| Human Molecular Genetics | Pages |
Acyl-CoA:dihydroxyacetonephosphate acyltransferase: cloning of the human cDNA and resolution of the molecular basis in rhizomelic chondrodysplasia punctata type 2
Introduction
Results
Identification of cDNA encoding human DHAPAT
Expression of DHAPAT in Saccharomyces cerevisiae
Mutation analysis of DHAPAT in RCDP type 2 patients
Discussion
Materials And Methods
RCDP type 2 patients
Identification of the human DHAPAT cDNA
Construction of DHAPAT expression plasmids
Expression of DHAPAT in S.cerevisiae
RT-PCR amplification of the DHAPAT cDNA and nucleotide sequence analysis
Acknowledgements
References
Acyl-CoA:dihydroxyacetonephosphate acyltransferase: cloning of the human cDNA and resolution of the molecular basis in rhizomelic chondrodysplasia punctata type 2
DDBJ/EMBL/GenBank accession no. AF043937
Rhizomelic chondrodysplasia punctata (RCDP) is a genetic disorder which is clinically characterized by rhizomelic shortening of the upper extremities, typical dysmorphic facial appearance, congenital contractures and severe growth and mental retardation. Patients with RCDP can be subdivided into three subgroups based on biochemical analyses and complementation studies. The largest subgroup contains patients with mutations in the PEX7 gene encoding the PTS2 receptor. This results in multiple peroxisomal abnormalities which includes a deficiency of acyl-CoA:dihydroxyacetonephosphate acyltransferase (DHAPAT), alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase), peroxisomal 3-ketoacyl-CoA thiolase and phytanoyl-CoA hydroxylase, although there are differences in the extent of the deficiencies observed. Patients in the two other subgroups have been reported to be either deficient in the activity of DHAPAT (RCDP type 2) or alkyl-DHAP synthase (RCDP type 3) while no other abnormalities could be observed. To examine whether the gene encoding DHAPAT is mutated in patients with RCDP type 2, we determined the N-terminal amino acid sequence of the enzyme isolated from human placenta. Using this sequence as a query, we identified a 2040 bp open reading frame (ORF) in the human database of expressed sequence tags. Expression of this ORF in the yeast Saccharomyces cerevisiae showed that we have identified the DHAPAT cDNA. The deduced amino acid sequence revealed no PTS2 consensus sequence. In contrast DHAPAT appears to contain a putative PTS1 at the extreme C-terminus. All RCDP type 2 patients analyzed were found to contain mutations in their DHAPAT cDNA. This demonstrates that RCDP type 2 is the result of mutations in DHAPAT.
INTRODUCTION
Peroxisomes were long thought to play only a minor role in cellular metabolism but the recognition of a group of genetic disorders in which one or more peroxisomal functions are impaired has changed this view dramatically. The cerebro-hepato-renal syndrome of Zellweger is generally considered as the prototype of this group of disorders. In these patients there is a generalized loss of peroxisomal functions due to genetic defects leading to a disturbed biogenesis of peroxisomes (1,2). Another major peroxisomal disorder is the rhizomelic form of chondrodysplasia punctata (RCDP) (3). Patients with this autosomal recessive disease show a variety of clinical abnormalities including rhizomelic shortening of the upper extremities, severe growth and mental retardation and cataract. In the majority of RCDP patients a set of four different biochemical abnormalities involving peroxisomes is found which includes (i) a partial deficiency of acyl-CoA:dihydroxyacetonephosphate acyltransferase (DHAPAT, EC 2.3.1.42); (ii) a deficiency of alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase), the first two enzymes in the biosynthetic route of ether-phospholipids (4); (iii) a deficiency of phytanoyl-CoA hydroxylase; and (iv) the absence of the mature form of peroxisomal 3-ketoacyl-CoA thiolase (3). Apart from this form of RCDP, two additional forms have been described with an isolated deficiency of DHAPAT (5-7) or alkyl-DHAP synthase (7,8) designated here as RCDP type 2 and 3, respectively.
