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Analysis of the Na+/Ca2+ exchanger gene family within the phylum Nematoda.

He C, O'Halloran DM - PLoS ONE (2014)

Bottom Line: The most notable feature of the resulting phylogenies was the heterogeneous evolution observed within exchanger subtypes.Specifically, in the case of the CCX exchangers we did not detect members of this class in three Clade III nematodes.We also provided re-annotation for predicted CCX gene structures from Heterorhabditis bacteriophora and Caenorhabditis japonica by RT-PCR and sequencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, The George Washington University, Washington, D.C., United States of America; Institute for Neuroscience, The George Washington University, Washington, D.C., United States of America.

ABSTRACT
Na+/Ca2+ exchangers are low affinity, high capacity transporters that rapidly transport calcium at the plasma membrane, mitochondrion, endoplasmic (and sarcoplasmic) reticulum, and the nucleus. Na+/Ca2+ exchangers are widely expressed in diverse cell types where they contribute homeostatic balance to calcium levels. In animals, Na+/Ca2+ exchangers are divided into three groups based upon stoichiometry: Na+/Ca2+ exchangers (NCX), Na+/Ca2+/K+ exchangers (NCKX), and Ca2+/Cation exchangers (CCX). In mammals there are three NCX genes, five NCKX genes and one CCX (NCLX) gene. The genome of the nematode Caenorhabditis elegans contains ten Na+/Ca2+ exchanger genes: three NCX; five CCX; and two NCKX genes. Here we set out to characterize structural and taxonomic specializations within the family of Na+/Ca2+ exchangers across the phylum Nematoda. In this analysis we identify Na+/Ca2+ exchanger genes from twelve species of nematodes and reconstruct their phylogenetic and evolutionary relationships. The most notable feature of the resulting phylogenies was the heterogeneous evolution observed within exchanger subtypes. Specifically, in the case of the CCX exchangers we did not detect members of this class in three Clade III nematodes. Within the Caenorhabditis and Pristionchus lineages we identify between three and five CCX representatives, whereas in other Clade V and also Clade IV nematode taxa we only observed a single CCX gene in each species, and in the Clade III nematode taxa that we sampled we identify NCX and NCKX encoding genes but no evidence of CCX representatives using our mining approach. We also provided re-annotation for predicted CCX gene structures from Heterorhabditis bacteriophora and Caenorhabditis japonica by RT-PCR and sequencing. Together, these findings reveal a complex picture of Na+/Ca2+ transporters in nematodes that suggest an incongruent evolutionary history of proteins that provide central control of calcium dynamics.

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Sequence and structural analysis of nematode exchangers.(A) Sliding window analysis of nucleotide diversity using DnaSP version 5 [47] with 100 bp windows and 25 bp steps for CCX (upper graph), NCKX (middle graph), and NCX exchanger (lower graph) gene pairs between C. elegans and C. briggsae. (B) Box plot analysis of molecular diversity for each ncx gene (ncx-1 to ncx-10) between C. elegans and C. briggsae. Whiskers extend to data points that are less than 1.5 × interquartile range away from 1st/3rd quartile; center lines show the medians and outliers are shown as dots. (C) Ribbon model of NCX-1 from C. elegans and an NCX-3 ortholog from A. suum (GS_14034). N and C termini are indicated, and red arrowheads refer to structural differences between each NCX. Numbers refer to the transmembrane (TM) domains. Extracellular side is up in left views. The position of two candidate sites in NCX-1 undergoing episodic diversifying selection are indicted - codon 455 and codon 925. In each case the right view is rotated by 90°. Structural predictions were made using Phyre [48] and visualized using RasMol [49]. In each case the single highest scoring modelling template was the resolved NCX (NCX_Mj) structure (PDB: 3V5U) from Liao et al. [8].
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pone-0112841-g003: Sequence and structural analysis of nematode exchangers.(A) Sliding window analysis of nucleotide diversity using DnaSP version 5 [47] with 100 bp windows and 25 bp steps for CCX (upper graph), NCKX (middle graph), and NCX exchanger (lower graph) gene pairs between C. elegans and C. briggsae. (B) Box plot analysis of molecular diversity for each ncx gene (ncx-1 to ncx-10) between C. elegans and C. briggsae. Whiskers extend to data points that are less than 1.5 × interquartile range away from 1st/3rd quartile; center lines show the medians and outliers are shown as dots. (C) Ribbon model of NCX-1 from C. elegans and an NCX-3 ortholog from A. suum (GS_14034). N and C termini are indicated, and red arrowheads refer to structural differences between each NCX. Numbers refer to the transmembrane (TM) domains. Extracellular side is up in left views. The position of two candidate sites in NCX-1 undergoing episodic diversifying selection are indicted - codon 455 and codon 925. In each case the right view is rotated by 90°. Structural predictions were made using Phyre [48] and visualized using RasMol [49]. In each case the single highest scoring modelling template was the resolved NCX (NCX_Mj) structure (PDB: 3V5U) from Liao et al. [8].

