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Originally published In Press as doi:10.1074/jbc.M405877200 on July 2, 2004

J. Biol. Chem., Vol. 279, Issue 35, 37185-37190, August 27, 2004
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Cofactor-independent Phosphoglycerate Mutase Has an Essential Role in Caenorhabditis elegans and Is Conserved in Parasitic Nematodes*

Yinhua Zhang, Jeremy M. Foster, Sanjay Kumar, Marjorie Fougere, and Clotilde K. S. Carlow{ddagger}

From the New England Biolabs, Beverly, Massachusetts 01915

Received for publication, May 26, 2004 , and in revised form, July 2, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosphoglycerate mutases catalyze the interconversion of 2- and 3-phosphoglycerate in the glycolytic and gluconeogenic pathways. They exist in two unrelated forms that are either cofactor (2,3-diphosphoglycerate)-dependent or cofactor-independent. The two enzymes have no similarity in amino acid sequence, tertiary structure, or catalytic mechanism. Certain organisms including vertebrates have only the cofactor-dependent form, whereas other organisms can possess the independent form or both. Caenorhabditis elegans has been predicted to have only independent phosphoglycerate mutase. In this study, we have cloned and produced recombinant, independent phosphoglycerate mutases from C. elegans and the human-parasitic nematode Brugia malayi. They are 70% identical to each other and related to known bacterial, fungal, and protozoan enzymes. The nematode enzymes possess the catalytic serine, and other key amino acids proposed for catalysis and recombinant enzymes showed typical phosphoglycerate mutase activities in both the glycolytic and gluconeogenic directions. The gene is essential in C. elegans, because the reduction of its activity by RNA interference led to embryonic lethality, larval lethality, and abnormal body morphology. Promoter reporter analysis indicated widespread expression in larval and adult C. elegans with the highest levels apparent in the nerve ring, intestine, and body wall muscles. The enzyme was found in a diverse group of nematodes representing the major clades, indicating that it is conserved throughout this phylum. Our results demonstrate that nematodes, unlike vertebrates, utilize independent phosphoglycerate mutase in glycolytic and gluconeogenic pathways and that the enzyme is probably essential for all nematodes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The genome of the free-living nematode Caenorhabditis elegans has been used widely as a model to understand molecular pathways common to nematodes and mammals on the basis of conserved evolution. However, nematodes as a group also have features that distinguish them from mammals. It is likely that some of these characteristics are the result of developmental and/or physiological processes potentially involving nematode-specific genes. Indeed, genome analysis has shown that C. elegans possesses many genes that do not have orthologs in mammals (1, 2). Although a few of these unique genes are known to participate in essential processes such as cuticle biosynthesis and sex determination (3), most of them remain uncharacterized.

With a view to gaining an improved understanding of nematode biology, we are focusing on genes absent in mammals that showed a defect when disrupted in high throughput RNA interference (RNAi)1 studies performed in C. elegans (4-7). One of the genes identified was cofactor-independent phosphoglycerate mutase (iPGM) (EC 5.4.2.1 [EC] ). PGMs catalyze the interconversion of 2- and 3-phosphoglycerate (2-PG and 3-PG) in the glycolytic and gluconeogenic pathways. Although these pathways are highly conserved among different organisms, two distinct PGM enzymes are known to exist, iPGM and the cofactor-dependent phosphoglycerate mutase, dPGM. The sizes, amino acid (aa) sequence composition, three-dimensional structure, and catalytic mechanisms of these two enzymes are vastly dissimilar. iPGM is comprised of ~500 aa and catalyzes the intramolecular transfer of the phospho group on monophosphoglycerates through a phosphoserine intermediate (8, 9). In contrast, dPGM is composed of ~250 amino acids and catalyzes the intermolecular transfer of the phospho group between the monophosphoglycerates and the cofactor (2,3-diphosphoglycerate) through a phosphohistidine intermediate (10). Vertebrates are known to possess the dPGM form exclusively, whereas in other organisms, the distribution of the two forms has been considered complex and unpredictable because either dPGM or iPGM or both forms may be present (11, 12). Whereas the two forms of PGM are distinct, the amino acid sequence of each form when present is conserved from bacteria to higher eukaryotes (13).

