INTRODUCTION

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An increasing number of inherited diseases have been identified in recent years in which peroxisomal functions are impaired. The prototype of this group of disorders is the Zellweger syndrome, first described in 1964 (1). Goldfischer and co-workers (2) discovered in 1973 that morphologically distinguishable peroxisomes were absent in hepatocytes of patients affected by this disorder, and this discovery was followed a decade later by the observation that there is an accumulation of very long chain fatty acids in the plasma of these patients (3) and that plasmalogens (a special kind of ether phospholipids) were deficient in tissues and erythrocytes from these patients (4).

The peroxisomal disorders identified today can be subdivided into three groups depending upon whether there is a generalized loss (A), a multiple loss (B), or a single loss (C) of peroxisomal functions (5). The Zellweger syndrome, as well as neonatal adrenoleukodystrophy (NALD),¹ are examples of disorders belonging to group A. Rhizomelic chondrodysplasia punctata (RCDP) is an example of a disorder belonging to group B, in which peroxisomes are present and only a limited number of peroxisomal functions is impaired. Four distinct biochemical abnormalities have been found in RCDP, including a deficiency in the activities of phytanoyl-CoA hydroxylase and the two peroxisomal enzymes involved in ether phospholipid synthesis, i.e. dihydroxyacetonephosphate (DHAP) acyltransferase and alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase) (6). Furthermore, the peroxisomal 3-oxoacyl-CoA thiolase is not imported and processed and hence is present as the 44-kDa precursor rather than as the 41-kDa mature form. The basic defect in RCDP is the inability to import proteins carrying the peroxisomal targeting signal (PTS) 2 (7, 8), which was first identified in the cleavable presequences of mammalian peroxisomal thiolases (9). Due to the deficient activities of the enzymes mentioned above, RCDP patients are deficient in plasmalogens and accumulate phytanic acid in plasma (5).

In recent years, enormous progress has been made in identifying genes whose products are involved in peroxisome biogenesis and mutations in these genes have been shown to be present in patients affected by peroxisomal disorders (10). The first gene identified involved in a peroxisomal disorder (Zellweger syndrome) was the peroxisome assembly factor 1 (PAF1, renamed as PEX2), which encodes a 35-kDa zinc binding integral membrane protein (11). Mutations in the gene encoding the human PTS1 receptor (PEX5) define complementation group 2 of the peroxisomal biogenesis disorders (12). Whereas a point mutation leading to a N489K substitution caused an isolated deficiency in PTS1 import, a mutation leading to a premature stop codon resulted in a deficiency in both PTS1 and PTS2 import, suggesting that the PTS1 receptor may also be involved in PTS2 import, at least in mammals. In RCDP, mutations in the PTS2 receptor (PEX7) are observed leading to deficiencies in PTS2 protein import (13-15).

A few patients have been identified that have an isolated deficiency in one of the peroxisomal enzymes involved in ether phospholipid synthesis, i.e. DHAP acyltransferase (16-18) or alkyl-DHAP synthase (19), respectively. These patients show severe clinical abnormalities comparable to RCDP stressing the importance of ether phospholipids for human physiology.

The molecular cloning of the cDNA encoding guinea pig and human alkyl-DHAP synthase (20, 21) revealed the presence of a cleavable presequence containing a PTS2 motif, a finding in line with its deficiency in RCDP. The availability of a specific antiserum against alkyl-DHAP synthase has now enabled us to examine the level and size of this enzyme in human peroxisomal disorders. Furthermore, the isolated alkyl-DHAP synthase deficiency reported in the only patient identified so far (19) has been examined at the cDNA level and led to the identification of a critical residue for enzymatic activity.

	EXPERIMENTAL PROCEDURES

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Materials

An enhanced chemiluminescense (ECL) detection kit was obtained from NEN Life Science Products. Horseradish peroxidase-conjugated goat anti-rabbit IgG was a product of Bio-Rad. Oligonucleotides were manufactured by Isogen, Maarssen, The Netherlands. M-Murine leukemia virus reverse transcriptase was a product of New England Biolabs. Taq DNA polymerase was from Promega, Madison, WI. Phenylglyoxal monohydrate was from Acros Organics, Geel, Belgium.

