* Departamento de Genética, Universidad de Valencia, Valencia, Spain
Laboratory of Genetics and Molecular Medicine, Instituto de Biomedicina, Consejo Superior de Investigaciones Científicas, Valencia, Spain
Correspondence: E-mail: ignacio.marin{at}uv.es.
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Abstract |
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Key Words: comparative genomics GSTs neuropathies
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Introduction |
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GDAP1 protein has sequence similarity with glutathione S-transferases (GSTs) (Cuesta et al. 2002; Baxter et al. 2002). GSTs belong to many different classes that are defined by all members of a class having high sequence similarity and also common structural and functional features (reviewed in Sheenan et al. 2001; and Sherratt and Hayes 2002). However, we already detected that GDAP1 sequence was different enough from those of known GST classes as to suggest that GDAP1 could belong to a novel, still undescribed, class (Cuesta et al. 2002). In addition to its distinctive primary sequence, GDAP1 protein shows structural features that are absent in canonical GSTs. Thus, two or three carboxyl-terminal transmembrane domains were predicted in GDAP1 protein (Baxter et al. 2002; Cuesta et al. 2002), whereas most GSTs are cytosolic enzymes and lack those domains. However, the precise characterization of the relationships among GDAP1 genes and GSTs is a complex task because GSTs are very numerous and highly heterogeneous. Thus, only a careful comparison to all the GST variants may establish whether GDAP1 truly can be considered a member of a novel class of GST-like genes. In this study, we performed a comprehensive analysis of all known classes of GSTs to determine the origin and evolution of the GDAP1 gene. We demonstrate that GDAP1 belongs to an ancient, clearly defined monophyletic group of genes, distinct from all other GSTs. In addition, precise structural analyses provide clues about the function of GDAP1 and other related proteins and the significance of known human GDAP1 mutations in a biochemical context.
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Materials and Methods |
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Results obtained from each of those searches were merged and protein sequences were aligned using ClustalX version 1.83 (Thompson et al. 1997). We then generated a preliminary phylogenetic tree using the Neighbor-Joining (NJ [Saitou and Nei 1987]) routine available in ClustalX 1.83. Once that primary tree was obtained, duplicates and partial sequences were detected and eliminated. We left only a few partial sequences for their relevance to this study, being clearly very similar to human GDAP1 (those sequences are detailed below). In some cases, only one or a few sequences were found together in an independent branch outside of the known GST families. Those sequences were then reanalyzed, generating new TBlastN searches, to determine whether additional members of a small family or subfamily previously undetected could be found. We were unable to generate a reliable alignment of canonical GSTs or GDAP1 proteins with members of two highly dissimilar classes of GSTs, namely the mitochondrial kappa class (Pemble, Wardle, and Taylor 1996; Jowsey et al. 2003) and the microsomal class (Morgenstern, Guthenberg, and DePierre 1982). Those classes were already known to be extremely different from the rest (Snyder and Maddison 1997). Particularly, microsomal GSTs are now included in a different superfamily of proteins known as MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism [Jakobsson et al. 1999; Hayes and Strange 2000]). Therefore, members of those two classes were excluded from our analyses.
Our final database was generated in April 2003 and contained 289 sequences. We then used the most conserved region of those sequences, which includes most of GST domains I and II (Hayes and Pulford 1995) corresponding to amino acids 26 to 283 of the 358-amino acid human GDAP1 protein (fig. 1), to generate a final multiple-protein alignment using again ClustalX 1.83. Alignments were then manually corrected using GeneDoc version 2.6 (Nicholas and Nicholas 1997). In some ambiguous cases, new Blast searches were performed to establish the most likely alignment for particular pairs or groups of sequences. Phylogenetic trees were obtained both by the NJ and maximum-parsimony (MP) methods, using the routines available in MEGA version 2.1 (Kumar et al. 2001). For NJ, sites with gaps were included and Kimura's correction was used, whereas for MP, the parameters were as follows: (1) all sites included, (2) 10 initial randomly-generated trees used as seeds, and (3) heuristic search using close-neighbor interchange with search level equal to 3. Support for the topologies obtained with those two methods was determined using bootstrap with 1,000 replicates. To check for the robustness of our results, we generated a second type of analysis using, instead of the full-length GST core, only the two most conserved regions found in all the GSTs included in our final multiple-protein alignment (corresponding to amino acids 26 to 101 and 216 to 283 in human GDAP1 protein; see fig. 1 for details). These conserved regions were used to generate NJ and MP phylogenetic trees using the same procedures as those for the full-length GST core trees.
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Phylogenetic trees shown in figures 2 and 3 were generated using MEGA 2.1. GeneDoc version 2.6 was used for displaying and highlighting the multiple-sequence alignments shown in figures 1 and 4.