The genetic defect in RCDP type 1 has recently been identified independently by three groups of investigators (9-11). The gene involved, hsPEX7, codes for the PTS2 receptor which plays a crucial role in the import of proteins with a peroxisomal targeting signal type 2 (PTS2) (12-16). The genes coding for alkyl-DHAP synthase (17), phytanoyl-CoA hydroxylase (18,19) and peroxisomal 3-ketoacyl-CoA thiolase (20) have all been cloned and comparison of their amino acid sequences indeed revealed a PTS2 consensus sequence (R/K-L/I/V-X5-H/Q-L/A) in all three proteins (17-20). DHAPAT activity is strongly diminished in RCDP type 1 patients which suggests that this enzyme is also routed to peroxisomes via the PTS2 import pathway. Patients with RCDP types 2 and 3 belong to different complementation groups which indicates that different genes cause RCDP. We suggested that the genes encoding DHAPAT or alkyl-DHAP synthase are mutated in RCDP types 2 and 3, respectively. So far, the gene coding for DHAPAT has remained unidentified, although the protein itself was purified from human placenta (21) and guinea pig liver (22) some years ago. In this paper we report the identification of the cDNA coding for human DHAPAT using the partial amino acid sequence information obtained from DHAPAT protein from human placenta. The cDNA encodes a 77.2 kDa protein with a typical PTS1 consensus sequence at its C-terminus (AKL). In addition, we show that all patients with RCDP type 2 have aberrant DHAPAT mRNA which establishes the molecular basis of the disease.
RESULTS
Identification of cDNA encoding human DHAPAT
We used the previously purified DHAPAT from human placenta (21) for N-terminal amino acid sequencing which revealed the following sequence: XVGPTXPXAVV(F/L)(L/Y)YKKELKN(Q/W)D. Using the BLAST algorithm (23) the database of expressed sequence tags (dbEST) was screened and one clone was identified containing a 2040 bp open reading frame (ORF) (Fig. 1) encoding a protein of 680 amino acids with a calculated mol. wt of 77.2 kDa. A start codon ATG was found in the context of a Kozak consensus sequence (24) preceded by an in-frame stop codon TAG. The deduced protein revealed a serine-rich stretch of 11 amino acids preceding the N-terminal amino acid sequence of the purified protein (Fig. 1). No PTS2 consensus sequence was observed in the deduced protein. Instead, a typical PTS1 was found at the extreme C-terminus of the deduced protein (alanine-lysine-leucine, AKL) (25). Considerable homology (36-42% identity) was observed between a region of 200 amino acids of DHAPAT and an enzyme with a strongly related reaction mechanism, acyl-CoA:glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15), from Escherichia coli, Caenorhabditis elegans and mouse (Fig. 2).
| Figure 1. Nucleotide sequence and deduced amino acid sequence of the human DHAPAT cDNA. A sequence for translation initiation in the context of a Kozak consensus (24) is found at position -42 preceded by an in-frame stop codon TAG. The amino acid sequence obtained by N-terminal protein sequencing is underlined. At the C-terminus of the deduced amino acid sequence a peroxisomal targeting signal type 1 (alanine-lysine-leucine) is found. In the 3[prime] untranslated region at position 2270 a putative polyadenylation signal (AATAAA) is observed. |
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Figure 2. Amino acid sequence alignment of the human DHAPAT with GPAT from different species. Amino acid sequence alignment of the human DHAPAT with GPAT from Escherichia coli (Ec, plsB; GenBank accesion no. M93136), Caenorhabditis elegans (Ce; GenBank accession no. U64847) and mouse (Mm; PIR locus A41672). Alignment was performed with the multiple sequence alignment utility from the Human Genome Center, Baylor College of Medicine, Houston. Only the region with high amino acid similarity is shown; amino acids conserved in DHAPAT and at least one of the GPAT sequences are blackened. The arginine residue marked with an asterisk was found to be substituted in patients 5, 6, 7 and 8. 
Expression of DHAPAT in Saccharomyces cerevisiae
To prove that the identified cDNA indeed encodes human DHAPAT, we expressed the complete ORF in the yeast S. cerevisiae under transcriptional control of the fatty acid-inducible CTA1 promoter (26). S. cerevisiae is able to acylate DHAP although it is disputed whether this is brought about by the same enzyme which acylates G3P (GPAT) (27,28) or via both GPAT and a distinct DHAPAT (29,30). GPAT exhibits no activity in our standard assay due to the presence of a high concentration of unlabeled G3P. In addition, the assay is performed at pH 5.6 at which the enzymes in S.cerevisiae show no activity (29; Fig. 3). The complete DHAPAT ORF was cloned and transformed into S.cerevisiae. After induction, lysates prepared from expressing strains contained considerable amounts of DHAPAT activity (Fig. 3) which confirmed that the cDNA does encode DHAPAT.
