Search PubMed⌕ Search

Biomedical subjects

G P Mannaerts

Publications and source records attributed to G P Mannaerts.

At least 37 records · Page 2Linked to original sources
PubMed2000

Peroxisomal lipid degradation via beta- and alpha-oxidation in mammals.

Peroxisomal beta-oxidation is involved in the degradation of long chain and very long chain fatty acyl-(coenzyme A)CoAs, long chain dicarboxylyl-CoAs, the CoA esters of eicosanoids, 2-methyl-branched fatty acyl-CoAs (e.g. pristanoyl-CoA), and the CoA esters of the bile acid intermediates di- and trihydroxycoprostanic acids (side chain of cholesterol). In the rat, straight chain acyl-CoAs (including the CoA esters of dicarboxylic fatty acids and eicosanoids) are beta-oxidized via palmitoyl-CoA oxidase, multifunctional protein-1 (which displays 2-enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase activities) and peroxisomal thiolase. 2-Methyl-branched acyl-CoAs are degraded via pristanoyl-CoA oxidase, multifunctional protein-2 (MFP-2) (which displays 2-enoyl-CoA hydratase and D-3-hydroxyacyl-CoA dehydrogenase activities) and sterol carrier protein-X (SCPX; displaying 2-methyl-3-oxoacyl-CoA thiolase activity). The side chain of the bile acid intermediates is shortened via one cycle of beta-oxidation catalyzed by trihydroxycoprostanoyl-CoA oxidase, MFP-2 and SCPX. In the human, straight chain acyl-CoAs are oxidized via palmitoyl-CoA oxidase, multifunctional protein-1, and peroxisomal thiolase, as is the case in the rat. The CoA esters of 2-methyl-branched acyl-CoAs and the bile acid intermediates, which also possess a 2-methyl substitution in their side chain, are shortened via branched chain acyl-CoA oxidase (which is the human homolog of trihydroxycoprostanoyl-CoA oxidase), multifunctional protein-2, and SCPX. The rat and the human enzymes have been purified, cloned, and kinetically and stereochemically characterized. 3-Methyl-branched fatty acids such as phytanic acid are not directly beta-oxidizable because of the position of the methyl-branch. They are first shortened by one carbon atom through the a-oxidation process to a 2-methyl-branched fatty acid (pristanic acid in the case of phytanic acid), which is then degraded via peroxisomal beta-oxidation. In the human and the rat, alpha-oxidation is catalyzed by an acyl-CoA synthetase (producing a 3-methylacyl-CoA), a 3-methylacyl-CoA 2-hydroxylase (resulting in a 2-hydroxy-3-methylacyl-CoA), and a 2-hydroxy-3-methylacyl-CoA lyase that cleaves the 2-hydroxy-3-methylacyl-CoA into a 2-methyl-branched fatty aldehyde and formyl-CoA. The fatty aldehyde is dehydrogenated by an aldehyde dehydrogenase to a 2-methyl-branched fatty acid while formyl-CoA is hydrolyzed to formate, which is then converted to CO2. The activation, hydroxylation and cleavage reactions, and the hydrolysis of formyl-CoA are performed by peroxisomal enzymes; the aldehyde dehydrogenation remains to be localized whereas the conversion of formate to CO2 occurs mainly in the cytosol.

Acyl Coenzyme A↗
PubMed2000

Phytanoyl-CoA hydroxylase: recognition of 3-methyl-branched acyl-coAs and requirement for GTP or ATP and Mg(2+) in addition to its known hydroxylation cofactors.