The parasitic nematodes of plants, animals, and humans are found worldwide and cause serious economic and health problems. Filarial nematodes, such as Brugia malayi, lead to particularly debilitating infections in humans and other mammals. Current treatments for nematode infections are inadequate. The identification of essential nematode genes that are absent from mammals may lead to the discovery of new drug targets and therapies.

In this study, we performed a detailed characterization of the function of C. elegans iPGM in vivo and report that iPGM is present in many parasitic nematode species. We provide molecular and biochemical studies on the iPGM enzymes from C. elegans and B. malayi, representing the first analysis of iPGM from metazoans.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Identification of iPGM as a Non-mammalian Gene with an RNAi Phenotype in C. elegansm—C. elegans iPGM (Ce-iPGM) was identified using a bioinformatic method relying on gene function data from large scale RNAi screens. Predicted proteins in Wormbase (www.wormbase.org, release version WS110, October, 2003) with a non-wild type entry in the RNAi phenotype field were retrieved and used to query the mouse and human genomes using BLASTP (14). Ce-iPGM (corresponding to predicted gene F57B10.3) was identified as a member of a group of genes that do not have orthologs in these mammalian genomes (E-value cut-off score = 10-10).

Identification of iPGM Enzymes in Parasitic Nematodes and Other Eukaryotes—Ce-iPGM was used to query NCBI databases for corresponding orthologs in Caenorhabditis briggsae, parasitic nematodes, and other eukaryotes. A number of genomic and cDNA sequences and ESTs were identified using the TBLASTN program (14) (E-value cut-off score = 10-10). Full-length iPGM sequences were retrieved for further analyses including multiple sequence alignment and construction of phylogenetic trees using the ClustalW program (15).

RNAi in C. elegans—C. elegans culture and handling were performed following standard procedures (16). A 995-bp fragment of Ce-iPGM was amplified from a mixed-stage C. elegans cDNA pool or a cloned and sequenced full-length cDNA using primers containing the T7 promoter sequence. The primer sequences were as follows: forward (5'-aatacgactcactataggACCAGTTATGGACAAGCTGTG-3') and reverse (5'-aatacgactcactataggTTGGACTGGGCACTAAACACC-3') with the T7 promoter sequence shown in lowercase. The PCR products were purified and used for in vitro transcription with T7 RNA polymerase using the HiScribeTM RNAi transcription kit (New England Biolabs). The synthesized dsRNA was purified using an RNeasy column (Qiagen) and eluted into H2O. The purified dsRNA was injected into the germ line of C. elegans young adult hermaphrodites (wild type N2) (17). Injected worms were allowed to recover on nematode growth medium plates overnight before singling. Each injected worm was transferred to a fresh plate every 8 or 16 h over the course of 3 days. The F1 embryos that were laid on each plate were counted immediately after transferring the injected worm. The L1 larvae that emerged from eggs on each plate were counted after 24 h. The plates were scored again 2 days later to assess worm growth and morphological defects. Embryos that failed to hatch were scored as embryonic lethal, and L1 larvae that failed to grow were scored as larval lethal. dsRNAs corresponding to fragments of the unc-22 (17) and pos-1 (18) genes were synthesized similarly and used as controls.

C. elegans iPGM Green Fluorescent Protein (GFP) Transcription Reporter—An intergenic region of 1.53 kb upstream of C. elegans predicted that iPGM gene F57B10.3b was used to drive the expression of GFP.