Mentions: The NCX Na+/Ca2+ exchangers were mostly conserved across all species examined (Figure 2A). NCX-1 and NCX-2 are most closely related in each case with NCX-3 representing a more diversified clade. The alpha repeat domains were highly conserved and adopted the consensus GSSAPE for the α1 repeat and GTS(I/V/L)PD for the α2 repeat. Together, these repeats comprise four transmembrane domains (TM2, TM3, TM7, TM8) that form a diamond shaped transport vestibule [8]. We identified three NCX genes for each Caenorhabditis species that we examined except in the case of C. brenneri where we only identified two NCX genes. For each of the NCX clusters, the Caenorhabditis orthologs grouped together (Figure 2A). For the NCX-1 and NCX-3 groups, the H. bacteriophora and H. contortus orthologs grouped together, and for NCX-3, the B. malayi, L. loa, and A. suum orthologs grouped close together (Figure 2A). Outside of the Caenorhabditis genus, we identified three NCX members for each species except P. pacificus, B. malayi and L. loa which each were assigned two NCX genes. In the case of P. pacificus, each NCX gene grouped into the NCX-1 cluster. Similarly, for S. ratti, two of its three NCX genes grouped into the NCX-2 cluster. In cases where multiple hits were detected within each cluster, we re-examined the alignment to ensure alternative splicing was not a misleading factor. We examined selection across the NCX Caenorhabditis taxa using MEME [41] and found a global dN/dS value  = 0.130, we also used SLAC [40] and found a global dN/dS value  = 0.138955 (p<0.01). We also conducted a sliding window analysis of nucleotide diversity between C. elegans and C. briggsae NCX genes using DnaSP ver. 5 [47], and observed similar polymorphic patterns across the ncx-1 and ncx-3 sequences (average π score  = 0.2075 for ncx-1, and 0.1955 for ncx-3), and slightly elevated levels of diversity for ncx-2 (average π score  = 0.369) (Figure 3A–3B). However, elevated nucleotide diversity for ncx-2 does not hold for other Caenorhabditis pairs: for example using C. elegans and C. japonica the average ncx-2 π score  = 0.173 (sampling variance  = 0.007), and using C. elegans and C. remanei the average ncx-2 π score  = 0.11 (sampling variance  = 0.003). Next, we tested specific sites for positive selection using REL [40] within the Caenorhabditis taxa, and from this analysis we did not detect any sites undergoing positive selection. We also tested for episodic diversifying selection using MEME and detected two significant (p<0.01) sites: codon 455, which is positioned close to the second calcium binding domain (CBD2) between TM5 and TM6, which detects local intracellular calcium levels, and codon 925 which is located after TM9 in the intracellular loop that connects with TM10 (see Figure 3C). The crystal structure for the NCX from Methanococcus jannaschii (NCX_Mj) has been resolved [8], and using this structure we examined structural diversity across NCX proteins in nematodes. The NCX-1 and NCX-3 clusters are the most divergent amongst the three NCX clusters (Figure 2A), and so to examine structural differences between diverse NCX proteins in nematodes we selected a representative from the NCX-1 cluster (C. elegans NCX-1) and a representative from the NCX-3 cluster (A. suum GS_14034) for in silico structural analysis. For NCX-1 from C. elegans 32% of the residues (285 residues in total) were modelled at 100% confidence and yielded 33% alpha helical and 20% beta strand structures. The A. suum GS_14034 NCX was modelled at 100% confidence for 36% of the sequence (289 residues) including 36% alpha helical and 22% beta strand structures. In each case the single highest scoring modelling template was the resolved NCX_Mj structure (PDB: 3V5U) from Liao et al. [8]. In the case of NCX-1 we observed a longer beta strand structure connecting TM8 and TM9 from residues 801–817 than is predicted for A. suum GS_14034 (see red arrowheads, Figure 3C), and similarly another lengthy beta structure is predicted for NCX-1 connecting TM4 to TM5, and also a shorter alpha helical structure within TM4 of NCX-1 (red arrowheads, Figure 3C), that is not predicted for A. suum GS_14034 (Figure 3C). In the case of A. suum GS_14034, TM4 is composed of consistent alpha helical structure from residues 165–185, and the linker connecting TM8 and TM9 is significantly shorter in A. suum GS_14034 from residues 742–748 (Figure 3C).