Figure 3. Functional expression of human DHAPAT cDNA in Saccharomyces cerevisiae. Transformed yeast cells were grown overnight in media supplemented with oleate to induce expression of DHAPAT. Total lysates were prepared as described in the Methods section and immediately used for DHAPAT activity measurements. WT, yeast transformed with pEL 26 containing an unrelated insert (n = 2); pDHAPAT, yeast transformed with pDHAPAT (n = 3); pDHAPATG632A, yeast transformed with pDHAPAT containing the 632G->A mutation (n = 5). Activity is given as mean ± SD for the number of independent experiments given in parentheses. 
Table 1
| Number | DHAPAT activity | ||
| (nmol/2h.mg) | Mutation | Consequence | |
| 1 | ND | 848 ins TT | premature stop |
| 2 | ND | 848 ins TT | premature stop |
| 3 | ND | 780 del | premature stop |
| 4 | ND | 1575 del | premature stop |
| 5 | 0.1 | 632 G->A | R211H |
| 6 | 0.2 | 632 G->A | R211H |
| 7 | 0.2 | 632 G->A | R211H |
| 8 | 0.1 | 631 C->T | R211C |
| Control | 8.1 ± 2.5 (78) | ||
| RCDP type 1 | 1.9 ± 0.9 (39) | ||
Mutation analysis of DHAPAT in RCDP type 2 patients
In order to establish whether the absence of DHAPAT activity in RCDP type 2 patients is indeed caused by mutations in the DHAPAT gene, we analyzed DHAPAT cDNAs isolated from eight patients. The results are summarized in Table 1. Mutation analysis in patients 1 and 2 revealed a homozygous insertion of two nucleotides at position 848-850 (Fig. 4A). This causes a frameshift and leads to a premature stop codon TAG at position 895 resulting in a truncated version of DHAPAT. Mutation analysis in patient 3 revealed a homozygous deletion of a single nucleotide at position 780 (Fig. 4B) which causes a frameshift leading to a premature stop codon TGA at position 787 and a truncated version of DHAPAT. In patient 4 a homozygous deletion of one nucleotide was found at position 1575 (Fig. 4C) which causes a frameshift leading to a premature stop codon TAA at position 1597 resulting in a truncated version of the enzyme. The DHAPAT cDNAs of patients 5, 6 and 7, who belong to a single family with consanguineous parents, contained a homozygous G->A mutation at position 632 (Fig. 4D) resulting in the substitution of the arginine residue at position 211 by a histidine. Interestingly, in the alignment of the protein sequences of human DHAPAT and GPAT from mouse, C.elegans and S.cerevisiae,this arginine residue is conserved suggesting an important role of this residue for the catalytic activity of the enzyme (Fig. 2). Expression of the cDNA carrying this mutation in S.cerevisiae leads to an almost fully inactive DHAPAT enzyme (Fig. 3). Mutation analysis in patient 8 showed a homozygous C->T mutation at position 631 (Fig. 4D) which results in the substitution of the arginine residue at postition 211 by a cysteine.
