Dihydroxyacetone Phosphate Acyltransferase Activity Research Articles

The Trypanosoma brucei dihydroxyacetonephosphate acyltransferase TbDAT is dispensable for normal growth but important for synthesis of ether glycerophospholipids.

Glycerophospholipids are the most abundant constituents of biological membranes in Trypanosoma brucei, which causes sleeping sickness in humans and nagana in cattle. They are essential cellular components that fulfill various important functions beyond their structural role in biological membranes such as in signal transduction, regulation of membrane trafficking or control of cell cycle progression. Our previous studies have established that the glycerol-3-phosphate acyltransferase TbGAT is dispensable for growth, viability, and ester lipid biosynthesis suggesting the existence of another initial acyltransferase(s). This work presents the characterization of the alternative, dihydroxyacetonephosphate acyltransferase TbDAT, which acylates primarily dihydroxyacetonephosphate and prefers palmitoyl-CoA as an acyl-CoA donor. TbDAT restores the viability of a yeast double null mutant that lacks glycerol-3-phosphate and dihydroxyacetonephosphate acyltransferase activities. A conditional null mutant of TbDAT in T. brucei procyclic form was created and characterized. TbDAT was important for survival during stationary phase and synthesis of ether lipids. In contrast, TbDAT was dispensable for normal growth. Our results show that in T. brucei procyclic forms i) TbDAT but not TbGAT is the physiologically relevant initial acyltransferase and ii) ether lipid precursors are primarily made by TbDAT.

Infantile Refsum Disease: Influence of Dietary Treatment on Plasma Phytanic Acid Levels.

Infantile Refsum disease (IRD) is one of the less severe of Zellweger spectrum disorders (ZSDs), a group of peroxisomal biogenesis disorders resulting from a generalized peroxisomal function impairment. Increased plasma levels of very long chain fatty acids (VLCFA) and phytanic acid are biomarkers used in IRD diagnosis. Furthermore, an increased plasma level of phytanic acid is known to be associated with neurologic damage. Treatment of IRD is symptomatic and multidisciplinary.The authors report a 3-year-old child, born from consanguineous parents, who presented with developmental delay, retinitis pigmentosa, sensorineural deafness and craniofacial dysmorphisms. While the relative level of plasma C26:0 was slightly increased, other VLCFA were normal. Thus, a detailed characterization of the phenotype was essential to point to a ZSD. Repeatedly increased levels of plasma VLCFA, along with phytanic acid and pristanic acid, deficient dihydroxyacetone phosphate acyltransferase activity in fibroblasts and identification of the homozygous pathogenic mutation c.2528G>A (p.Gly843Asp) in the PEX1 gene, confirmed this diagnosis. Nutritional advice and follow-up was proposed aiming phytanic acid dietary intake reduction. During dietary treatment, plasma levels of phytanic acid decreased to normal, and the patient's development evaluation showed slow progressive acquisition of new competences.This case report highlights the relevance of considering a ZSD in any child with developmental delay who manifests hearing and visual impairment and of performing a systematic biochemical investigation, when plasma VLCFA are mildly increased. During dietary intervention, a biochemical improvement was observed, and the long-term clinical effect of this approach needs to be evaluated.

Characterization of a compensatory mutant of Leishmania major that lacks ether lipids but exhibits normal growth and G418 resistance

Ether glycerolipid biosynthesis in Leishmania major initiates with the acylation of dihydroxyacetonephosphate by the glycosomal dihydroxyacetonephosphate acyltransferase LmDAT. We previously reported that a null mutant of LmDAT is severely affected in logarithmic growth, survival during stationary phase, and in virulence in mice. In addition, it lacks all ether glycerolipids, produces altered forms of the ether‐lipid based virulence factors lipophosphoglycan and increased levels of GPI‐anchored protein gp63. Here, we describe the characterization of a compensatory mutant of a null strain of LmDAT, Δlmdat/Δlmdatrev. Similarly to the null mutant, the Δlmdat/Δlmdatrev strain formed altered forms of lipophosphoglycan and increased levels of gp63, and was avirulent in mice infection. Further, dihydroxyacetonephosphate acyltransferase activity was absent in the revertant clone, indicating that a mutation in another acyltransferase gene did not confer dihydroxyacetonephosphate specificity. In contrast, the revertant grew normally but still exhibited poor survival during stationary phase. In addition, agarose gel analysis of its genomic DNA failed to detect any amplified DNA. Surprisingly, its sensitivity to aminoglycoside based antibiotic G418 was three times lower than that of the null mutant, while resistance to blasticidin and hygromycin was similar to that of the null mutant.