Phytanoyl-CoA hydroxylase is a peroxisomal alpha-oxidation enzyme that catalyzes the 2-hydroxylation of 3-methyl-branched acyl-CoAs. A polyhistidine-tagged human phytanoyl-CoA hydroxylase was expressed in E. coli and subsequently purified as an active protein. The recombinant enzyme required GTP or ATP and Mg(2+), in addition to its known cofactors Fe(2+), 2-oxoglutarate, and ascorbate. The enzyme was active towards phytanoyl-CoA and 3-methylhexadecanoyl-CoA, but not towards 3-methylhexadecanoic acid. Racemic, R- and S-3-methylhexadecanoyl-CoA were equally well hydroxylated. Hydroxylation of R- and S-3-methylhexadecanoyl-CoA yielded the (2S, 3R) and (2R,3S) isomers of 2-hydroxy-3-methylhexadecanoyl-CoA, respectively. Human phytanoyl-CoA hydroxylase did not show any activity towards 2-methyl- and 4-methyl-branched acyl-CoAs or towards long and very long straight chain acyl-CoAs, excluding a possible role for the enzyme in the formation of 2-hydroxylated and odd-numbered straight chain fatty acids, which are abundantly present in brain. In conclusion, we report the unexpected requirement for ATP or GTP and Mg(2+) of phytanoyl-CoA hydroxylase in addition to the known hydroxylation cofactors. Due to the fact that straight chain fatty acyl-CoAs are not a substrate for phytanoyl-CoA hydroxylase, 2-hydroxylation of fatty acids in brain can be allocated to a different enzyme/pathway.

Acyl Coenzyme A↗
PubMed2000

Mitochondrial and peroxisomal targeting of 2-methylacyl-CoA racemase in humans.

2-Methylacyl-CoA racemase is an auxiliary enzyme required for the peroxisomal beta-oxidative breakdown of (2R)-pristanic acid and the (25R)-isomer of C(27) bile acid intermediates. The enzyme activity is found not only in peroxisomes but also is present in mitochondria of human liver and fibroblasts. The C terminus of the human racemase, a protein of 382 amino acids with a molecular mass of 43,304 daltons as deduced from its cloned cDNA, consists of KASL. Hitherto this sequence has not been recognized as a peroxisomal targeting signal (PTS1). From the in vitro interaction between recombinant racemase and recombinant human PTS1 receptor (Pex5p), and the peroxisomal localization of green fluorescent protein (GFP) fused to the N terminus of full-length racemase or its last six amino acids in tranfected Chinese hamster ovary (CHO) cells, we concluded that ASL is a new PTS1 variant. To be recognized by Pex5p, however, the preceding lysine residue is critical. As shown in another series of transfection experiments with GFP fused to the C terminus of the full-length racemase or racemase with deletions of the N terminus, mitochondrial targeting information is localized between amino acids 22 and 85.Hence, our data show that a single transcript gives rise to a racemase protein containing two topogenic signals, explaining the dual cellular localization of the activity.

Amino Acid Sequence↗
PubMed1999

Purification, molecular cloning, and expression of 2-hydroxyphytanoyl-CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during alpha-oxidation of 3-methyl-branched fatty acids.

In the third step of the alpha-oxidation of 3-methyl-branched fatty acids such as phytanic acid, a 2-hydroxy-3-methylacyl-CoA is cleaved into formyl-CoA and a 2-methyl-branched fatty aldehyde. The cleavage enzyme was purified from the matrix protein fraction of rat liver peroxisomes and identified as a protein made up of four identical subunits of 63 kDa. Its activity proved to depend on Mg(2+) and thiamine pyrophosphate, a hitherto unrecognized cofactor of alpha-oxidation. Formyl-CoA and 2-methylpentadecanal were identified as reaction products when the purified enzyme was incubated with 2-hydroxy-3-methylhexadecanoyl-CoA as the substrate. Hence the enzyme catalyzes a carbon-carbon cleavage, and we propose calling it 2-hydroxyphytanoyl-CoA lyase. Sequences derived from tryptic peptides of the purified rat protein were used as queries to recover human expressed sequence tags from the databases. The composite cDNA sequence of the human lyase contained an ORF of 1,734 bases that encodes a polypeptide with a calculated molecular mass of 63,732 Da. Recombinant human protein, expressed in mammalian cells, exhibited lyase activity. The lyase displayed homology to a putative Caenorhabditis elegans protein that resembles bacterial oxalyl-CoA decarboxylases. Similarly to the decarboxylases, a thiamine pyrophosphate-binding consensus domain was present in the C-terminal part of the lyase. Although no peroxisome targeting signal, neither 1 nor 2, was apparent, transfection experiments with constructs encoding green fluorescent protein fused to the full-length lyase or its C-terminal pentapeptide indicated that the C terminus of the lyase represents a peroxisome targeting signal 1 variant.