Methods

Cell Culture-- Fibroblasts were cultured and harvested by gentle trypsinization and washing by low speed centrifugation as described before (22). The NALD patient with an isolated PTS1 import deficiency (complementation group 2 of the Kennedy Krieger Institute or group 4 of the Amsterdam nomenclature) has been described before (7). The alkyl-DHAP synthase deficient patient has also been described before (19). The RCDP patients studied in this paper belong both to complementation group 11 of the Kennedy Krieger Institute or group 1 of the Amsterdam nomenclature (23). The two Zellweger patients studied in this paper belong both to complementation group 1 of the Kennedy Krieger Institute or group 2 of the Amsterdam nomenclature (23).

Western Blotting and in Vitro Transcription/Translation-- The antiserum used was raised against recombinant guinea pig alkyl-DHAP synthase as described before (24). SDS-polyacrylamide gel electrophoresis on 9% acrylamide gels was performed according to Laemmli (25), and proteins were transferred to nitrocellulose membranes. Nonspecific binding sites were blocked overnight at 4 °C with 4% milk powder and 0.4% Tween-20 in phosphate-buffered saline. Subsequent manipulations were done at room temperature. The membranes were incubated with the antiserum for 1 h at a dilution of 1:2000 in phosphate-buffered saline containing 0.1% milk powder and 0.01% Tween-20. After washing, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG for 1 h. Peroxidase activity was detected with ECL.

cDNA Analysis of Alkyl-DHAP Synthase-deficient Patient-- Total RNA was isolated from fibroblasts derived from the alkyl-DHAP synthase-deficient patient using the method of Chomczynski and Sacchi (26). First strand cDNA was prepared as described before (17), and PCR was performed with primers tagged with M13 sequences to allow sequencing with M13 primers (Table I). In previous experiments we were not able to amplify a large part of the presequence probably due to an extremely high GC content so only the mature protein was sequenced. PCR was performed essentially as described before (17). DNA Sequencing was performed on an Applied Biosystems 377A automated DNA sequencer following ABI protocols.

View this table: [in this window] [in a new window]   Table I Primers used for amplication of human alkyl-DHAP synthase cDNA Primers contain either a 21M13 or a M13rev extension to allow sequencing with the corresponding sequencing primers, respectively.

Phenylglyoxal Inhibition of Alkyl-DHAP Synthase-- The recombinant His-tagged alkyl-DHAP synthase described in Ref. 20 was used as enzyme source. E. coli cells expressing this protein were resuspended in either 50 mM borate buffer (pH 8.3), 125 mM bicarbonate buffer (pH 7.5), or 20 mM Tris-HCl (pH 7.4) each containing 1 mM EDTA. Cells were lysed by adding 0.2% Triton X-100 and 0.2 mg/ml lysozyme to these suspensions and incubating the samples for 1 h at 0 °C. After centrifugation (10 min at 22000 × g), the supernatants containing the active soluble alkyl-DHAP synthase were incubated without or with the indicated concentrations of inhibitors and substrate. Aliquots of these samples were withdrawn after the indicated time periods, diluted, and assayed for alkyl-DHAP synthase activity (27).

Chromosomal Mapping-- A 1850-base pair NcoI fragment of human alkyl-DHAP synthase cDNA (21) was used as probe. Metaphase spreads were prepared from human lymphocytes, and FISH detection was performed as described before (28, 29) by SeeDNA Biotech Inc., North York, Canada.

	RESULTS

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In Fig. 1A a Western blot analysis is shown from fibroblast homogenates derived from controls and patients with the indicated peroxisomal disorders using the antiserum against recombinant guinea pig alkyl-DHAP synthase. Human alkyl-DHAP synthase in control fibroblasts runs at a slightly higher molecular weight than the guinea pig counterpart (compare lanes 1, 2, and 3), as has been observed before for liver (24). In Zellweger syndrome and RCDP, alkyl-DHAP synthase is present at strongly reduced levels, although some residual mature protein is observed as well as protein running at a higher molecular weight, which may correspond to the precursor form of alkyl-DHAP synthase. In a NALD patient having an isolated PTS1 import deficiency, alkyl-DHAP synthase is present at a level comparable to that in control fibroblasts but running at a higher molecular weight than in controls. Presumably, the protein is not proteolytically processed in this patient and most likely represents the precursor form of alkyl-DHAP synthase. This is supported by the observation that a cDNA construct containing the complete open reading frame encoding human alkyl-DHAP synthase precursor transcribed in an in vitro transcription/translation assay yields a protein of the same size as detected in this NALD patient (Fig. 2). Alkyl-DHAP synthase activity measurement in a fibroblast homogenate of this patient yielded a value comparable to control fibroblasts, indicating that the presence of the presequence does not greatly affect enzymatic activity (Fig. 1B).