Characterization of a compensatory mutant of Leishmania major that lacks ether lipids but exhibits normal growth, and G418 and hygromycin resistance

Ether glycerolipid biosynthesis in Leishmania major initiates with the acylation of dihydroxyacetonephosphate by the glycosomal dihydroxyacetonephosphate acyltransferase LmDAT. We previously reported that a null mutant of LmDAT is severely affected in logarithmic growth, survival during stationary phase, and in virulence in mice. In addition, it lacks all ether glycerolipids, produces altered forms of the ether-lipid based virulence factors lipophosphoglycan and increased levels of GPI-anchored protein gp63. Here, we describe the characterization of a compensatory mutant of a null strain of LmDAT, Δlmdat/Δlmdatrev. Similarly to the null mutant, the Δlmdat/Δlmdatrev strain formed altered forms of lipophosphoglycan and increased levels of gp63, and was avirulent in mice infection. Further, dihydroxyacetonephosphate acyltransferase activity was absent in the revertant clone, indicating that a mutation in another acyltransferase gene did not confer dihydroxyacetonephosphate specificity. In contrast, the revertant grew normally but still exhibited poor survival during stationary phase. In addition, agarose gel analysis of its genomic DNA failed to detect any amplified DNA. Surprisingly, its sensitivity to aminoglycoside based antibiotics G418 and hygromycin was lower than that of the null mutant, wild type and complemented line.

Leishmania major Expresses a Single Dihydroxyacetone Phosphate Acyltransferase Localized in the Glycosome, Important for Rapid Growth and Survival at High Cell Density and Essential for Virulence

Despite major advances in the understanding of pathogenesis of the human protozoan parasite Leishmania major, little is known about the enzymes and the primary precursors involved in the initial steps of synthesis of its major glycerolipids including those involved in virulence. We have previously demonstrated that the initial step of acylation of the precursor glycerol 3-phosphate is not essential for the synthesis of ester and ether phospholipids in this parasite. Here we show that Leishmania expresses a single acyltransferase with high specificity for the precursor dihydroxyacetone phosphate and shows the best activity in the presence of palmitoyl-CoA. We have identified and characterized the LmDAT gene encoding this activity. LmDAT complements the lethality resulting from the loss of both dihydroxyacetone phosphate and glycerol-3-phosphate acyltransferase activities in yeast. Recombinant LmDAT exhibits biochemical properties similar to those of the native enzyme of the promastigote stage parasites. We show that LmDAT is a glycosomal enzyme and its loss in a delta lmdat/delta lmdat null mutant results in complete abrogation of the parasite dihydroxyacetone phosphate acyltransferase activity. Furthermore, lack of LmDAT causes a major alteration in parasite division during the logarithmic phase of growth, an accelerated cell death during stationary phase, and loss of virulence. Together, our results demonstrate that LmDAT is the only dihydroxyacetone phosphate acyltransferase of the L. major localized in the peroxisome, important for growth and survival and essential for virulence.

Plasmalogen metabolism-related enzymes in rat brain during aging: influence of n-3 fatty acid intake