Figure 4. Nucleotide sequence analysis of RCDP type 2 DHAPAT cDNA: electropherograms showing the regions containing the mutations. (A) Patient 1: insertion of two nucleotides (TT) at position 848 results in a frameshift and a premature stop codon. (B) Patient 3: deletion of one nucleotide (G) at position 780 results in a frameshift and a premature stop codon. (C) Patient 4: deletion of one nucleotide (C) at position 1575 resulting in a frameshift and a premature stop codon. (D) Patient 5: a 632G->A mutation results in the substitution of the arginine residue at position 211 for a histidine. Patient 8: a 631C->A mutation results in the substitution of the same arginine residue for a cysteine. The results described in this paper show that we have identified the cDNA for human DHAPAT. This is not only clear from functional expression of the cDNA in yeast but also from the identification of mutations in the cDNA from patients with an isolated DHAPAT deficiency. The DHAPAT cDNA was identified in the dbEST using a 22 amino acid sequence determined by N-terminal amino acid sequencing of purified DHAPAT. Comparison of this empirically determined sequence with the primary sequence revealed several mismatches. Since extensive screening of the dbEST did not identify other DHAPAT (iso)forms, we believe that these mismatches are due to the low yield of some amino acids during Edmann degradation. The primary sequence also revealed 11 additional amino acids preceding the determined sequence. It is not clear whether these amino acids were lost due to proteolytic degradation during the purification or whether they reflect proteolytic processing after peroxisomal import. Unexpectedly, the primary amino acid sequence of DHAPAT does not contain a PTS 2 consensus sequence which was predicted from the observation that RCDP type 1 patients, who are affected in the PTS2 import pathway, have strongly decreased DHAPAT activity. In contrast, a PTS1 consensus sequence, alanine-lysine-leucine (AKL), was found at the extreme C-terminal end suggesting that DHAPAT is imported via the PTS1 pathway. In line with this finding is the fact that DHAPAT was found to be fully deficient in a patient with a defect in the PTS1 import pathway and a normal PTS2 import pathway (31,32). In all patients we detected only one mutation, suggesting that they are homozygous. Because we performed sequence analysis at the mRNA level, we cannot exclude compound heterozygosity with one allele being a null allele. Final proof has to come from studies at the genomic level which requires elucidation of the gene structure. The underlying basis for the finding that DHAPAT activity is decreased in RCDP type 1 patients with an isolated defect in the PTS2 import pathway, remains unclear. The identification of DHAPAT cDNA is not only important for future prenatal diagnosis and carrier detection but will also allow us to shed more light on the physiological role of ether-phospholipids in mammals which has so far remained enigmatic with the exception of platelet activating factor which has been implicated in a variety of pathophysiological conditions like hypotension and asthma (33). Creation of a knock-out mouse which is now underway, will allow us to study the functional role of ether-phospholipids in detail including its proposed role in scavenging of reactive oxygen species (34,35). Patients 1 and 2. Patient 1 (female) has been described by Wanders et al. (5) and showed all the clinical symptoms described for RCDP. Biochemical investigations revealed a strong deficiency of erythrocyte plasmalogens. Subsequent studies in cultured fibroblasts showed that the biochemical abnormalities were restricted to an isolated DHAPAT deficiency. Patient 2 was a fetus from the same parents. Prenatal diagnosis revealed a severe DHAPAT deficiency in the amniotic fluid cells. After abortion the diagnosis was confirmed by activity measurement in fetal skin fibroblasts (Table 1. ).
DISCUSSION
MATERIALS AND METHODS
RCDP type 2 patients
Patient 8. This male patient from unrelated parents suffered from a mild form of RCDP.
Identification of the human DHAPAT cDNA
DHAPAT was purified from human placenta as described (21) and the N-terminal amino acid sequence was determined using a Procise 494 protein sequencer which revealed the following sequence: XVGPTXPXAVV(F/L)(L/Y)YKKELKN(Q/W)D. The BLAST algorithm (23) was used to screen the human dbEST and a single EST clone was found which had 89% identity with the N-terminal amino acid sequence (GenBank accession no. AA112015). Using the nucleotide sequence of this EST clone, additional EST clones were found in the database (GenBank accession nos AA102248, T77274, AA086418, AA086392, R39556, Z39614, N83157, AA216692 and N55660). Alignment of all the EST clone sequences resulted in three distinct blocks containing (i) the 5[prime]-end, (ii) a middle part, and (iii) the 3[prime]-end of the putative cDNA. Two clones were requested at the UK HGMP Resource Centre at Cambridge (clones 561789 and 562691). Polymerase chain reaction (PCR) experiments using primers based on the EST sequences revealed that clone 561789 contained an insert of ~2.4 kb comprising all three blocks of the alignment while clone 562691 only contained the second half of the cDNA (results not shown). Clone 561789 was subsequently sequenced in its entirety.