Amino Acid Sequence↗
PubMed1999

N-acetyl-sphingenine-1-phosphate is a potent calcium mobilizing agent.

Calcium mobilization induced by phosphorylated sphingoid bases was analyzed in calf pulmonary artery endothelial cells by confocal microscopy. A sphingenine-1-phosphate (SeP) analogue, N-acetyl-sphingenine-1-phosphate (N-C2-SeP), exogenously added to these cells, caused a fast and transient intracellular rise in calcium and was as potent as SeP. A minimal concentration of 0.6 nM for N-C2-SeP versus 1 nM for SeP was determined. The N-C2-SeP-induced Ca2+-signaling, like the response to SeP, was due to a release from thapsigargin-sensitive, ryanodine-insensitive, intracellular Ca2+-stores and not to a Ca2+-influx. N-C2-SeP can be considered as a truncated ceramide-phosphate, a lipid already reported to be mitogenic (Gomez-Munoz, A., Duffy, P.A., Martin, A., O'Brien, L., Byun, H.S., Bittman, R. and Brindley, D.N. (1995) Mol. Pharmacol. 47, 833-839), an effect that might be secondary to Ca2+-mobilization.

Animals↗
PubMed1999

Comparison of the stability and substrate specificity of purified peroxisomal 3-oxoacyl-CoA thiolases A and B from rat liver.

The specific activities and substrate specificities of 3-oxoacyl-CoA thiolase A (thiolase A) purified from normal rat liver peroxisomes and 3-oxoacyl-CoA thiolase B (thiolase B) isolated from livers of rats treated with the peroxisome proliferator clofibrate were virtually identical. The enzymes could be distinguished by their N-terminal amino acid sequences, their isoelectric points and their stability, the latter being higher for thiolase A. Contrary to thiolase B, which showed a marked cold lability in the presence of KCl by dissociating into monomers with poor activity, thiolase A retained its full activity and its homodimeric structure under these conditions.

Acetyl-CoA C-Acyltransferase↗
PubMed1999

Role and organization of peroxisomal beta-oxidation.

In mammals, peroxisomes are involved in breakdown of very long chain fatty acids, prostanoids, pristanic acid, dicarboxylic fatty acids, certain xenobiotics and bile acid intermediates. Substrate spectrum and specificity studies of the four different beta-oxidation steps in rat and/or in man demonstrate that these substrates are degraded by separate beta-oxidation systems composed of different enzymes. In both species, the enzymes acting on straight chain fatty acids are palmitoyl-CoA oxidase, an L-specific multifunctional protein (MFP-1) and a dimeric thiolase. In liver, bile acid intermediates undergo one cycle of beta-oxidation catalyzed by trihydroxycoprostanoyl-CoA oxidase (in rat), or branched chain acyl-CoA oxidase (in man), a D-specific multifunctional protein (MFP-2) and SCPX-thiolase. Finally, pristanic acid is degraded in rat tissues by pristanoyl-CoA oxidase, the D-specific multifunctional protein-2 and SCPX-thiolase. Although in man a pristanoyl-CoA oxidase gene is present, so far its product has not been found. Hence, pristanoyl-CoA is believed to be desaturated in human tissues by the branched chain acyl-CoA oxidase. Due to the stereospecificity of the oxidases acting on 2-methyl-branched substrates, an additional enzyme, 2-methylacyl-CoA racemase, is required for the degradation of pristanic acid and the formation of bile acids.

Acyl-CoA Dehydrogenase, Long-Chain↗
PubMed1999

Hepatic alpha-oxidation of phytanic acid. A revised pathway.