Plasmalogens (Pls) are phospholipids containing a vinyl–ether bond at the sn-1 position of the glycerol backbone. They represent between 1/2 and 2/3 of the ethanolamine phospholipids in the brain. During aging, the Pls content in human brain falls down. However, the role of Pls metabolism-related enzymes in the regulation of Pls levels remains to be determined. Dihydroxyacetone phosphate acyltransferase (DHAP-AT) is the enzyme involved in the first step of Pls biosynthesis. In the brain, a phospholipase A 2, which selectively acts on Pls, has been isolated (Pls-PLA 2s). In this work, we aimed to evaluate the impact of DHAP-AT (a key enzyme of Pls biosynthesis) and Pls-PLA 2 (a specific Pls degradation enzyme) on the evolution of Pls content in the rat brain during aging. The influence of n-3 fatty acid intake was also evaluated. Littermates from two generations of n-3 deficient rats were fed an equilibrated diet containing either α-LNA alone or with two doses of DHA. After weaning, 3, 9 or 21 months of diet, rats were sacrificed. Enzymatic assays were performed, Pls levels were assessed and the sn-2 position of ethanolamine Pls was analyzed. DHAP-AT activity significantly increased between weaning and 3 months with a concomitant increase of brain Pls, which reached maximal levels after 9 months. Then, Pls levels and DHAP-AT activity significantly decreased while Pls-PLA 2s activity significantly increased. Dietary n-3 fatty acids had no effect on DHAP-AT activity and on Pls levels. In conclusion, the increase of brain Pls content in the first part of the life may be related to the high increase of DHAP-AT activity, probably stimulated by DHA. In aged animals, the decrease of Pls levels may mainly be caused to an increase of their degradation by Pls-PLA 2. Dietary DHA may not oppose the physiologic aging.

Role of dihydroxyacetonephosphate acyltransferase in the biosynthesis of plasmalogens and nonether glycerolipids

The variant CHO-K1 cell line, NRel-4, is unable to synthesize plasmalogens because of a severe reduction in dihydroxyacetonephosphate acyltransferase (DHAPAT) activity (Nagan, N., A. K. Hajra, L. K. Larkins, P. Lazarow, P. E. Purdue, W. B. Rizzo, and R. A. Zoeller. 1998. Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol. Biochem. J. 332: 273-279). Northern analysis demonstrated that the loss of this activity was attributable to a severe reduction in mRNA levels for DHAPAT. Transfection of NRel-4 cells with a plasmid bearing the human DHAPAT cDNA recovered DHAPAT activity and plasmalogen biosynthesis. Examination of clonal isolates from the transfected population showed that recovery of as little as 10% of wild-type DHAPAT activity restored plasmalogen levels to 55% of normal, whereas in one isolate, NRel-4.15, which overexpressed DHAPAT activity by 6-fold over wild-type cells, plasmalogen levels were returned only to wild-type values. Although the rate of plasmenylethanolamine biosynthesis was restored in NRel-4.15, the biosynthesis of nonether glycerolipids was either decreased or unaffected, suggesting that peroxisomal DHAPAT does not normally contribute to nonether glycerolipid biosynthesis. These data demonstrate that a defect in the gene that codes for peroxisomal DHAPAT is the primary lesion in the NRel-4 cell line and that the peroxisomal DHAPAT is essential for the biosynthesis of plasmalogens in animal cells.

2P-0550 Possible mechanisms for the antiobesity effects of a novel compound OT-13540

Treatment of normolipidemic rats by alkylthiopropionic acid (CETTD), resulted in a dose- and time-dependent increase in total dihydroxyacetone phosphate acyltransferase (DHAPAT) activity, in extent comparable to that of 3-thiadicarboxylic acid (BCMTD) and alkylthioacetic acid (CMTTD). Thus, in CETTD- and CMTTD-treated rats, the specific DHAPAT activity increased in the microsomal, peroxisomal and mitochondrial fractions. In contrast, repeated administration of the peroxisome proliferator, BCMTD, decreased the specific DHAPAT activity both in the peroxisomal fraction and in purified peroxisomes. A three-fold increase in specific activity was, however, revealed in the mitochondrial fraction. Whether the variation of the DHAPAT activity in the mitochondrial and microsomal fractions among the feeding groups can be explained by increased number of enlarged and small peroxisomes sedimenting in the fractions, are to be considered. Subcellular fractionation studies confirmed previous findings that rat liver glycerophosphate acyltransferase (GPAT) was located both in mitochondria and the microsomal fraction. BCMTD was considerably more potent than CMTTD in stimulating the microsomal and mitochondrial GPAT activities. Administration of CETTD marginally affected the isoenzymes of GPAT. Diacylglycerol acyltransferase (DGAT) activity was increased by 35% in BCMTD and CMTTD treated rats, but by administration of CETTD the enzyme activity was decreased by more than 80%. The acyl-CoA cholesterol acyltransferase (ACAT) activity was marginally affected in animals treated with BCMTD, CMTTD and CETTD. Thus, the results indicate that the initial steps in the synthesis of triacylglycerols and ether glycerolipids as well as the last step in triacylglycerol synthesis could not be identified as mediating the fat accumulation or the lowering of triacylglycerol content in liver of CETTD, or BCMTD and CMTTD treated rats. On the other hand, CMTTD increased the palmitoyl-CoA oxidation in mitochondria, and CETTD considerably inhibited the activity. Therefore, it is conceivable that the development of fatty liver with CETTD is mostly due to inhibition of mitochondrial β-oxidation.