Construction of DHAPAT expression plasmids
In order to construct pDHAPAT the complete DHAPAT ORF was amplified from cDNA clone 561789 using the Expand[trade] High Fidelity PCR System (Boehringer Mannheim) according to the manufacturer's protocol with the following primers: BamHI-tagged forward primer: 5[prime]-tctggatccaaaATGGAGTCTTCCAGTTCATC-3[prime] plus HindIII-tagged reverse primer: 5[prime]-tctaagcttTTAAAGTTTTGCAGTGGCTGG-3[prime]. The ORF was subsequently cloned into the BamHI and HindIII sites of pEL 26, a yeast expression plasmid containing the oleate-inducible CTA1 promoter (26) yielding pDHAPAT. The complete ORF was sequenced to exclude the presence of nucleotide substitutions introduced by PCR. To construct pDHAPATG632A the complete DHAPAT ORF was amplified from skin fibroblasts cDNA from patient 5 as described above. Next, a 543 bp BglII fragment from pDHAPAT was replaced by the fragment containing the 632G->A mutation. Nucleotide sequence analysis confirmed the introduction of the mutation and the absence of PCR-introduced nucleotide substitutions.
Expression of DHAPAT in S.cerevisiae
Saccharomyces cerevisiae wild-type strain BJ1991 was transformed as described (36). To prepare lysates, yeast cells were harvested and resuspended in 250 µl 100 mM KPi, pH 7.4, 2 mM EDTA, 5 mM dithiothreitol, 1 mg/ml Pefabloc and 1 µg/ml leupeptin. After addition of 200 µl glass beads, cells were vortexed for 30 min at 4°C. Glass beads and cell debris were removed by centrifugation at 12.000 g for 2 min at 4°C. DHAPAT activity was measured as described previously (21).
RT-PCR amplification of the DHAPAT cDNA and nucleotide sequence analysis
First strand cDNA synthesis was performed as described (37) using 5-10 µg of total RNA isolated from cultured human skin fibroblasts. DHAPAT encoding cDNA was amplified from first strand cDNA as template in four overlapping fragments using primer sets based on the sequence obtained from EST clone 561789. Fragment 1: `-21M13 forward'-tagged primer DHAP25f (sense 5[prime]-tgtaaaacgacggccagtGAGCACCACCACGGTTTAGC-3[prime] ) plus `M13 reverse'-tagged DHAP692r (antisense 5[prime]-caggaaacagctatgaccGACATTCGTAGCAGCTCACC-3[prime]); fragment 2: `-21M13 forward'-tagged primer DHAP540f (sense 5[prime]-tgtaaaacgacggccagtGCATCCTGTTGTTCTGCTGC-3[prime] ) plus `M13 reverse'-tagged primer DHAP1258r (antisense 5[prime]-caggaaacagctatgaccGAACAGCAACTATTAGGGTC-3[prime] ); fragment 3: `-21M13 forward'-tagged primer DHAP1078f (sense 5[prime]-tgtaaaacgacggccagtTCACTTCGATCTTTGGCAGC-3[prime] ) plus `M13 reverse'-tagged primer DHAP1764r (antisense 5[prime]-caggaaacagctatgaccTTCTCTGTAACTAGGATGTC-3[prime] ); fragment 4: `-21M13 forward'-tagged primer DHAP1612f (sense 5[prime]-tgtaaaacgacggccagtCTACAGTTGCTTTCGCTTCC-3[prime] ) plus `M13 reverse'-tagged primer DHAP2225r (antisense 5[prime]-caggaaacagctatgaccTGAGAGGATGTGTCTCTTCC-3[prime] ). PCR conditions for each primer set were 95°C for 2 min followed by 30 cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 1 min. Subsequent sequence analysis of these PCR fragments using both sense and antisense strands was performed using `-21M13 forward' and `M13 reverse' fluorescent primers on an Applied Biosystems 377A automated sequencer according to the manufacturer's protocol.
ACKNOWLEDGEMENTS
We would like to thank Nico Ponne and co-workers for operating the ABI automated sequencer, and Drs Hans R. Waterham, Henk van den Bosch and Ronald Oude Elferink for critical reading of the manuscript. The Procise protein sequencer was largely financed by the Dutch Organization for Scientific Research (NWO), via the Foundation for Medical and Health Research (MW).
REFERENCES
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