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Alkyl-DHAP synthase in human fibroblasts analyzed with Western blot immunostaining (A) and activity measurements (B). A, 150 µg of protein was loaded in each lane; from left to right, Guinea pig liver homogenate, two independent control fibroblast cell lines, a NALD patient having an isolated PTS1 import deficiency, a patient with an isolated alkyl-DHAP synthase deficiency, two RCDP patients and two Zellweger syndrome patients. B, alkyl-DHAP synthase activity was measured as described (27). Results are expressed as mean ± S.D. for three determinations on each homogenate.

View larger version (92K): [in this window] [in a new window]   Fig. 2.   Size of alkyl-DHAP synthase in human fibroblasts compared with the size of the enzyme translated in vitro. M, markers; lane 1, control fibroblast homogenate; lane 2, NALD (PTS1 deficient) fibroblast homogenate; lane 3, in vitro transcription/translation reaction mixture with no added DNA; lane 4, in vitro transcription/translation reaction mixture with cDNA for human alkyl-DHAP synthase precursor.

Fibroblasts from the patient with an isolated alkyl-DHAP synthase deficiency (19) were found to contain a normally processed protein at a level comparable to that of control fibroblasts, suggesting that this patient has a point mutation affecting a critical residue for enzymatic activity. To investigate this possibility, the cDNA sequence encoding alkyl-DHAP synthase from this patient was determined by direct sequencing of the reverse transcriptase-PCR products using fluorescently labeled M13 primers as described under "Experimental Procedures." Only one difference was observed between the deduced amino acid sequence and the human wild-type sequence (21), i.e. amino acid 419, which is an arginine in the wild-type sequence, was changed into a histidine as a result of a G to A transition at nucleotide 1256 (Fig. 3A). This transition creates a HphI site in the mutant cDNA, and the sensitivity of the mutant cDNA to this restriction enzyme confirms that the G to A transition is not a sequencing artifact (Fig. 3B). To obtain further proof that this substitution is responsible for the inactivity of the enzyme in this patient, and that it does not represent a natural polymorphism, recombinant pControl and pMutant constructs were prepared as indicated in Fig. 4A (compare legend). Both constructs were expressed in E. coli. As can be seen in Fig. 4 the recombinant mutant protein and its control counterpart were expressed at very similar levels both when assayed with Coomassie staining (Fig. 4B) or immunostaining (Fig. 4C). However, no alkyl-DHAP synthase activity was detected in E. coli homogenates expressing the recombinant mutant protein (encoded by the pMutant construct) with His-419, whereas the expression of the recombinant control protein (encoded by the pControl construct) with Arg-419 resulted in an enzymatic activity of 1.87 milliunit/mg E. coli homogenate protein. These results provide convincing proof that this R419H mutation is responsible for the inactivity of the enzyme in this patient and indicates that this arginine is essential for alkyl-DHAP synthase activity.

View larger version (50K): [in this window] [in a new window]   Fig. 3.   Mutation in the patient with an isolated deficiency in alkyl-DHAP synthase. Panel A, mutation in the alkyl-DHAP synthase-deficient patient as determined by nucleotide sequencing. The HphI restriction site that is created by this mutation is underlined. Panel B, presence of the HphI site in the alkyl-DHAP synthase patient. A DNA fragment was amplified with primers flanking the mutation (sense nucleotides 1072-1092 and antisense 1450-1468) from both control and the alkyl-DHAP synthase-deficient patient. The PCR reactions were subjected to HphI cleavage.

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Expression of a recombinant protein in E. coli carrying the R419H mutation. Panel A, cloning strategy. The pET-15b guinea pig alkyl-DHAP synthase expression construct (line 1) was used as described in (20). For construction of pControl (line 4), the SacI-Bsu36I (nucleotides 553-1299) in the guinea pig construct (line 1) was replaced by the corresponding human fragment (line 2) previously isolated from a gt11 library (21). For construction of pMutant (line 5) carrying the R419H mutation, the SacI-Bsu36I fragment in construct (line 1) was replaced by the corresponding fragment amplified from cDNA derived from the alkyl-DHAP synthase-deficient patient (line 3). Panel B, Coomassie stain of E. coli homogenates expressing the pControl and pMutant constructs. As a negative control, an empty pET-15b vector was used. Panel C, immunodetection of alkyl-DHAP synthase in these E. coli homogenates. Panel D, alkyl-DHAP synthase activity in these E. coli homogenates.