The promoter was amplified from C. elegans genomic DNA using the primer pair: forward (5'-ATGCGTCGACTTCTCACAAGGATCTGAGG-3', where the SalI site is underlined) and reverse (5'ATGCGGTACCTCAATAACGATGAGACAGACC-3', where the KpnI site is underlined). The PCR product was cloned into pPD95.752 using KpnI and SalI to generate the transcription reporter (pIP83). Transgenic C. elegans carrying the reporter on extra-chromosomal arrays (IP159: nbEx50) were identified following the co-injection of the reporter (~17 µg/ml) with the pRF4 plasmid (~57 µg/ml) that carries a dominant Rol-6(su1006) gene as the transgenic marker (19).

Cloning and Overexpression of iPGM Enzymes from C. elegans and B. malayi—Full-length Ce-iPGM was amplified from a C. elegans mixed-stage cDNA pool. Initially, primers were designed according to the C. elegans predicted iPGM open reading frame (ORF) F57B10.3a in Wormbase as follows: forward (5'-ACGTGGATCCATGTTCGTAGCCCTGGGCGCTC-3', where the BamHI site is underlined) and reverse (5'-ACGTAAGCTTCTAGATCTTCTGAACAATCG-3', where the HindIII site is underlined). The amplified cDNA was cloned into the pMAL-c2X vector (New England Biolabs) using the HindIII and BamHI sites, and the insert was sequenced. Subsequently, the cDNA corresponding to the predicted ORF F57B10.3b was PCR-amplified from this construct and cloned into the BamHI and HindIII sites of pET21a(+) (Novagen) to express a fusion protein with a His6 tag at the C terminus. The primers were as follows: forward (5'-AGTCGGATCCATGGCGATGGCAAATAAC-3', where the BamHI site is underlined) and reverse (5'-AGTCAAGCTTGATCTTCTGAACAATG-3', where the HindIII site is underlined).

Based on BLAST analysis, two putative B. malayi iPGM ESTs were identified (GenBankTM accession numbers 5510517 and 1912539) and the corresponding cDNA clones were obtained from the Filarial Genome Project Resource Center (genome{at}smith.edu). The clones were further sequenced at the 5' and 3' ends. Based on these sequences, a complete cDNA (Bm-iPGM) was amplified from an adult female cDNA pool and cloned into the pMAL-c2x vector using the following primers: forward (5'-ATGCGGATCCATGGCCGAAGCAAAGAATCGAGTATGTCTGGTAGTGATTGATGGT-3', where the BamHI site is underlined) and reverse (5'-ATGCCTGCAGGGCTTCATTAACCAATGGC-3', where the PstI site is underlined). The Bm-iPGM was subsequently re-amplified and cloned into the BamHI and XhoI of pET21a(+) using the following primers: forward (5'-AGTCGGATCCATGGCCGAAGCAAAGAATCG-3', where the BamHI site is underlined) and reverse (5'-ATGCCTCGAGGGCTTCATTAACCAATGGC-3', where the XhoI site is underlined) to express a fusion protein with a C-terminal His tag.

Both C. elegans and B. malayi iPGM in pET21a(+) were expressed in the Escherichia coli strain ER2566 (fhuA2 lacZ::T7 gene1 [lon] ompT gal sulA11 [dcm] R (zgb-210::Tn10-TetS) endA1 D(mcrC-mrr)114::IS10 R(mcr-73::miniTn10-TetS)2) (New England BioLabs). Conditions were optimized to maximize expression, solubility, and yield of each recombinant protein. For Ce-iPGM, cultures were grown at 30 °C and induced with 0.1 mM isopropyl-1-thio-{beta}-D-galactopyranoside (Sigma) at 15 °C overnight. Bm-iPGM was produced by growing cultures at 37 °C and inducing with 0.1 mM isopropyl-1-thio-{beta}-D-galactopyranoside for 3 h at 37 °C. The His-tagged proteins were extracted and purified on nickel columns (Qiagen) using native conditions according to the manufacturer's instructions. An elution buffer (40 mM NaH2PO4, 300 mM NaCl, pH 8.0) containing 60 mM imidazole was found to be optimal in releasing both His-tagged proteins from the nickel resin with a high level of purity.