Biochemical markers predicting survival in peroxisome biogenesis disorders.

To identify prognostic markers reflecting the extent of peroxisome dysfunction in primary skin fibroblasts from patients with peroxisome biogenesis disorders (PBD). PBD are a genetically heterogeneous group of disorders due to defects in at least 11 distinct genes. Zellweger syndrome is the prototype of this group of disorders, with neonatal adrenoleukodystrophy and infantile Refsum disease as milder variants. Common to these three disorders are liver disease, variable neurodevelopmental delay, retinopathy, and perceptive deafness. Because genotype-phenotype studies are complicated by the genetic heterogeneity among patients with PBD, the authors evaluated a series of biochemical markers as a measure of peroxisome dysfunction in skin fibroblasts. Multiple peroxisomal functions including de novo plasmalogen synthesis, dihydroxyacetonephosphate acyltransferase (DHAPAT) activity, C26:0/C22:0 ratio, C26:0 and pristanic acid beta-oxidation, and phytanic acid alpha-oxidation were analyzed in fibroblasts from a series of patients with defined clinical phenotypes. A poor correlation with age at death was found for de novo plasmalogen synthesis, C26:0/C22:0 ratio, and phytanic acid alpha-oxidation. A fairly good correlation was found for pristanic acid beta-oxidation, but the best correlation was found for DHAPAT activity and C26:0 beta-oxidation. A mathematic combination of DHAPAT activity and C26:0 beta-oxidation showed an even better correlation. DHAPAT activity and C26:0 beta-oxidation are the best markers in predicting life expectancy of patients with PBD. Combination of both markers gives an even better prediction. These results contribute to the management of patients with PBD.

Peroxisomal Proliferation in Rat Liver and Essential Fatty Acid Deficiency

The effects of essential fatty acid (EFA) deficiency were studied on male Wistar rats membrane lipid composition, enzyme activities and proliferation of liver peroxisome by feeding the animals, during 4 weeks, with fat free diets supplemented or not with a peroxisome proliferator, clofibrate (CLO).In deficient (E) goup, peroxisomal membrane was characterized by higher level of total de novo synthesized unsaturated (n-9 and n-7 series) fatty acids and lower content of EFA (n-6 and n-3 series) and exhibited cholesterol (CHOL) to phospholipids (PLs) ratio diminution compared with that of control (C) group. Co-administration of CLO with the fat-free diet (EC group) enhanced the membrane uptake of n-9 fatty acids. Slight increase of membrane dihydroxyacetone-phosphate acyl-transferase (DHAP-AT) and decrease of palmitoyl-CoA synthetase activities were found in E group which featured normal-shaped peroxisomes.In EC group, enzyme inductions of peroxisomal pamitoyl-CoA oxidase and palmitoyl-CoA synthetase were slightly higher and significantly lower respectively compared with those of C group treated with CLO (CC group) whereas activity of DHAP-AT was stable. Identical marked proliferation of peroxisomes were observed in both groups.It is inferred that, despite the parallel alterations of membrane lipid composition and of enzyme activities of peroxisomes upon 4 weeks of EFA deficientdiet, the biogenesis and proliferation of the organelles were independently processed at a normal level. The possibility of homeostatic regulation and its physiological importance were discussed.

Restoration of biochemical function of the peroxisome in the temperature-sensitive mild forms of peroxisome biogenesis disorder in humans.