Fig. 5A shows that alkyl-DHAP synthase activity is hardly affected by the serine-modifying agent phenylmethanesulfonyl fluoride at concentrations of 0.5 or 1 mM. Only at higher concentrations some inhibition of enzyme activity is observed. By contrast, alkyl-DHAP synthase activity is efficiently inhibited by concentrations of 0.5 and 1 mM of the arginine-modifying agent phenylglyoxal. This inhibition appears to be most efficient in incubations in bicarbonate buffer. Subsequent experiments (Fig. 5B) indicate that the inhibition by 2 mM phenylglyoxal follows pseudo-first order kinetics with k = 0.067 min¹. The presence of the substrate palmitoyl-DHAP protects the enzyme against phenylglyoxal-mediated inactivation resulting in an inactivation constant (k = 0.006 min¹) that is even somewhat lower than the one observed in the absence of inhibitor (k = 0.009 min¹).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Effect of phenylglyoxal on alkyl-DHAP synthase activity. A, effect of phenylglyoxal concentration and incubation buffer. Enzyme was incubated for 45 min at 25 °C without or with the indicated concentrations of phenylmethanesulfonyl fluoride or phenylglyoxal as described under "Experimental Procedures" prior to activity assay. Symbols: , phenylmethylsulfonyl fluoride, Tris buffer; , phenylglyoxal, Tris buffer; , phenylglyoxal, borate buffer; , phenylglyoxal, bicarbonate buffer. Enzyme activity is expressed as the percentage of activity measured in the absence of inhibitor. B, effect of incubation time and palmitoyl-DHAP. Enzyme was incubated for the indicated time periods without inhibitor (), 2 mM phenylglyoxal (), or 2 mM phenylglyoxal in the presence of 0.3 mM palmitoyl-DHAP () in Tris buffer as described under "Experimental Procedures." Activities are expressed as the logarithm of the percentage of the activity measured at zero time.

FISH mapping of a 1850-base pair human alkyl-DHAP synthase cDNA probe resulted in signals on one pair of chromosomes, i.e. the long arm of chromosome 2, region q31 (Fig. 6). No additional locus is revealed by FISH detection under the conditions used. Therefore, we conclude that the gene encoding human alkyl-DHAP synthase is located on chromosome 2q31.

View larger version (112K): [in this window] [in a new window]   Fig. 6.   Chromosomal assignment of the gene encoding human alkyl-DHAP synthase. The chromosomal mapping of human alkyl-DHAP synthase was determined by FISH on human metaphase spreads of human blood lymphocytes. Panel A, FISH signals on chromosome; panel B, 4,6-diamidino-2-phenylindole stain of the same mitotic figure. The gene for human alkyl-DHAP synthase was localized on chromosome 2, region q31.

	DISCUSSION

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The discovery that plasmalogens are deficient in the tissues from patients suffering from Zellweger syndrome stressed unambiguously the indispensible role of peroxisomes for ether phospholipid biosynthesis (4). The activities of the first two (peroxisomal) enzymes in this biosynthetic route, DHAP acyltransferase and alkyl-DHAP synthase, are deficient in Zellweger syndrome, as well as in several other peroxisomal disorders including RCDP (5, 6). Recently, we succeeded in cloning of the cDNA encoding alkyl-DHAP synthase, and this led to the discovery that this enzyme is synthesized as a precursor protein containing a PTS2 motif in a cleavable presequence (20, 21). The availability of an antiserum against alkyl-DHAP synthase enabled us to study the molecular basis of the deficiency of this enzyme in more detail. The strongly reduced levels in fibroblasts derived from patients affected by Zellweger syndrome and RCDP suggest that alkyl-DHAP synthase is not stable in the cytoplasm when it is not imported into the peroxisome due to the absence of peroxisomes (Zellweger syndrome) or defective import (RCDP) (Fig. 1). Instabiliy of peroxisomal proteins in Zellweger syndrome is also observed for the enzymes involved in -oxidation that become rapidly degraded in the absence of functional peroxisomes (30). However, fibroblasts derived from patients suffering from Zellweger syndrome have invariably been shown to retain some residual alkyl-DHAP synthase activity, which generally amounts to approximately 10% of control fibroblasts (31), as well as a residual capacity to synthesize plasmalogens (32). These results indicate that alkyl-DHAP synthase can nevertheless fold into an active enzyme in this disorder. In this respect, it is remarkable that in Zellweger syndrome all residual signals on Western blots correspond to the precursor form rather than the mature form, already suggesting that the precursor may be active and responsible for the residual activities observed. The reduced levels of alkyl-DHAP synthase in Zellweger syndrome and RCDP fibroblasts are in line with the observations made in liver samples from patients suffering from these disorders, although in these samples actually no residual enzyme protein could be observed (24).