Synthetic 3-methyl-branched chain fatty acids were used to decipher the breakdown of phytanic acid. Based on results obtained in intact or permeabilized rat hepatocytes, rat liver homogenates or subcellular fractions, a revised alpha-oxidation pathway is proposed which appears to be functioning in man as well. In a first step, the 3-methyl-branched chain fatty acid is activated by an acyl-CoA synthetase. This reaction requires CoA, ATP and Mg2+. Subsequently, the acyl-CoA ester is hydroxylated at position 2 by a peroxisomal dioxygenase. This step is dependent on alpha-oxoglutarate, ascorbate (or glutathione), Fe2+ and O2. The 2-hydroxy-3-methylacyl-CoA intermediate is cleaved by a peroxisomal lyase to formyl-CoA and a 2-methyl-branched fatty aldehyde. Formyl-CoA is (partly enzymically) hydrolyzed to formate, which is then converted, most likely in the cytosol, to CO2. In the presence of NAD+, the aldehyde is dehydrogenated to a 2-methyl-branched fatty acid, presumably by a peroxisomal aldehyde dehydrogenase. This acid can--after activation--be degraded via a D-specific peroxisomal beta-oxidation system.

Animals↗
PubMed1999

Stereochemistry of the alpha-oxidation of 3-methyl-branched fatty acids in rat liver.

The stereochemistry of the alpha-oxidation of 3-methyl-branched fatty acids was studied in rat liver. R- and S-3-methylhexadecanoic acid were equally well alpha-oxidized in intact hepatocytes and homogenates. Subcellular fractionation studies showed that alpha-oxidation of both isomers is confined to peroxisomes. Dehydrogenation of 2-methylpentadecanal, the end-product of the peroxisomal alpha-oxidation of 3-methylhexadecanoic acid, to 2-methylpentadecanoic acid, followed by derivatization with R-1-phenylethylamine and subsequent separation of the stereoisomers by gas chromatography, revealed that the configuration of the methyl-branch is preserved throughout the whole alpha-oxidation process. Metabolism and formation of the 2-hydroxy-3-methylhexadecanoyl-CoA intermediate were also investigated. Separation of the methyl esters of the four isomers of 2-hydroxy-3-methylhexadecanoic acid was achieved by gas chromatography after derivatization of the hydroxy group with R-2-methoxy-2-trifluoromethylphenylacetic acid chloride and the absolute configuration of the four isomers was determined. Although purified peroxisomes are capable of metabolizing all four isomers of 2-hydroxy-3-methylhexadecanoyl-CoA, they can only form the (2S,3R) and the (2R,3S) isomers. Our experiments exclude the racemization of the 3-methyl branch during the alpha-oxidation process. The configuration of the 3-methyl branch does not influence the rate of alpha-oxidation, but determines the side of the 2-hydroxylation, hence the configuration of the 2-hydroxy-3-methylacyl-CoA intermediates formed during the process.

Animals↗
PubMed1999

[Lipid degradation by way of beta and alpha oxidation in peroxisomes of mammals].

Recently, it has become clear that the peroxisomal beta-oxidation system in rat and man consists of multiple pathways. In rat and man straight chain fatty acids, dicarboxylic fatty acids and prostaglandins are oxidized via the L-specific pathway catalyzed by palmitoyl-CoA oxidase, multifunctional protein-1 and thiolase. 2-Methyl-branched fatty acids and the bile acid intermediates are oxidized via the D-specific pathway. In the rat this pathway is catalyzed by prostanoyl-CoA oxidase, multifunctional protein-2 and sterol carrier protein-X (branched fatty acids) and by trihydroxycoprostanoyl-CoA oxidase, multifunctional protein-2 and sterol carrier protein-X (bile acid intermediates). In the human, branched fatty acids and bile acid intermediates are oxidized via branched chain acyl-CoA oxidase, multifunctional protein-2 and sterol carrier protein-X. All enzymes of these pathways have been purified, cloned and characterized. Also the reactions that constitute the alpha-oxidation pathway for 3-methyl-branched fatty acids, have recently been identified in rat and man. The revised pathway consists of the following reactions: 1) an activation reaction catalyzed by an acyl-CoA synthetase, that forms a 3-methylacyl-CoA; 2) a hydroxylation (dioxygenase) reaction catalyzed by a 3-methylacyl-CoA 2-hydroxylase, that converts the CoA ester to a 2-hydroxy-3-methylacyl-CoA; 3) a cleavage reaction catalyzed by a 2-hydroxy-3-methylacyl-CoA lyase, that releases a 2-methyl fatty aldehyde and formyl-CoA. The branched aldehyde is dehydrogenated by an aldehyde dehydrogenase to a 2-methyl branched fatty acid that can be degraded by peroxisomal beta-oxidation. Formyl-CoA is enzymatically hydrolyzed to formate which is then converted to CO2.