PGM Enzyme Assay—iPGM activity was determined in forward and reverse directions using standard enzyme-coupled assays (12, 20). In the forward reaction (glycolytic), the conversion of 3-PG to 2-PG was measured, whereas in the reverse direction (gluconeogenic), the conversion of 2-PG to 3-PG was assayed. In both cases, PGM activity was determined indirectly by measuring the consumption of NADH, which was monitored at 340 nm. The amount of NADH being oxidized to NAD corresponds to the amount of enzyme product (2-PG in the forward direction or 3-PG in the reverse direction) produced in the PGM reaction. Reactions were performed at 30 °C for 5 min with data collected at 10-s intervals using a Beckman DU 640 spectrophotometer. In the forward reaction, iPGM was added to a 1-ml assay buffer (30 mM Tris-HCl, pH 7.0, 5 mM MgSO4, 20 mM KCl, 0.15 mM NADH) containing 1 mM ADP, 10 mM 3-PG (Sigma P8877), and 2.5 units each of enolase (Sigma E6126; EC 4.2.1.11 [EC] ), pyruvate kinase (Sigma P7768; EC 2.7.1.40 [EC] ), and lactate dehydrogenase (Sigma L2518; EC 1.1.1.27 [EC] ). In the reverse reaction, iPGM was added to a 1-ml assay buffer containing 1 mM ATP, 10 mM 2-PG (Sigma P0257), and 2.5 units each of phosphoglycerate kinase (Sigma P7634; EC 2.7.2.3 [EC] ) and glyceraldehyde 3-phosphate dehydrogenase (Sigma G0763; EC 1.2.1.12 [EC] ). One unit of PGM activity is defined as the amount of activity that is required for the conversion of 1.0 µM NADH to NAD/min in the above assay conditions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification and Cloning of iPGM from C. elegans and B. malayi—Ce-iPGM was identified in this study as a member of a set of C. elegans genes that has no apparent similarity to any mammalian genes and showed a visible phenotype in high throughput RNAi studies (4-7). Ce-iPGM is encoded by a single predicted gene F57B10.3, which potentially can produce two splice forms, F57B10.3a and F57B10.3b, based on existing C. elegans ESTs (Wormbase). They encode ORFs of 539 and 521 aa, respectively, differing only in 18 aa at the N terminus. The cDNA corresponding to the predicted ORF of F57B10.3a was amplified by PCR and sequenced and found to match the predicted intron-exon junctions. Ce-iPGM was used to query the nematode EST databases, and partial cDNA sequences encoding B. malayi iPGM (Bm-iPGM) were identified. A full-length Bm-iPGM cDNA was amplified and cloned from a cDNA pool and subsequently sequenced. The Bm-iPGM was predicted to encode a 515 aa protein.

Both C. elegans and B. malayi deduced proteins are typical in size of iPGMs and appear to be canonical iPGM enzymes based on aa sequence comparisons. The sequence alignment (Fig. 1) shows that they possess the catalytic serine and 13 other residues predicted to be involved in catalysis based on the structural studies of the biochemically characterized Bacillus stearothermophilus iPGM (8, 9, 21). The nematode enzymes share ~40-42% amino acid identity and 59-62% similarity to B. stearothermophilus iPGM (22) and have a 70% amino acid identity and ~82% similarity to each other (Fig. 1). They are less related to the experimentally verified iPGM enzymes from the kinetoplastid protozoans Trypanosoma brucei and Leish-mania mexicana (26-28% identical and 47-50% similar) (23, 24), although the similarity extends over the full length of the proteins.