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Results |
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As shown in figures 1 and 2, we have found genes very similar to GDAP1 in several vertebrate species, including mammals, birds, and fishes. This result strongly suggests that all those genes are orthologs, and, therefore, the origin of this gene predates the fish-tetrapod split. In addition, figures 2A and 2B also show the close similarity of GDAP1 to an obvious paralog that has been called, in humans, GDAP1L1 (Ganglioside-induced differentiation-associated protein 1-like 1 [UniGene Cluster Hs.20977, located at chromosome 20q12-q13]) This gene also originated before the fish-tetrapod split, as we can deduce because a very similar sequence is found in the genome of the zebrafish, Danio rerio. A few additional sequences from other species had clear similarities with GDAP1 or GDAP1L1 but were too short to be included in our study. Among them, we found sequences from the mammals Sus scrofa (GenBank accession number BG833972) and Bos taurus (GenBank accession number AL212783), the amphibian Xenopus laevis (GenBank accession number BG813460), and the fish Tetraodon nigroviridis (GenBank accession number AL212873). Although bootstrap support is low, the position in our general phylogenetic trees of single genes in the invertebrate model species Drosophila melanogaster (CG4623 gene) and Anopheles gambiae (agCP6058 gene) suggests they could also be GDAP1 orthologs in those organisms. These two sequences appear also as the closest relatives of GDAP1 and GDAP1L1 genes among all animal sequences when only the most conserved region is used to generate phylogenetic trees (fig. 2B). To avoid the inherent difficulties in obtaining well-supported topologies when a very large data set of highly heterogeneous sequences are included, as well as to show more clearly the relative positions of some of the most significant and extensively studied GST proteins, we generated a second analysis that included only the sequences in our main database that belonged to model animal organisms with fully or very extensively sequenced genomes (Homo sapiens, Mus musculus, D. melanogaster, Anopheles gambiae, and Caenorhabditis elegans). The results of this second analysis are detailed in figure 3, where the NJ tree is presented and where again both the NJ and the MP bootstrap data are shown together. As we can see comparing figures 2 and 3, the topologies for the significant animal GSTs in both trees are almost identical, and when they differ, topologies are only weakly supported. In particular, the GDAP1 group proteins appear as clearly different from all the other GSTs and the close similarity of Drosophila CG4623 and Anopheles agCP6058 with GDAP1 proteins (and not with other GSTs) is much more obvious. We conclude that GDAP1 belongs to a particular class of GST-like genes, having relatives in both vertebrates and invertebrates. However, figure 2A also shows that some other sequences appear also quite close to GDAP1 proteins in our phylogenetic trees. They are single genes in the plants Arabidopsis and Oryza and sequences of eubacterial origin, corresponding to the related genes PcpC (encoding tetrachlorohydroquinone [TCHQ] dehalogenase [Orser et al. 1993; Cai and Xun 2002; Habash et al. 2002]) and LinD (encoding 2,5-dichlorohydroquinone dehalogenase [Miyauchi et al. 1998]) of sphingomonad -proteobacterial species (reviewed in Copley 1998, 2000). To determine whether the similarity of these proteins and GDAP1 is significant, we must first consider the information provided by structural analyses.
In figure 4, we summarize the features detected by the secondary structure prediction programs on the sequences of GDAP1 and GDAP1L1 genes and the invertebrate GDAP1-related sequences. As shown in figure 1, a particular feature of GDAP1, GDAP1L1, and its relatives is the presence of nucleotides encoding a long stretch of additional amino acids between the two most conserved regions of the protein. In figure 4, we show that this long additional region is located by secondary structure prediction programs to lie between what in canonical GSTs are two alpha helices, respectively called 4 and
5 (see e.g., Board et al. 2000; Thom et al. 2001). We will thus refer to the additional region as the "
4-
5 loop." The proteins encoded by the closest GDAP1 relatives in invertebrates also possess an
4-
5 loop, which lends credibility to the hypothesis that GDAP1 genes may have originated before the protostome-deutorostome split. Additionally, Baxter et al. (2002) and Cuesta et al. (2002) mentioned that two or perhaps three transmembrane domains were predicted at the C-terminal end of human GDAP1 proteins. We have searched, using three different programs that detect those domains, whether the proteins that appear as close relatives to human GDAP1 in our trees also are predicted to have transmembrane domains (see Materials and Methods). We have found that all three algorithms coincide in making the strongest prediction for a transmembrane domain at the most distal C-terminus of both GDAP1 and GDAP1L1 proteins in all vertebrate species for which those regions are available. This domain would correspond to amino acids 319 to 340 in human GDAP1 and to amino acids 339 to 362 in human GDAP1L1 (fig. 4). In addition, a second transmembrane domain, located N-terminally with respect to the first one, is predicted by the three programs in both GDAP1 and GDAP1L1 proteins (see also fig. 4), although support for this prediction is weaker. Finally, the program TMpred predicts a third transmembrane domain in GDAP1L1 genes, just upstream of the other two, but this result is not confirmed by the other two programs. When considering the Drosophila putative GDAP1 orthologs, both TMpred and HMMTOP 2.0 also strongly support the presence in the C-terminus of its product of a single transmembrane domain, whereas TMHMM detects some evidence, but it is considered not significant. This discrepancy may be the result of TMHMM being more restrictive than the other two programs (Käll and Sonnhammer 2002). For the Anopheles sequence, only TMPred predicts a transmembrane domain. However, the putative C-terminus of the Anopheles protein lacks similarity with that of the Drosophila protein, and we cannot exclude at present that it may be incorrectly determined. In fact, the putative N-terminal end of this sequence is most likely incorrect (fig. 4).
