important question as to how its antipathogenic function has been maintained. Without the VL regions, the antipathogenic function of the heavy-chain Ig is now carried out by the VHH regions. VHH genes are apparently recent duplicates of VH genes, but their gene products have different characteristics with respect to solubility, efficiency of antigen binding, and the antigens recognized (Sheriff and Constantine 1996
; Nguyen et al. 2000
). Therefore, it seems that adaptive evolution after duplication of VH genes has led to the dichotomy of these two types of Ig's. One way of studying adaptive evolution in Ig's is to examine the extent of positive Darwinian selection in the antigen-binding regions because the need to bind a diverse spectrum of antigens may be aided by natural selection (e.g., Tanaka and Nei 1989
; Sitnikova and Nei 1998
). We have therefore examined the effect of positive selection in the antigen-binding regions of VHH genes. However, a more direct way of studying this problem would be to compare the extent of positive selection or purifying selection at each codon site of VHH genes in comparison with that of VH genes. This is because the structural change of the heavy-chain Ig is likely to have shifted the antigen-binding regions of VHH genes (reviewed in Muyldermans, Cambillau, and Wyns 2001
), and a difference in selective force at a given amino acid site between VHH and VH genes may be viewed as an indication of adaptive change that has occurred in VHH genes. Here, a site-by-site examination is important because adaptive evolution can theoretically occur at any site, and examination of the average selective force over a region may obscure the effect of selection at single sites or at a subset of sites.
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Materials and Methods |
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For the purpose of the phylogenetic tree construction, we used the same VH sequences from humans, mice, rabbits, sheep, cattle, and pigs as those used by Sitnikova and Su (1998)
because they are representatives of the mammalian VH repertoire including three major VH gene groups (A, B, and C) (Ota and Nei 1994
). The VH data set from Sitnikova and Su (1998)
included primarily germline genes. Only for the swine species Sus scrofa did they use cDNA sequences. However, we recently found seven pig germline genes published (accession numbers AF06469286), and included them in the present study.
Phylogenetic Analysis
The VHH and VH nucleotide sequences were aligned using the computer program Clustal W (Thompson, Higgins, and Gibson 1994
), with additional minor modifications by visual inspection. Phylogenetic analyses were conducted using the MEGA computer program (Kumar, Tamura, and Nei 1993
), except when parsimonious trees were constructed (see later). We estimated the evolutionary distances between nucleotide sequences using the uncorrected p-distance. The p-distance was used because it is known to give better results when the number of sequences is large and the number of nucleotides used is relatively small (Nei and Kumar 2000
; Takahashi and Nei 2000
). The phylogenetic tree was constructed using the neighbor-joining (NJ) method (Saitou and Nei 1987
). Sites with alignment gaps (indels) in each pair of sequences under comparison were omitted (the pairwise deletion option in MEGA). However, we obtained essentially the same result when the option of complete deletion (i.e., omitting all sites with indels) was used.
To examine the reliability of the tree topology, we constructed a parsimony consensus tree using PAUP* (Swofford 1998
). In this case, the full heuristic search (standard stepwise addition + tree bisectionreconnection [TBR]) method was done for 500 bootstrap replications, and for each replication the TBR search was repeated 100 times. The resultant bootstrap 50% majority-rule consensus tree was compared with the NJ tree.
Selection on the Antigen-binding Regions of Camelid VHH Genes
In general, the Ig variable region genes can be divided into five regions: two CDRs, CDR1 and CDR2, and three framework regions (FRs), FR1, FR2, and FR3. The CDRs were antigen-binding sites and highly variable (Davies, Padlan, and Sheriff 1990
). A number of previous studies showed that positive selection is operating on the CDRs of VH genes, as indicated by the fact that the average nonsynonymous (amino acid altering) substitutions per site (
N) is higher than the average synonymous (silent) substitutions per site (
S) in the CDRs, and that the reverse is true for the FRs (e.g., Tanaka and Nei 1989
; Rothenfluh et al. 1994
; Ota and Nei 1995
; Sitnikova and Nei 1998
). We first studied adaptive evolution in the heavy-chain Ig by examining if there is positive selection operating in the CDRs of VHH genes. For this purpose, we computed
N and
S for CDRs and FRs separately. Here we used the definition of CDRs and FRs presented by Hamers-Casterman et al. (1993)
. It should be noted that this definition for the VHH genes may not be appropriate because it was based on the 3D structure of the conventional Ig and on the alignment of VHH genes to VH genes. However, a number of crystal-structural studies of the heavy-chain Ig's (Desmyter et al. 1996
; Spinelli et al. 2000
) showed that this definition is generally acceptable. We therefore used it in our study. We deleted 21 llama VHH genes, 9 camel VHH genes, and 5 camel VH genes from the aligned data set because they contained long alignment gaps. As a result, a total of 211 sequences were used in this analysis. In addition, we used the original Nei and Gojobori method (Nei and Gojobori 1986
) rather than the modified Nei and Gojobori method (Zhang, Rosenberg, and Nei 1998
) to obtain the values of
S and
N because our estimate showed that the transition-transversion bias in the VHH genes (R = 0.99) was low, and because the original method is more conservative than the modified method. The variance of
N and
S was computed using the method of Nei and Jin (1989)
.