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  		FIG. 1.
Alignment of the deduced amino acid sequences of nematode iPGMs with experimentally verified enzymes. The aa sequences of nematode iPGM enzymes from Ce (GenBankTM accession number AY594354 [GenBank] corresponding to the ORF of F57B10.3b) and Bm (GenBankTM accession number AY330617 [GenBank] ) are aligned with an extensively studied and structurally characterized iPGM from the bacterium B. stearothermophilus (Bs, GenBankTM accession number 27734396) and experimentally verified eukaryotic iPGM enzymes from T. brucei (Tb, GenBankTM accession number 7380854) and L. mexicana (Lm, GenBankTM accession number 28400786). Residues that are identical in at least three sequences are highlighted black, and the conserved amino acid changes are shaded gray. The catalytic serine (@) and 13 other residues (asterisk) involved in catalysis (8) were found in all of these enzymes. The alignment was generated with ClustalW and displayed with BOXSHADE (www.ch.embnet.org/software/BOX_form.html).

 
Ce-iPGM Plays an Essential Role in Development—To determine the role of Ce-iPGM in vivo, its endogenous activity was reduced by RNAi (17) and the effects on C. elegans development were monitored at various time intervals following injection. Several defects were observed, most notably embryonic lethality (Fig. 2). The degree of embryonic lethality at different time periods after dsRNA injection was carefully monitored. The embryos that were laid 18-26 h after injection of Ce-iPGM dsRNA hatched normally and continued to develop normally (data not shown). The embryos that were laid 26-42 h after injection showed only a low level of embryonic lethality (~22%). Strikingly, the embryonic lethality increased significantly after 42 h with most embryos laid 42-66 h post-injection, failing to hatch (94-97% embryonic lethality) (Fig. 2A). In contrast, RNAi inactivation of control unc-22 (17) and pos-1 (18) genes resulted in full penetrant phenotypes in progeny produced by 18-h post-injection (Fig. 2B). The phenotype severity and time course of the effects observed with iPGM RNAi were comparable following the administration of dsRNA at 1 mg/ml (Fig. 2A) and at higher (3 mg/ml) or lower (0.4 mg/ml) concentrations (data not shown).



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  		FIG. 2.
The Ce-iPGM RNAi effects are delayed (panels A and B) and cause multiple developmental defects (panels C-J). Monitoring of the occurrence of the Ce-iPGM (A) and control (B) RNAi phenotypes over time is shown. The percentages of F1 progeny displaying RNAi phenotypes (embryonic lethality for Ce-iPGM and pos-1, or uncoordinated movement in larvae for unc-22) are shown at various time intervals post-dsRNA injection. For each RNAi experiment, the percentage of progeny displaying the phenotype shown was based on three injected P0 hermaphrodites, although similar results were obtained with more worms. A, for Ce-iPGM, the percentage of embryonic lethality was 0% (0/45, 0/36, and 0/34 embryos failed to hatch for individual worms) at 18-26 h post-injection, 22% (31/116, 11/68, and 14/70 embryos) at 26-42 h post-injection, 97% (34/35, 25/26, and 13/13) at 42-50 h post-injection, and 94% (7/7, 9/9, and 1/2) at 50-60 h post-injection. Embryonic lethality is shown for three uninjected worms at the same four time periods: 2% (total, 3/172); 0% (0/291); 3.4% (2/88); and 0% (0/82), respectively. For worms injected with control dsRNAs (B), 100% of the progeny displayed unc-22 (168/168 and 143/143) or pos-1 (332/332 and 120/120) phenotypes at 18-42 and 42-60 h post-injection, respectively. Differential interference contrast images of abnormal embryos and larvae resulting from RNAi knockdown of Ce-iPGM (panels C-D, F-G, and I-J) and wild type worms of similar stages (panels E and H). Embryos that failed to hatch were arrested at various stages during embryogenesis including early (C) and late stages (D). Wild type embryos of similar stages are shown for comparison (E). Variable phenotypes were observed in larvae including degenerating intestinal cells that contain large vacuoles (F, arrows) and variable abnormal body morphologies (G, arrowhead) compared with wild type larvae (H). Some larvae arrested at L1 (I) or died (J). Images C-E were obtained using a x63 objective, and images F-J were obtained using a x40 objective.