Analysis of the Na+/Ca2+ exchanger gene family within the phylum Nematoda.

He C, O'Halloran DM - PLoS ONE (2014)

Sequence and structural analysis of nematode exchangers.(A) Sliding window analysis of nucleotide diversity using DnaSP version 5 [47] with 100 bp windows and 25 bp steps for CCX (upper graph), NCKX (middle graph), and NCX exchanger (lower graph) gene pairs between C. elegans and C. briggsae. (B) Box plot analysis of molecular diversity for each ncx gene (ncx-1 to ncx-10) between C. elegans and C. briggsae. Whiskers extend to data points that are less than 1.5 × interquartile range away from 1st/3rd quartile; center lines show the medians and outliers are shown as dots. (C) Ribbon model of NCX-1 from C. elegans and an NCX-3 ortholog from A. suum (GS_14034). N and C termini are indicated, and red arrowheads refer to structural differences between each NCX. Numbers refer to the transmembrane (TM) domains. Extracellular side is up in left views. The position of two candidate sites in NCX-1 undergoing episodic diversifying selection are indicted - codon 455 and codon 925. In each case the right view is rotated by 90°. Structural predictions were made using Phyre [48] and visualized using RasMol [49]. In each case the single highest scoring modelling template was the resolved NCX (NCX_Mj) structure (PDB: 3V5U) from Liao et al. [8].
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pone-0112841-g003: Sequence and structural analysis of nematode exchangers.(A) Sliding window analysis of nucleotide diversity using DnaSP version 5 [47] with 100 bp windows and 25 bp steps for CCX (upper graph), NCKX (middle graph), and NCX exchanger (lower graph) gene pairs between C. elegans and C. briggsae. (B) Box plot analysis of molecular diversity for each ncx gene (ncx-1 to ncx-10) between C. elegans and C. briggsae. Whiskers extend to data points that are less than 1.5 × interquartile range away from 1st/3rd quartile; center lines show the medians and outliers are shown as dots. (C) Ribbon model of NCX-1 from C. elegans and an NCX-3 ortholog from A. suum (GS_14034). N and C termini are indicated, and red arrowheads refer to structural differences between each NCX. Numbers refer to the transmembrane (TM) domains. Extracellular side is up in left views. The position of two candidate sites in NCX-1 undergoing episodic diversifying selection are indicted - codon 455 and codon 925. In each case the right view is rotated by 90°. Structural predictions were made using Phyre [48] and visualized using RasMol [49]. In each case the single highest scoring modelling template was the resolved NCX (NCX_Mj) structure (PDB: 3V5U) from Liao et al. [8].
Mentions: The NCX Na+/Ca2+ exchangers were mostly conserved across all species examined (Figure 2A). NCX-1 and NCX-2 are most closely related in each case with NCX-3 representing a more diversified clade. The alpha repeat domains were highly conserved and adopted the consensus GSSAPE for the α1 repeat and GTS(I/V/L)PD for the α2 repeat. Together, these repeats comprise four transmembrane domains (TM2, TM3, TM7, TM8) that form a diamond shaped transport vestibule [8]. We identified three NCX genes for each Caenorhabditis species that we examined except in the case of C. brenneri where we only identified two NCX genes. For each of the NCX clusters, the Caenorhabditis orthologs grouped together (Figure 2A). For the NCX-1 and NCX-3 groups, the H. bacteriophora and H. contortus orthologs grouped together, and for NCX-3, the B. malayi, L. loa, and A. suum orthologs grouped close together (Figure 2A). Outside of the Caenorhabditis genus, we identified three NCX members for each species except P. pacificus, B. malayi and L. loa which each were assigned two NCX genes. In the case of P. pacificus, each NCX gene grouped into the NCX-1 cluster. Similarly, for S. ratti, two of its three NCX genes grouped into the NCX-2 cluster. In cases where multiple hits were detected within each cluster, we re-examined the alignment to ensure alternative splicing was not a misleading factor. We examined selection across the NCX Caenorhabditis taxa using MEME [41] and found a global dN/dS value  = 0.130, we also used SLAC [40] and found a global dN/dS value  = 0.138955 (p<0.01). We also conducted a sliding window analysis of nucleotide diversity between C. elegans and C. briggsae NCX genes using DnaSP ver. 5 [47], and observed similar polymorphic patterns across the ncx-1 and ncx-3 sequences (average π score  = 0.2075 for ncx-1, and 0.1955 for ncx-3), and slightly elevated levels of diversity for ncx-2 (average π score  = 0.369) (Figure 3A–3B). However, elevated nucleotide diversity for ncx-2 does not hold for other Caenorhabditis pairs: for example using C. elegans and C. japonica the average ncx-2 π score  = 0.173 (sampling variance  = 0.007), and using C. elegans and C. remanei the average ncx-2 π score  = 0.11 (sampling variance  = 0.003). Next, we tested specific sites for positive selection using REL [40] within the Caenorhabditis taxa, and from this analysis we did not detect any sites undergoing positive selection. We also tested for episodic diversifying selection using MEME and detected two significant (p<0.01) sites: codon 455, which is positioned close to the second calcium binding domain (CBD2) between TM5 and TM6, which detects local intracellular calcium levels, and codon 925 which is located after TM9 in the intracellular loop that connects with TM10 (see Figure 3C). The crystal structure for the NCX from Methanococcus jannaschii (NCX_Mj) has been resolved [8], and using this structure we examined structural diversity across NCX proteins in nematodes. The NCX-1 and NCX-3 clusters are the most divergent amongst the three NCX clusters (Figure 2A), and so to examine structural differences between diverse NCX proteins in nematodes we selected a representative from the NCX-1 cluster (C. elegans NCX-1) and a representative from the NCX-3 cluster (A. suum GS_14034) for in silico structural analysis. For NCX-1 from C. elegans 32% of the residues (285 residues in total) were modelled at 100% confidence and yielded 33% alpha helical and 20% beta strand structures. The A. suum GS_14034 NCX was modelled at 100% confidence for 36% of the sequence (289 residues) including 36% alpha helical and 22% beta strand structures. In each case the single highest scoring modelling template was the resolved NCX_Mj structure (PDB: 3V5U) from Liao et al. [8]. In the case of NCX-1 we observed a longer beta strand structure connecting TM8 and TM9 from residues 801–817 than is predicted for A. suum GS_14034 (see red arrowheads, Figure 3C), and similarly another lengthy beta structure is predicted for NCX-1 connecting TM4 to TM5, and also a shorter alpha helical structure within TM4 of NCX-1 (red arrowheads, Figure 3C), that is not predicted for A. suum GS_14034 (Figure 3C). In the case of A. suum GS_14034, TM4 is composed of consistent alpha helical structure from residues 165–185, and the linker connecting TM8 and TM9 is significantly shorter in A. suum GS_14034 from residues 742–748 (Figure 3C).