Selection on Individual Codon Sites of Camelid VHH Genes
To study adaptive evolution in the heavy-chain Ig in more detail, we compared the selective force operating at each codon site of VHH and VH genes. In this analysis, we chose 32 representative VHH genes from camels and 38 representative genes from llamas. The 70 VHH sequences used were chosen from 180 camel and llama VHH sequences by first constructing an NJ tree of the 180 sequences and then choosing randomly one sequence from each tight cluster found in the tree. In this way, we excluded the closely related sequences to speed up the computational process (see later).
In this analysis we used the method of Suzuki and Gojobori (1999)
, which can be briefly summarized as follows. First, the ancestral nucleotide sequence of each interior node was inferred by using a parsimony criterion for a given tree topology, which was generated by the NJ method. The numbers of synonymous (st) and nonsynonymous (nt) sites were then computed for each codon site by taking into account the extant sequences as well as the ancestral sequences. Next, for each codon site the observed numbers of synonymous and nonsynonymous changes for all branches were summed up to obtain the total numbers of synonymous (sc) and nonsynonymous (nc) changes, respectively. The probability of occurrence of nc nonsynonymous changes when the total number of changes was tc (=sc + nc) and all the changes were assumed to be neutral is then given by
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For the purpose of comparison, we also applied the same procedures to a combined data set of human VH3 genes and camel VH genes. Here we used genes from the human VH3 subfamily because these genes are known to be closely related to the camelid VHH genes (Vu et al. 1997
; Nguyen et al. 2000
). We also included camel VH genes for comparison because they appear to share the last common ancestor with VHH genes (see Results). Another important reason for using both human and camel VH genes is that the statistical power of the method of Suzuki and Gojobori increases as the number of sequences used or the total number of nucleotide changes observed for a certain site increases. In general, at least 5060 fairly divergent sequences should be used (T. Gojobori, personal communication). Therefore, the use of either human VH3 genes or camel VH genes alone is unlikely to be sufficient. Lastly, our preliminary study indicated that if examined separately, the human VH3 and the camel VH genes showed quite similar profiles of selective forces, so that it seemed reasonable to combine these two homologous data sets. In this analysis, the number of human VH3 genes used was 58, and the number of camel VH genes used was 38. All human VH3 genes were retrieved from the international ImMunoGeneTics (IMGT) database (http://imgt.cines.fr:8104; Lefranc et al. 1999
), whereas camel VH genes were retrieved from GenBank, as mentioned earlier. The 58 human sequences were chosen from a total of 113 human VH3 genes and the 38 camel sequences from 50 camel VH genes, using the same criterion as that used for VHH genes. The aligned sequences for these genes are available from the World Wide Web at http://mep.bio.psu.edu/databases.
The above computation was done by using the computer program SGI written by C.S., except for the reconstruction of ancestral sequences, which was done by using the computer program pamp in the PAML package (Yang 2000
). The SGI program is available at http://mep.bio.psu.edu/.
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Results |
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The llama VHH sequences show longer root-to-tip branch lengths than the camel VHH genes (fig. 1 ). However, this does not necessarily mean that positive selection has occurred in the FRs of the llama VHH genes. As the llama genes used were obtained from cDNA libraries, the high diversity among the llama VHH sequences could be the result of the effect of somatic mutations in the FRs, the relaxation of functional constraint, or random chance. However, to understand the cause of the longer branches of llama VHH genes, it is necessary to use germline VHH sequences from L. glama. Note that some human and mouse germline genes appear as long root-to-tip branch lengths like those of llama cDNA.