In addition to GDAP1 and its closest relatives, the above-mentioned plant and eubacterial genes generate products that also present an 4-
5 loop (fig. 1). This might suggest again an evolutionary relationship, but the presence of this loop could also be interpreted as a convergent feature; that is, the sequences that encode this extra loop may have independently evolved in totally unrelated genes. This putative convergence could generate a spurious proximity of all those sequences in our phylogenetic trees when the full-length GST core is analyzed. The fact is that only a few GSTs have an extended region between helices
4 and
5, and those presenting it would have a greater chance to appear clustered together. Thus, the analyses performed using only the most conserved regions of the GST core (fig. 2B), which obviously do not include the
4-
5 loop (fig. 1), became crucial to determining whether the plant and eubacterial sequences and GDAP1 sequences are indeed related. As it can be seen in figure 2B, the relative proximity in the phylogenetic trees of GDAP1 proteins and the plant and eubacterial sequences holds true even when the extra region is eliminated. Thus, a relationship between GDAP1 genes and these sequences is supported, although certainly not strongly, by our data. Interestingly, neither the plant nor bacterial putative relatives of GDAP1 present any potential transmembrane region.
The currently known mutations in human GDAP1 that cause CMT4A neuropathy are also shown in figure 4 (data derived from Baxter et al. 2002; Cuesta et al. 2002; Nelis et al. 2002; Azzedine et al. 2003; Boerkoel et al. 2003; De Sandre-Giovannoli et al. 2003; and Senderek et al. 2003). These mutations can be grouped into a few types: (1) those that generate truncated versions of the protein, affecting the GST domain (W31X, Q163X, S194X, L223X, and G262fsX284); (2) single amino acidic changes in the GST-like core domain (R120Q; R282C); (3) a single mutation in the predicted first transmembrane domain (R310Q); (4) a mutation that generates a truncated version of the protein that lacks the C-terminal end (T288fsX290); (5) a single amino acidic change that affects a residue that is conserved in both GDAP1 and GDAP1L1 genes, found in the 4-
5 loop (R161H); and (6) splice site mutations ( IVS4 + 1G > A; IVS3 2A > G. This last mutation apparently eliminates exon 4, being therefore equivalent to a deletion of part of the GST domain (see De Sandre-Giovannoli et al. 2003). These highly heterogeneous results demonstrate that all the characteristic regions of this protein (GST domain; C-terminal, putative transmembrane domain; and
4-
5 loop) can be mutated to generate an autosomal recessive CMT pathology.
Despite considerable primary sequence divergence, members of different GST classes have similar three-dimensional structures (summarized in Sheenan et al. 2001; and Board et al. 2000). We were interested in determining whether GDAP1 proteins may have a similar fold. We found that the sequence of GDAP1 is similar enough to those of GSTs to generate a prediction of most of its three-dimensional structure. We used Swiss-model to reconstruct the three-dimensional structure of GDAP1 based on the most similar GST structure available, a plant phi-class GST (Neuefeind et al. 1997 [see Materials and Methods]). As shown in figure 5, GDAP1 is modeled as having the thioredoxin fold at its N-terminus (domain I), characterized by four beta sheets and three alpha helices (1 to
3, left side of fig. 5). This fold is highly conserved in all GSTs and GST-related proteins (Senderek et al. 2003). The structure of the C-terminally located domain II is more difficult to predict. Part of helix
5 and helices
6 and
7 could also acquire a three-dimensional structure similar to that found in GSTs (fig. 5, right). On the other hand, helix
4, part of
5, and, logically, the
4-
5 loop, cannot be properly modeled using canonical GSTs as templates (fig. 5). Because canonical GSTs dimerize, we examined further whether the
4-
5 loop would be situated in the interaction surface by properly juxtaposing two models as the one shown in figure 5. We found that, if indeed the
4-
5 loop is located more or less as the interhelical region between
4 and
5 in canonical GSTs (represented in white in fig. 5), it would be clearly not be part of the interaction surface (not shown).