Animals↗
PubMed1998

C-terminal tripeptide Ser-Asn-Leu (SNL) of human D-aspartate oxidase is a functional peroxisome-targeting signal.

The functionality of the C-terminus (Ser-Asn-Leu; SNL) of human d-aspartate oxidase, an enzyme proposed to have a role in the inactivation of synaptically released d-aspartate, as a peroxisome-targeting signal (PTS1) was investigated in vivo and in vitro. Bacterially expressed human d-aspartate oxidase was shown to interact with the human PTS1-binding protein, peroxin protein 5 (PEX5p). Binding was gradually abolished by carboxypeptidase treatment of the oxidase and competitively inhibited by a Ser-Lys-Leu (SKL)-containing peptide. After transfection of mouse fibroblasts with a plasmid encoding green fluorescent protein (GFP) extended by PKSNL (the C-terminal pentapeptide of the oxidase), a punctate fluorescent pattern was evident. The modified GFP co-localized with peroxisomal thiolase as shown by indirect immunofluorescence. On transfection in fibroblasts lacking PEX5p receptor, GFP-PKSNL staining was cytosolic. Peroxisomal import of GFP extended by PGSNL (replacement of the positively charged fourth-last amino acid by glycine) seemed to be slower than that of GFP-PKSNL, whereas extension by PKSNG abolished the import of the modified GFP. Taken together, these results indicate that SNL, a tripeptide not fitting the PTS1 consensus currently defined in mammalian systems, acts as a functional PTS1 in mammalian systems, and that the consensus sequence, based on this work and that of other groups, has to be broadened to (S/A/C/K/N)-(K/R/H/Q/N/S)-L.

Amino Acid Oxidoreductases↗
PubMed1998

Identification and characterization of human PMP34, a protein closely related to the peroxisomal integral membrane protein PMP47 of Candida boidinii.

The cDNA of a human protein displaying 33% identity to the peroxisomal integral membrane protein PMP47 of Candida boidinii (CbPMP47) was cloned. It encodes a protein of 307 amino acids, with a calculated molecular mass of 34,566 Da. The protein, which we name HsPMP34, contains six membrane spans and belongs, like PMP47, to the family of mitochondrial solute carriers. Based on overlapping available expressed sequence tags (ESTs) combined with the sequencing of one EST, the amino acid sequence of the mouse counterpart (molecular mass of 34,422 Da) could also be deduced. By means of reverse-transcription PCR, the presence of PMP34 mRNA was revealed in liver of man and in liver and other tissues of mouse and rat. The human gene is localized to chromosome 22q13. Given the similarity of HsPMP34 not only to CbPMP47 but also to known or putative mitochondrial transporters, its subcellular localization was investigated. Fluorescence microscopy of HepG2 cells or mouse fibroblasts transfected with a plasmid encoding a HsPMP34/green fluorescent fusion protein revealed a punctate pattern, reminiscent of a peroxisomal distribution. By indirect immunofluorescence, the HsPMP34/green fluorescent fusion protein clearly colocalized with the dimeric peroxisomal thiolase. Upon transfection of mouse fibroblasts lacking functional peroxisomes due the knock-out of PEX5 [Baes, M., Gressens, P., Baumgart, E., Carmeliet, P., Casteels, M., Fransen, M., Evrard, P., Fahimi, D., Declercq, P. E., Collen, D., Van Veldhoven, P. P. & Mannaerts, G. P. (1997) Nat. Genet. 17, 49-53], fluorescence was associated with larger and less-abundant structures. Taken together, the data indicate that HsPMP34 is a peroxisomal membrane protein and is either the human counterpart of CbPMP47 or closely related to it. According to its structure, the protein is most probably involved in transport.

Acetyl-CoA C-Acetyltransferase↗
PubMed1998

Maturation of peroxisomes in differentiating human hepatoblastoma cells (HepG2): possible involvement of the peroxisome proliferator-activated receptor alpha (PPAR alpha).