Bottom Line: The most notable feature of the resulting phylogenies was the heterogeneous evolution observed within exchanger subtypes.Specifically, in the case of the CCX exchangers we did not detect members of this class in three Clade III nematodes.We also provided re-annotation for predicted CCX gene structures from Heterorhabditis bacteriophora and Caenorhabditis japonica by RT-PCR and sequencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, The George Washington University, Washington, D.C., United States of America; Institute for Neuroscience, The George Washington University, Washington, D.C., United States of America.

ABSTRACT
Na+/Ca2+ exchangers are low affinity, high capacity transporters that rapidly transport calcium at the plasma membrane, mitochondrion, endoplasmic (and sarcoplasmic) reticulum, and the nucleus. Na+/Ca2+ exchangers are widely expressed in diverse cell types where they contribute homeostatic balance to calcium levels. In animals, Na+/Ca2+ exchangers are divided into three groups based upon stoichiometry: Na+/Ca2+ exchangers (NCX), Na+/Ca2+/K+ exchangers (NCKX), and Ca2+/Cation exchangers (CCX). In mammals there are three NCX genes, five NCKX genes and one CCX (NCLX) gene. The genome of the nematode Caenorhabditis elegans contains ten Na+/Ca2+ exchanger genes: three NCX; five CCX; and two NCKX genes. Here we set out to characterize structural and taxonomic specializations within the family of Na+/Ca2+ exchangers across the phylum Nematoda. In this analysis we identify Na+/Ca2+ exchanger genes from twelve species of nematodes and reconstruct their phylogenetic and evolutionary relationships. The most notable feature of the resulting phylogenies was the heterogeneous evolution observed within exchanger subtypes. Specifically, in the case of the CCX exchangers we did not detect members of this class in three Clade III nematodes. Within the Caenorhabditis and Pristionchus lineages we identify between three and five CCX representatives, whereas in other Clade V and also Clade IV nematode taxa we only observed a single CCX gene in each species, and in the Clade III nematode taxa that we sampled we identify NCX and NCKX encoding genes but no evidence of CCX representatives using our mining approach. We also provided re-annotation for predicted CCX gene structures from Heterorhabditis bacteriophora and Caenorhabditis japonica by RT-PCR and sequencing. Together, these findings reveal a complex picture of Na+/Ca2+ transporters in nematodes that suggest an incongruent evolutionary history of proteins that provide central control of calcium dynamics.

Show MeSH
Related in: MedlinePlus