Adaptive Evolution: Selection in the CDRs and FRs of Camelid VHH and VH Genes
In this analysis, we examined whether or not positive selection operates on the CDRs of camelid VHH genes. We also included the camel VH genes in the analysis because no such study has been done before. We divided all camel and llama genes into three groups: camel VHH, llama VHH, and camel VH genes (Table 1 ). The values of S and
N were calculated separately for three different comparisons: within each group, between different groups, and for the entire data set. The results obtained are presented in Table 1
and may be summarized as follows: (1) When CDRs are considered,
N is greater than
S in all within- and between-group comparisons, although the difference is statistically significant in only one comparison. It is interesting that
N is greater than
S even in the comparison between VH and VHH genes. This suggests that the CDRs of the VH and VHH genes are located in the same DNA regions, as discussed earlier. Table 1
shows that if we consider the entire data set of all sequences, the difference between
N and
S is highly significant. This result is consistent with the conclusion obtained by Tanaka and Nei (1989)
and suggests that the CDRs are subject to positive Darwinian selection in both VHH and VH genes. (2) In contrast, in FRs
N is almost always smaller than
S, and if we consider the entire set of sequences, the former is significantly smaller than the latter. (3) Comparison of
S values between the CDRs and the FRs in each within- and between-group study shows that
S is more or less the same for all CDRs and FRs, and none of the differences is statistically significant. In contrast,
N in the CDRs is always greater than
N in the FRs, and the difference is always significant. This result also suggests that positive selection operates primarily in the CDRs. This conclusion appears to apply to both VH and VHH genes.
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A closer examination of the substitutions occurring in the FR2 of VHH genes reveals that many nonsynonymous substitutions were from hydrophobic amino acids to hydrophilic ones. For example, the ancestral amino acid at site 44 was presumably hydrophobic because a great majority of the camel VH and human VH3 genes have the amino acid glycine (G) at this site. However, this glycine has changed to hydrophilic amino acids (E, K, and D) in 65 out of the 70 sites studied in the camel and llama VHH genes. To explain this observation, we propose that mutations from hydrophobic amino acids to hydrophilics might have been fixed by directional selection at some sites in the FR2 region for the following two reasons. First, in the case of the conventional Ig, it is known that before the Ig is assembled in the endoplasmic reticulum, some hydrophobic amino acids in FR2 are bound by the chaperon protein BiP (Knarr et al. 1995
), and that during the antibody assembly process, this BiP protein has to be replaced by an L chain before the Ig can be secreted to the circulatory system. In the heavy-chain Ig, once the hydrophobic amino acids mutate to hydrophilic ones, it would be impossible for the BiP to bind to them. Therefore, the heavy-chain Ig can be secreted from the cell without the L chain (Muyldermans and Lauwereys 1999
). Second, these mutations would increase the solubility of the heavy-chain Ig and would probably compensate for the loss of solubility without the VL region (Ghahroudi et al. 1997
). With regard to these two aspects, the high proportions of nonsynonymous changes in FR2 of VHH genes might be an adaptation to the loss of the L chain.
Another difference between VHH and VH genes was observed in the region immediately preceeding (including the beginning of) CDR1 (positions 2432; fig. 2
). Sites of this region showed much higher proportions of nonsynonymous changes in VHH genes than in VH genes (see fig. 2A
). Interestingly, this region corresponds to the first hypervariable region (the H1 loop; Chothia et al. 1992
) and is where the main-chain atoms of the first structural loop locate. Crystallographic studies of the VHH-antigen complexes showed that the amino acids located in this region interact with the antigens (Desmyter et al. 1996
; Decanniere et al. 1999
; Spinelli et al. 2000
). The nonsynonymous changes in this region of the VHH genes are likely to reflect an additional expansion of the antigen-binding repertoire of the heavy-chain Ig to compensate for the loss of the antigen-binding specificity without the VL regions.