 
When the embryos that failed to hatch were examined further, it was observed that the development was arrested at different time points during embryogenesis (Fig. 2, C-D). In some of the larvae that escaped embryonic lethality, additional defects were noted including sluggish movement (data not shown), variable abnormal body morphology, developmental arrest, and lethality (Fig. 2, F-J). In a total of 31 larvae that developed from embryos collected at 42-50 h post-injection, larval lethality was 19%. The larval lethality rate increased to 37% in the 19 surviving larvae that resulted from embryos collected at 50-65 h post-injection.

To further address the in vivo function of Ce-iPGM, its spatial and developmental expression was examined in transgenic C. elegans carrying an iPGM::GFP transcription reporter construct. GFP expression was observed in most cells with higher levels in the nerve ring, intestine, and muscles of the body wall (Fig. 3). The distribution pattern and expression levels were consistent in larval stages and adult worms.



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  		FIG. 3.
Expression of transcription reporter Ce-iPGM::GFP in transgenic C. elegans. A-D, transgenic animals were examined using differential interference contrast (A and C) and fluorescence microscopy (B and D). The GFP reporter was expressed throughout the worm at different stages. Panels A and B show late embryos. The center embryo shows GFP expression from the reporter gene, whereas the rest are probably not transgenic. Panels C and D show a L2 larva expressing the GFP reporter with highest levels of expression in the nerve ring region (arrow) and body wall muscles (arrowheads). Images were acquired using x63 (A and B) or x40 objectives (C and D). Scale bar is 50 µm.

 
Recombinant Ce-iPGM and Bm-iPGM Have PGM Activity—To verify their PGM activities, Ce-iPGM and Bm-iPGM were overexpressed in E. coli as C-terminal His6-tagged proteins for enzymatic analysis. The ORF corresponding to the C. elegans shorter splice variant that was selected for expression as the 18 aa extension present in the longer form does not match any known iPGM sequence. Ce-iPGM and Bm-iPGM recombinant proteins (~60 kDa in size) were abundantly expressed and subsequently purified (Fig. 4A). PGM activity was measured using standard enzyme-coupled assays (12, 20). Both Ce-iPGM and Bm-iPGM showed PGM activity in the glycolytic (conversion of 3-PG to 2-PG) (Fig. 4B) and gluconeogenic directions (2-PG to 3-PG) (Fig. 4C) as evidenced by the consumption of NADH by the coupling enzymes. The slope resulting from the change in NADH concentration was used to calculate specific activities. The specific activities in the forward reaction for Ce-iPGM and Bm-iPGM were ~40 and 93 units/mg, respectively. For the reverse reaction, the activities for Ce-iPGM and Bm-iPGM were 72 and 256 units/mg, respectively. The addition of 2,3-diphosphoglycerate, the cofactor for dPGM (11), did not alter these activities. Likewise, the enzyme activities were not affected by the addition of vanadate, a known inhibitor of dPGM (data not shown) (25).



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  		FIG. 4.
Purification and activity of recombinant nematode iPGMs. Panel A, expression and purification of Ce-iPGM. Lane 1, soluble E. coli protein following induction with isopropyl-1-thio-{beta}-D-galactopyranoside; lane 2: soluble E. coli protein without induction; lane 3, flow-through from the nickel column; lanes 4-5, washes from column prior to elution; lanes 6-11, sequential elution of His-tagged Ce-iPGM; M, protein marker (New England Biolabs P7708). The Bm-iPGM was expressed and purified with similar purity (data not shown). Panel B, conversion of 3-PG to 2-PG (glycolytic direction) by Ce-iPGM (0.9 µg) and Bm-iPGM (0.9 µg). Panel C, conversion of 2-PG to 3-PG (gluconeogenic direction) by Ce-iPGM (1.14 µg) and Bm-iPGM (0.35 µg). Purified Ce-iPGM and Bm-iPGM recombinant proteins were assayed for PGM activity in both directions. In both reactions, PGM activity was determined indirectly by measuring a decrease of NADH concentration by its absorbance at 340 nm. The consumption of NADH is directly proportional to PGM activity.