We have found that peroxisome assembly is temperature-sensitive (ts) in mild forms of peroxisome biogenesis disorders (PBDs), that is all infantile Refsum disease (IRD) patients and a few neonatal adrenoleukodystrophy patients of several complementation groups. The number of peroxisomes increased daily in incubation at 30 degrees C in the ts cells. Oxidation of very long-chain fatty acids, processing of acyl-CoA oxidase and dihydroxyacetonephosphate acyltransferase activity also improved after 8 days incubation at 30 degrees C in the IRD fibroblasts. These biochemical functions of the peroxisome did not change at 30 degrees C in Zellweger fibroblasts. Number of peroxisomes gradually decreased after 4 days when the temperature shifted from 30 to 37 degrees C in the ts cells. These results indicate that the biochemical functions of peroxisome are also restored by incubation at 30 degrees C in the mild and ts phenotype of PBDs, and the results will aid to predict the severity and the prognosis of affected children.

Ether lipid biosynthesis: alkyl-dihydroxyacetonephosphate synthase protein deficiency leads to reduced dihydroxyacetonephosphate acyltransferase activities

Recent studies have indicated that two peroxisomal enzymes involved in ether lipid synthesis, i.e., dihydroxyacetonephosphate acyltransferase and alkyl-dihydroxyacetonephosphate synthase, are directed to peroxisomes by different targeting signals, i.e., peroxisomal targeting signal type 1 and type 2, respectively. In this study, we describe a new human fibroblast cell line in which alkyl-dihydroxyacetonephosphate synthase was found to be deficient both at the level of enzyme activity and enzyme protein. At the cDNA level, a 128 base pair deletion was found leading to a premature stop. Remarkably, dihydroxyacetonephosphate acyltransferase activity was strongly reduced to a level comparable to the activities measured in fibroblasts from patients affected by the classical form of rhizomelic chondrodysplasia punctata (caused by a defect in peroxisomal targeting signal type 2 import). Dihydroxyacetonephosphate acyltransferase activity was completely normal in another alkyl-dihydroxyacetonephosphate synthase activity-deficient patient. Fibroblasts from this patient showed normal levels of the synthase protein and inactivity results from a point mutation leading to an amino acid substitution. These results strongly suggest that the activity of dihydroxyacetonephosphate acyltransferase is dependent on the presence of alkyl-dihydroxyacetonephosphate synthase protein. This interpretation implies that the deficiency of dihydroxyacetonephosphate acyltransferase (targeted by a peroxisomal targeting signal type 1) in the classic form of rhizomelic chondrodysplasia punctata is a consequence of the absence of the alkyl-dihydroxyacetonephosphate synthase protein (targeted by a peroxisomal targeting signal type 2).

Zellweger syndrome in Saudi Arabia and its distinct features.

Clinical and laboratory findings of Zellweger syndrome (ZS) patients diagnosed at King Faisal Specialist Hospital and Research Center (KFSH & RC), Riyadh, Saudi Arabia over a period of 10 years are presented in this report. Eleven patients (nine females and two males) from 2 to 4 months old were referred to KFSH & RC for evaluation of hypotonia, seizures, and dysmorphic features. The common clinical findings included high forehead, large fontanelle, shallow orbit ridges, micrognathia, upslanting palebral fissures, epicanthal folds, severe hypotonia, hyporeflexia, pigmentary retinopathy, optic nerve atrophy, complete or partial agenesis of corpus callusum, and failure to thrive. We did not observe any Brushfield spots, any renal and brain cysts, or adrenal insufficiency. Some unique clinical findings were the presence of gallstones, club feet, or bilateral knee or hip dislocation in some patients. All patients had markedly elevated plasma levels of very long chain fatty acids (VLCFA). Electron microscopy performed on liver biopsies of two patients revealed absence of peroxisomes. Biochemical studies of dermal fibroblasts from three patients showed deficient beta-oxidation of lignoceric acid and dihydroxyacetone phosphate acyltransferase (DHAPATase) activity. The tribal living in Saudi Arabia and our observation that 10 of the 11 parents in this study were first-degree relatives and, except for families 1 and 3, each family had at least another baby who died of the same disease. This suggests that the incidence of ZS in Saudi Arabia may actually be higher than our experience at KFSH & RC.

Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol.