The residual alkyl-DHAP synthase content of RCDP fibroblasts consists of both the precursor and the mature form of the enzyme (Fig. 1). This finding is different from the observations made with peroxisomal 3-oxoacyl-CoA thiolase (the other known PTS2 protein in humans), which is only present as the 44-kDa precursor form (6). A clear explanation for this discrepancy is lacking.

Alkyl-DHAP synthase is present in the NALD patient with a specific PTS1 import defect (described in Ref. 7) at a level comparable to control fibroblasts, which is in line with the expectation that alkyl-DHAP synthase, being a PTS2 protein, is imported into the peroxisome in this patient. However, alkyl-DHAP synthase is present as its precursor form rather than as its mature form in this disorder. An analogous observation has been made with peroxisomal 3-oxoacyl-CoA thiolase, which, although imported, is also not processed in this patient (7). These findings can be explained by assuming that the protease responsible for processing of both alkyl-DHAP synthase and 3-oxoacyl-CoA thiolase is a PTS1 protein, and is thus not present in the peroxisome in this disorder. Indeed, evidence has been reported which suggests that pp110, a protein with a consensus PTS1 at its C terminus, is the protease that cleaves the peroxisomal thiolase (33). Since alkyl-DHAP synthase and peroxisomal thiolase contain sequence homology at the cleavage site (20), pp110 may as well be responsible for cleavage of alkyl-DHAP synthase. The alkyl-DHAP synthase activity in the homogenate of this patient is not impaired (even somewhat higher than the two controls) indicating that processing of the precursor is not needed for the enzyme to become active. Nevertheless, this patient still exhibits a relatively mild impairment in de novo plasmalogen synthesis when compared with controls (about a factor two lower than controls, see patient PBD018 in Ref. 34), which may point to an involvement of a PTS1 protein in ether lipid synthesis. Unfortunately, the DHAP acyltransferase activity in this patient has not been reported.

The discovery of a patient with an isolated deficiency in alkyl-DHAP synthase activity with the clinical symptoms of RCDP indicates that ether phospholipids are extremely important for human physiology and that the deficiency in ether phospholipids is actually responsible for the RCDP phenotype (5, 6). The observation that fibroblasts of this patient contain normal levels of mature alkyl-DHAP synthase protein suggests that the protein is properly incorporated into the peroxisome and that an amino acid residue essential for enzymatic activity has been mutated. The subsequent identification of this mutation responsible for the inactivity (R419H) gives us a first example of an essential residue for enzymatic activity. In agreement with this result is the observation that the enzyme can be inactivated by the arginine-modifying agent phenylglyoxal (Fig. 5). The finding that the enzyme is efficiently protected against this inactivation when substrate (palmitoyl-DHAP) is present at a saturating concentration provides a strong indication that this inactivation results from modification of (an) arginine residue(s) in the active site. However, experimental proof is lacking that the inhibition of enzymatic activity by phenylglyoxal is indeed a result of a modification of arginine 419. Arginines are known to be ideally suited for interaction with negatively charged phosphate groups due to their ability to form multiple hydrogen bonds with this moiety (35). Because of resonance stabilization of the guanidinium group in the side chains of these residues, arginines are poor proton donors and would probably not function as general acid catalysts (36). In this respect it is noteworthy that acyl-dihydroxyacetone lacking the phosphate group appears not to be a substrate for alkyl-DHAP synthase, stressing the importance of the phosphate group (37). However, from the data presented in this paper it is not strictly proven that this arginine residue is actually an active site residue involved in substrate binding or catalysis, since such a mutation may as well disturb the secondary or tertiary structure of a protein to the extent that it becomes inactive. The conclusion that the R419H substition does not reflect a natural occurring polymorphism is also stressed by the observation that this arginine residue is also present in at least two independent human expressed sequence tags clones (21) and guinea pig alkyl-DHAP synthase (20).