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Discussion |
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Obvious orthologs of GDAP1 exist in many vertebrate species, including fishes. GDAP1 paralogs, corresponding to the closely related GDAP1L1 human gene and its orthologs, also are found in different vertebrates. Moreover, GDAP1-related genes, most likely with a common evolutionary origin, are detected in invertebrates. These results suggest that the GDAP1 class of GSTs may have originated before the protostome-deuterostome split, perhaps 700 MYA. In addition, we have detected genes notably similar in sequence and also in structure to GDAP1 in a few plants and, even more surprisingly, bacterial species. To explain the plant results, we favor the hypothesis that GDAP1-class genes may in fact have originated very early in eukaryotes, before the plant-animal split. Thus, we predict that GDAP1 genes could be found in other eukaryotic lineages, and that lack of these genes in some eukaryotes would be the result of secondary losses.
The classification of the PcpC and LinD genes has always been problematic. Their products were originally classified as theta-class GSTs. They were later ascribed to the zeta class, despite a low level of sequence similarity. This reassignment was based on two facts. First, the presence of two catalytically essential serine and cysteine residues at the N-terminal ends of the proteins encoded by both these genes and in zeta-class GSTs. Second, the possibility that TCHQ dehalogenase (encoded by PcpC) works like maleylacetoacetate isomerase, a role performed by some zeta-class GSTs (Anandarajah et al. 2000 [reviewed in Copley 2000]). However, our results clearly show that both in sequence and structurally, PcpC and LinD are more similar to GDAP1-class genes than to zeta-class GST genes, suggesting their classification should be re-evaluated. If these bacterial genes are confirmed to be significantly related to GDAP1-class genessomething that our results do not fully prove and that probably will require the characterization of more genes of this classthis may be difficult to explain. We think that the presence of GDAP1-related genes in a few closely related proteobacteria could not be explained by conventional vertical transmission. We should hypothesize a relatively recent horizontal gene transfer event, perhaps from a eukaryote containing a GDAP1-like gene to an eubacteria. Evidence for horizontal transfer events of genes that are involved in glutathione biosynthesis have been recently obtained (Copley and Dhillon 2002). Phylogenetic analyses of bacterial GSTs also suggest that horizontal gene transfers may have occurred (summarized in Vuilleumier and Pagni 2002). Thus, eukaryotes and those prokaryotes that use glutathione may have been able in the past to share significant biochemical novelties, through horizontal gene transfer, to better utilize that molecule. In any case, the study of those bacterial proteins is unlikely to provide insights into GDAP1 functions in animals. They have highly specific functions that are unlikely to exist in eukaryotes (reviewed in Copley 1998, 2000; and Vuilleumier and Pagni 2002).
The distribution of the known mutations in the GDAP1 gene that have been associated with CMT disease suggest that not only the characteristic GST homology region but also the most C-terminal end of the molecule, a short region that is predicted to contain two transmembrane domains, is important for the proper function of the GDAP1 protein. This is shown by the fact that truncations or amino acidic substitutions in that region have pathological consequences. In addition, the existence of a single amino acidic substitution (R161H) generating CMT disease that affects a conserved residue located in the middle of the 4-
5 loop characteristic of GDAP1 and GDAP1-related genes demonstrates that this peculiar region is also very significant for protein function. The large amount of available GST sequences gives obvious clues about significant, and thus highly conserved, amino acids in the GST core domain. Similarly, evolutionary conservation in both GDAP1 and GDAP1L1 may provide useful information about which residues in the
4-
5 loop are functionally significant.
Our analyses do not offer much novel information about GST evolution. Most of the results shown here were already anticipated by Snyder and Maddison (1997). Those authors, who used a much smaller set of sequences, attributed the lack of resolution of their phylogenetic trees to the limited amount of available data, especially the fact that the phylogenetic range of the species included was narrow. This interpretation now appears to be incorrect, because neither the inclusion of many more sequences nor the combined use of different analytical methods solves the relationships among the innermost branches of the tree. As far as we know, ours is the most comprehensive analysis of GST proteins ever performed, and we have even expanded it to include close to 500 GST sequences (unpublished data), but, in all cases, the relationships among GST classes are largely unresolved. We conclude that the information provided by the GST core is insufficient to generate well-supported trees for all GST classes. The alternative approach of concentrating the analyses on closely related classes, to understand the evolution of particular groups of GSTs, may be thus more fruitful. Finally, the fact that we have not found any other novel GST classes suggests that GDAP1 genes may be the last type of GST-like proteins to be described in mammals.
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Acknowledgements |
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Footnotes |
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