We have developed a two-step selection protocol to generate a population of Chinese hamster ovary (CHO) cell variants that are plasmalogen-deficient, but contain intact, functional peroxisomes (plasmalogen-/peroxisome+). This involved sequential exposures of a mutagenized cell population to photodynamic damage by using two different pyrene-labelled sensors, 9-(1'-pyrene)nonanol and 12-(1'-pyrene)dodecanoic acid. By this procedure we generated several isolates, all except one of which displayed a severe decrease in plasmalogen biosynthesis. Further characterization of one of the plasmalogen-deficient isolates, NRel-4, showed that it contained intact, functional peroxisomes. Whole-cell homogenates from NRel-4 displayed severely decreased dihydroxyacetone phosphate acyltransferase, which catalyses the first step in plasmalogen biosynthesis. NRel-4 and another, recently described, plasmalogen-deficient cell line, NZel-1 [Nagan, Hajra, Das, Moser, Moser, Lazarow, Purdue and Zoeller (1997) Proc. Natl. Acad. Sci. U.S. A. 94, 4475-4480] were hypersensitive to singlet oxygen, supporting the notion of plasmalogens as radical oxygen scavengers. Wild-type-like resistance could be conferred on NRel-4 upon restoration of plasmalogen content by supplementation with a bypass compound, sn-1-hexadecylglycerol. NRel-4 and other plasmalogen-/peroxisome+ strains will allow us to examine further the role of ether lipids in cellular functions without complications associated with peroxisome deficiency, and might serve as an animal cell model for certain forms of the human genetic disorder rhizomelic chondrodysplasia punctata.

Acyl-CoA:dihydroxyacetonephosphate acyltransferase: cloning of the human cDNA and resolution of the molecular basis in rhizomelic chondrodysplasia punctata type 2.

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.

Copurification of dihydroxyacetone-phosphate acyl-transferase and other peroxisomal proteins from liver of fenofibrate-treated rats

Dihydroxyacetone-phosphate acyl-transferase (DHAP-AT), a peroxisomal membrane-bound enzyme that catalyzes the first step of ether-glycerolipid synthesis, was purified from liver of rats treated with fenofibrate, a peroxisome proliferator. The protocol first included isolation of peroxisomes, their purification through a discontinuous gradient and solubilization of membranes in CHAPS. DHAP-AT was further purified by four chromatographic steps, namely low-pressure size-exclusion, cation-exchange, hydroxylapatite and chromatofocusing. The chromatofocusing step led to a 4000-fold increase in the specific activity of DHAP-AT with respect to the liver homogenate with a yield of about 0.2%. Trypsin digestion of a 64-kDa protein band upon SDS-PAGE resulted in a peptide sequence unknown in databases. A corresponding degenerated oligonucleotide was used as a probe in Northern blotting, and a transcript of 3.3 kb was detected in some rat tissues. Moreover, the overall procedure allowed co-purification of four major peroxisomal enzymes: urate-oxidase, catalase, multifunctional enzyme and palmitoyl-CoA oxidase, respectively.

Rhizomelic chondrodysplasia punctata is caused by deficiency of human PEX7, a homologue of the yeast PTS2 receptor.

The rhizomelic form of chondrodysplasia punctata (RCDP) is an autosomal recessive disease of peroxisome biogenesis characterized by deficiencies in several peroxisomal proteins, including the peroxisomal enzymes of plasmalogen biosynthesis and peroxisomal 3-ketoacyl thiolase. In cultured fibroblasts from patients with this disorder, both the peroxisomal targeting and proteolytic removal of the amino-terminal type 2 peroxisomal targeting sequence (PTS2) of thiolase are defective, whereas the biogenesis of proteins targeted by carboxyterminal type 1 peroxisomal targeting sequences (PTS1) is unimpaired. We have previously isolated a Saccharomyces cerevisiae peroxisomal biogenesis mutant, pex7 (formerly peb1/pas7), which demonstrates a striking similarity to the cellular phenotype of RCDP fibroblasts in that PTS1 targeting is functional, but the peroxisomal packaging of PTS2 targeted thiolase is lacking. Complementation of this mutant has led to the identification of the protein ScPex7p, a PTS2 receptor. In this paper we report cloning of the human orthologue of ScPEX7, and demonstrate that this is the defective gene in RCDP. We show that expression of human PEX7 in RCDP cells rescues PTS2 targeting and restores some activity of dihydroxyacetone phosphate acyltransferase (DHAP-AT), a peroxisomal enzyme of plasmalogen biosynthesis, and we identify the mutations responsible for loss of function of PEX7 in a compound heterozygote RCDP patient. These results imply that several peroxisomal proteins are targeted by PTS2 signals and that the various biochemical and clinical defects in RCDP result from a defect in the receptor for this class of PTS.