 
Distribution and Conservation of iPGM and dPGM Enzymes in Nematodes and Other Eukaryotes—To investigate whether iPGM is present in other nematodes, Ce-iPGM was used as a query to search the recently sequenced genome of the closely related free-living nematode C. briggsae (26) and the ~300,000 EST sequences available from 30 different parasitic nematode species (27). The C. briggsae genome was found to contain a predicted gene (CBG12654 that encodes a protein with 95% amino acid identity to Ce-iPGM. In addition, 38 nematode ESTs obtained from a highly diverse group of parasitic nematodes were found to match Ce-iPGM. These included 16 non-overlapping ESTs from a total of 12 parasitic species found in humans (B. malayi, Trichinella spiralis, Onchocerca volvulus, Strongyloides stercoralis, and Necator americanus), animals (Trichuris muris, Litomosoides sigmodontis, Haemonchus contortus, and Ostertagia ostertagi), and plants (Globodera rostochiensis, Meloidogyne incognita, and Heterodera glycines). When these matching sequences were sorted among the five major clades of the phylum Nematoda (3), they were found in all four clades that have existing EST sequences (Fig. 5A). In contrast, no dPGM sequences were found in the C. elegans and C. briggsae genomes or the nematode EST data base when human dPGM (GenBankTM gi130353) (28) was used as the query. The same human dPGM query identified a large number of orthologs in other organisms ranging from bacteria to mammals (data not shown).



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  		FIG. 5.
Evidence for the presence of iPGM enzymes in nematodes and conservation of iPGMs among eukaryotes. Panel A, animal (a), human (h), and plant (p) parasitic nematode species with ESTs that matched Ce-iPGM by TBLASTN are shown in groups according to the classification of major nematode clades (3). Panel B, a phylogenetic tree of predicted full-length iPGM protein sequences is shown as a phylogram. An iPGM from B. stearothermophilus was included as a bacterial representative. The percentage of amino acid sequence identity to C. elegans iPGM is shown. The scale bar indicates the number of substitutions per residue. The GenBankTM accession numbers of the EST sequences (A) or eukaryotic iPGM sequences (B) are shown in brackets.

 
The relationship of the nematode iPGMs to other eukaryotic IPGMs was examined using multiple sequence alignments. Ce-iPGM was used to query available databases, and orthologs were identified in fungi, protozoa, and plants. A phylogenetic tree was constructed using full-length iPGM sequences from diverse species that represent the major taxa (Fig. 5B) with a distantly related iPGM from an Archaea Pyrococcus furiosus (29) included as an outgroup in displaying the tree. The iPGM enzymes from C. elegans and B. malayi clustered together as expected from the high degree of conservation between them (70% aa identity). The C. elegans enzyme was more closely related to putative fungal iPGM enzymes (41-53% identity) than those found in protozoa (26-30% identity) and plants (~32% identity). It was 42% identical to the B. stearothermophilus enzyme chosen to represent bacteria


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
iPGM has been found mainly in bacteria and plants and predicted to exist in C. elegans (12, 30). In this study, we provide biochemical characterization of C. elegans iPGM and confirmation of its essential role in development. We also cloned and characterized iPGM from the parasitic nematode B. malayi. We discovered that iPGM is probably present in all of the nematodes as indicated by the presence of numerous matching ESTs from a diverse collection of parasitic species. In sharp contrast, dPGM, the form of the enzyme found in vertebrates, was not detected among any of the available nematode sequence databases.