We have studied the alterations of peroxisomes in the human hepatoblastoma cell line HepG2, induced to differentiate by long-term cultivation (20 days without passaging) using morphological and biochemical techniques as well as mRNA analysis. Ultrastructural studies revealed alterations in shape and size of peroxisomes, with significant increases in mean diameter and formation of small clusters exhibiting heterogeneous staining for catalase after 20 days in culture. These alterations of peroxisomes correspond to the changes described during the maturation process from prenatal to adult human hepatocytes. As revealed by Northern and Western blotting there was marked elevation of the mRNA (190%) and protein (180%) of the peroxisomal branched-chain acyl-CoA oxidase. This protein is the key regulatory enzyme for the side chain oxidation of cholesterol for bile acid synthesis, a pathway associated with mature hepatocytes. Concomitantly a marked increase of bile canaliculi was noted by light and electron microscopy. This differentiation process was confirmed also by the increase of albumin synthesis (mRNA: 160%; protein: 190%) which is generally used as a differentiation marker of hepatocytes in culture. Interestingly, the mRNA for peroxisome proliferator-activated receptor alpha (PPAR alpha) increased drastically by almost 390% and its corresponding protein by 150%, suggesting its involvement in maturation of the peroxisomal compartment in differentiating HepG2 cells. In contrast to the wellknown increases during the drug-induced peroxisome proliferation of cytochrome P450 4A, multifunctional enzyme 1, palmitoyl-CoA oxidase and the 70-kDa peroxisomal membrane protein, those proteins were either not altered or only slightly elevated during the differentiation process, suggesting that peroxisome proliferation and maturation are two distinct and differentially regulated processes.

Albumins↗
PubMed1997

Substrate specificities of 3-oxoacyl-CoA thiolase A and sterol carrier protein 2/3-oxoacyl-CoA thiolase purified from normal rat liver peroxisomes. Sterol carrier protein 2/3-oxoacyl-CoA thiolase is involved in the metabolism of 2-methyl-branched fatty acids and bile acid intermediates.

The two main thiolase activities present in isolated peroxisomes from normal rat liver were purified to near homogeneity. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the first enzyme preparation displayed a single band of 41 kDa that was identified as 3-oxoacyl-CoA thiolase A (thiolase A) by N-terminal amino acid sequencing. The second enzyme preparation consisted of a 58- and a 46-kDa band. The 58-kDa polypeptide reacted with antibodies raised against either sterol carrier protein 2 or the thiolase domain of sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCP-2/thiolase), formerly also called sterol carrier protein X, whereas the 46-kDa polypeptide reacted only with the antibodies raised against the thiolase domain. Internal peptide sequencing confirmed that the 58-kDa polypeptide is SCP-2/thiolase and that the 46-kDa polypeptide is the thiolase domain of SCP-2/thiolase. Thiolase A catalyzed the cleavage of short, medium, and long straight chain 3-oxoacyl-CoAs, medium chain 3-oxoacyl-CoAs being the best substrates. The enzyme was inactive with the 2-methyl-branched 3-oxo-2-methylpalmitoyl-CoA and with the bile acid intermediate 24-oxo-trihydroxycoprostanoyl-CoA. SCP-2/thiolase was active with medium and long straight chain 3-oxoacyl-CoAs but also with the 2-methyl-branched 3-oxoacyl-CoA and the bile acid intermediate. In peroxisomal extracts, more than 90% of the thiolase activity toward straight chain 3-oxoacyl-CoAs was associated with thiolase A. Kinetic parameters (Km and Vmax) were determined for each enzyme with the different substrates. Our results indicate the following: 1) the two (main) thiolases present in peroxisomes from normal rat liver are thiolase A and SCP-2/thiolase; 2) thiolase A is responsible for the thiolytic cleavage of straight chain 3-oxoacyl-CoAs; and 3) SCP-2/thiolase is responsible for the thiolytic cleavage of the 3-oxoacyl-CoA derivatives of 2-methyl-branched fatty acids and the side chain of cholesterol.

Acetyl-CoA C-Acetyltransferase↗