A third difference is observed at position 14, which is well conserved in VH genes and yet subject to strong positive selection in the VHH genes (fig. 2
). We found that most nonsynonymous substitutions occurring at this site were conservative rather than radical because (1) the ancestral amino acid at this site was probably proline (P), if we consider the fact that a majority of the camel VH and human VH3 genes have a proline at this site; and (2) in VHH genes almost all amino acids (A, S, and T) that replaced proline at this site are small and neutral, just like proline, and in only one sequence was a relatively large and charged amino acid (H) found. It is puzzling why different small and neutral amino acids are positively selected at this site. This site is unlikely to be involved in antigen recognition because crystal-structure study indicates that this amino acid site resides at the region opposite the antigen-binding pocket (Desmyter et al. 1996
). Examining the crystal structure, we found that this site is located extremely close to a small hydrophobic socket (formed by site 11 in FR1 and sites 110 and 112 in FR4 encoded by the gene JH; Lesk and Chothia 1988
), which is well conserved at the sequence level and contacts the CH1 region in the conventional Ig. In the heavy-chain Ig, however, this function of the socket must have changed because of the loss of the CH1 domain. This functional change may require conformational changes in the flanking regions. For example, as the socket is hydrophobic, it may have to be buried inside the molecule to minimize exposure to the solvent. Examining the indexes of solvent accessibility (the tendency to have access to the solvent) of these amino acids (Bordo and Argos 1991
; Karplus 1997
), we found that all new amino acids (A, S, and T) at position 14 have considerably lesser solvent accessibility and smaller nonpolar surface area compared with the ancestral amino acid (P). This suggests that these mutations are directional and may have led to a conformational change of the flanking region of the socket. If this is the case, the high frequency of nonsynonymous substitutions at site 14 might be an adaptation to the loss of the CH1 region in the heavy-chain Ig.
The profile of the extent of purifying selection (fig. 3 ) is negatively correlated to that of positive selection (fig. 2 ). In particular, (1) the FR2 region of VHH genes shows a lesser extent of purifying selection than the FR2 of VH genes, (2) in VHH genes, codon sites 2432 show considerably lower proportions of synonymous changes than those in VH genes, and (3) in VH genes, purifying selection operates at site 14, whereas positive selection occurs at this site in the VHH genes, as mentioned earlier.
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Discussion |
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In the present paper, we examined the adaptive changes of codons at individual amino acid sites using the method of Suzuki and Gojobori (1999)
. This method appears to give results that are more reliable than the previous ones (e.g., Fitch et al. 1997
; Nielsen and Yang 1998
) because it is based on more realistic assumptions and models (Suzuki and Nei 2001
). For example, in the method of Fitch et al., the computation of pn in equation (1) is done under the assumption that st/(st + nt) is the same for all variable amino acid sites. However, as a majority of amino acid sites are subject to purifying selection rather than to positive selection with st > nt, this method tends to underestimate P for many sites, and consequently it tends to overestimate the number of sites with positive selection. In contrast, Nielsen and Yang assumed that the ratio of nonsynonymous to synonymous substitution rates is the same for all positively selected codon sites. In reality, this ratio is expected to vary from codon to codon.
To see whether different statistical methods give different results, we applied the codeml program of the PAML package (Yang 2000
) to identify positively selected amino acid sites by the maximum likelihood method of Nielsen and Yang (1998)
. The results obtained by this method were different from those obtained by the Suzuki-Gojobori method, except for the VHH CDR1 region where both methods identified the same three sites. Moreover, the results of the codeml program depend on the initial value of
, the ratio of nonsynonymous changes to synonymous changes. Different initial
values will generate different results, suggesting that the method often fails to find the global maximum likelihood value. It is also known that the Nielsen-Yang method often gives false positive results (Suzuki and Nei 2001
). In fact, the Nielsen-Yang method suggested more positively selected sites, especially in the FR regions, where it was difficult to offer a biological explanation. However, the identification of selective sites by the current statistical methods is subject to sampling errors (Suzuki and Gojobori 1999
). Therefore, the validity of the results obtained by these methods should eventually be examined experimentally by using site-directed mutagenesis or some other molecular techniques (e.g., Jermann et al. 1995
).
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Acknowledgements |
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Footnotes |
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Keywords: adaptive evolution
positive selection
phylogenetic tree
Camelidae
VHH genes
immunoglobulin
Address for correspondence and reprints: Chen Su, Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285. su_chen{at}lilly.com
.
Present address: Department of Ultrastructure, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint Genesius Rode, Belgium
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References |
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