We demonstrate that recombinant iPGM enzymes from C. elegans and B. malayi are active in both glycolytic and gluconeogenic directions. The specific activities were similar to those reported for the recombinant iPGM from T. brucei (23), but they were ~10-fold lower than the enzyme from the thermophilic bacterium B. stearothermophilus (22). Similar to other iPGMs, the activity of the nematode enzymes was not affected by the addition of 2,3-diphosphoglycerate, the cofactor for dPGM or vanadate, a known inhibitor of dPGM. The nematode enzymes were clearly active in neutral buffer containing magnesium ions like the enzymes from E. coli (12) and T. brucei (31). However, it would be of interest in the future to evaluate the effect of other ions on activity because cobalt is preferred by the iPGM from T. brucei (31) and L. mexicana (24), whereas manganese is optimal for iPGM from Bacillus species (22, 32) and present in E. coli iPGM (12).

As expected from its involvement in a fundamental metabolic pathway, we found Ce-iPGM expressed throughout the worm in all of the developmental stages. The enzyme was most abundant in cells that would be expected to have a higher metabolic rate, such as the frequently contracting body wall muscles, nerve ring, and intestinal cells. Disruption of Ce-iPGM by RNAi resulted in variable defects including embryonic and larval lethality. These defects were initially detected in high throughput RNAi screens (4) and characterized in more detail in this study. An interesting finding from our detailed analysis was the apparent delay in the onset of the RNAi effect compared with other genes and an increasing potency over time. For other genes, the progeny produced from the microinjected mother start to show potent and consistent phenotypes ~6 h after injecting dsRNA (17). For iPGM, a low level effect was detected only by 26 h and then it reached maximum potency at 42-66 h post-injection. This delay and incomplete penetrance may be the result of a slower depletion of iPGM transcripts or a stable abundant reservoir of the protein or both. Alternatively, it may reflect the involvement of a non-sustainable salvage regulation in response to the depletion of iPGM. The delayed RNAi effect peculiar to this gene may have partly contributed to the different phenotypes observed for iPGM in the various large-scale RNAi screens (4, 33-35). Nevertheless, it is likely that 100% embryonic lethality would be observed when iPGM activity is completely deleted in a strain carrying a genetic null mutation of iPGM.

Our studies clearly demonstrate an essential role for iPGM in C. elegans. Similarly, essential roles for iPGM have also been reported in two bacterial species. Deletion of the iPGM gene in a spore-forming bacterium, Bacillus subtilis, resulted in extremely slow growth and in an inability to produce spores (36). Inactivation of the iPGM locus by a transposon insertion in the tomato bacterial pathogen Pseudomonas syringae resulted in a mutant strain that could not grow or infect tomatoes (37). The demonstration of the essential roles of iPGM in these organisms suggests that it is possible to target this enzyme in certain pathogens. Our discovery that nematodes have only iPGM in their genomes indicates that it represents an excellent drug target against parasitic nematodes because it is absent from their human and other vertebrate hosts.


   FOOTNOTES
 
* This work was supported by Donald Comb (New England Biolabs). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: New England Biolabs, 32 Tozer Road, Beverly, MA 01915. Tel.: 978-927-5054 (ext. 263); Fax: 978-921-1350; E-mail: carlow{at}neb.com.

1 The abbreviations used are: RNAi, RNA interference; iPGM, cofactor-independent phosphoglycerate mutase; PGM, phosphoglycerate mutase; dPGM, cofactor-dependent phosphoglycerate mutase; aa, amino acid; GFP, green fluorescent protein; ds, double-stranded; ORF, open reading frame; Ce, C. elegans; Bm, B. malayi; 2-PG and 3-PG, 2- and 3-phosphoglycerate; EST, expressed sequence tag. Back

2 A. Fire, personal communication. Back


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the encouragement of Donald Comb (New England Biolabs). We thank Nicole Nichols for advice on PGM assays. We also thank Chris Gissendanner, Francine Perler, Catherine Poole, and Rich Roberts for critical reading of the paper.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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