Peroxisomes and Essential Fatty Acid Deficiency.

The effect of dietary essential fatty acid (EFA) deficiency on the lipid composition of peroxisomal extracts was studied in Wistar rats. After 21 days of EFA deficiency, no difference was noted in protein levels, but a significant change had occurred in the level of phospholipids and fatty acid composition of phospholipids. In EFA-deficient rat peroxisomes, phosphatidylcholine and phosphatidylinositol were both significantly higher than in controls. This alteration was more marked in peroxisomes than in microsomes. The peroxisomal fatty acid composition was markedly affected as well. In EFA-deficient rats, linoleic (18:2 n-6), arachidonic (C20:4 n-6), and docosahexaenoic (C22:6 n-3) acids were lower whereas oleic (C18:1 n-9), octadecenoic (C18:1 n-7), and eicosatrienoic (C20:3 n-9) acids were higher than in controls. This alteration was also more prominent in the peroxisomal than the microsomal extracts. Catalase activity was weakly reduced, whereas dihydroxyacetone-phosphate acyltransferase activity was strongly increased in EFA-deficient rats. We conclude that the composition of phospholipids and fatty acids may be a factor contributing to peroxisome function and that a diet poor in essential fatty acids or defective unsaturated fatty acid metabolism could amplify peroxisomal dysfunction of genetic origin.

Measurement of dihydroxyacetone-phosphate acyltransferase (DHAPAT) in chorionic villous samples, blood cells and cultured cells.

Dihydroxyacetone-phosphate acyltransferase (DHAPAT) is a peroxisomal enzyme catalysing the first step in ether-phospholipid biosynthesis. DHAPAT is deficient in cells from patients suffering from a variety of peroxisomal disorders. Accurate measurement of the activity of this enzyme is of great importance, especially since it is a central parameter in the prenatal diagnosis of the disorders of peroxisome biogenesis, rhizomelic chondrodysplasia punctata and DHAPAT-deficiency. We describe a straightforward and accurate assay allowing the activity of DHAPAT to be measured reliably in chorionic villus samples, blood cells, cultured skin fibroblasts, cultured chorionic villus fibroblasts and cultured amniocytes.

Regulation of phosphatidic acid biosynthetic enzymes in Saccharomyces cerevisiae.

Phosphatidic acid is the biosynthetic precursor of all glycerolipids. To understand how phosphatidic acid biosynthesis is controlled in Saccharomyces cerevisiae, we studied the regulation of three enzyme activities involved in the synthesis of this glycerolipid precursor, i.e., glycerophosphate acyltransferase (GPAT), dihydroxyacetone phosphate acyltransferase (DHAPAT), and acyl DHAP reductase. GPAT activity was increased 3-fold, while DHAPAT activity was increased up to 9-fold in wild type cells grown in a nonfermentable carbon source compared to that of glucose-grown cells. The ratio of GPAT/DHAPAT activity was 12 in glucose-grown cells but only 4 in cells grown in glycerol/ethanol. In the previously characterized tpa1 mutant, (T. S. Tillman and R. M. Bell. 1986. J. Biol. Chem. 261: 9144-9149), GPAT was decreased 2-fold and DHAPAT 27-fold compared to activities in the wild type. Acyl DHAP reductase activity in both wild type and tpa1 cells grown on a nonfermentable carbon source was increased approximately 2-fold over that of glucose-grown cells. All three enzymatic activities increased as wild type cells grown on glucose entered the stationary phase of growth. Therefore, GPAT, DHAPAT, and acyl DHAP reductase activities appear to be regulated by the respiratory state of the cell. None of the activities was affected to a great extent by inositol, which is a key regulator of many enzymes involved in the synthesis of PtdOH-derived phospholipids in S. cerevisiae, nor by deletion of the mitochondrial genome.(ABSTRACT TRUNCATED AT 250 WORDS)