Presence of thiamine pyrophosphate in mammalian peroxisomes
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Thiamine pyrophosphate (TPP) is a cofactor for 2-hydroxyacyl-CoA lyase 1 (HACL1), a peroxisomal enzyme essential for the α-oxidation of phytanic acid and 2-hydroxy straight chain fatty acids. So far, HACL1 is the only known peroxisomal TPP-dependent enzyme in mammals. Little is known about the transport of metabolites and cofactors across the peroxisomal membrane and no peroxisomal thiamine or TPP carrier has been identified in mammals yet. This study was undertaken to get a better insight into these issues and to shed light on the role of TPP in peroxisomal metabolism.
Because of the crucial role of the cofactor TPP, we reanalyzed its subcellular localization in rat liver. In addition to the known mitochondrial and cytosolic pools, we demonstrated, for the first time, that peroxisomes contain TPP (177 ± 2 pmol/mg protein). Subsequently, we verified whether TPP could be synthesized from its precursor thiamine, in situ, by a peroxisomal thiamine pyrophosphokinase (TPK). However, TPK activity was exclusively recovered in the cytosol.
Our results clearly indicate that mammalian peroxisomes do contain TPP but that no pyrophosphorylation of thiamine occurs in these organelles, implying that thiamine must enter the peroxisome already pyrophosphorylated. Consequently, TPP entry may depend on a specific transport system or, in a bound form, on HACL1 translocation.
KeywordsThiamine Phytanic Acid Peroxisomal Membrane Potassium Hexacyanoferrate Thiamine Pyrophosphate
2-hydroxyacyl-CoA lyase 1
Thiamine (vitamin B1) is a water-soluble micronutrient essential for normal cellular functions, growth and development. Humans, and other higher eukaryotes, cannot synthesize thiamine but depend on an appropriate dietary intake and absorption of this vitamin. Its plasma concentration is regulated by intestinal and renal mechanisms which play a crucial role in regulating body thiamine homeostasis. Upon entry into cells, thiamine is quickly converted to its biologically active form, thiamine pyrophosphate (TPP) by thiamine pyrophosphokinase (TPK). TPP plays a critical role in the carbohydrate and energy metabolism. It functions as a prosthetic group for the mitochondrial enzyme complexes like pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and branched-chain α-keto acid dehydrogenase. In addition, TPP is involved in the cytosolic pentose pathway functioning as coenzyme for transketolase. Recently, it became clear that TPP is also important for a less well known pathway, namely the α-oxidation of 3-methyl-branched and straight chain 2-hydroxy long chain fatty acids [1, 2, 3]. In this pathway, which is confined to peroxisomes, TPP, together with Mg2+, is required for the proper functioning of 2-hydroxyphytanoyl-CoA lyase (2-HPCL), recently renamed to 2-hydroxyacyl-CoA lyase 1 (approved gene symbol and protein product HACL1) . This peroxisomal matrix protein acts as a tetramer and catalyzes the cleavage of 2-hydroxyphytanoyl-CoA and 2-hydroxy long chain acyl-CoA into formyl-CoA and an aldehyde shortened by one carbon [1, 5]. So far, HACL1 is the only known peroxisomal TPP-dependent enzyme in mammals.
The importance of TPP/thiamine in the α-oxidation pathway is stressed by the deleterious effects seen in rats given a thiamine-deficient diet enriched in phytol . Phytol is the precursor of phytanic acid and is, under normal conditions, converted to pristanic acid and further β-oxidized in peroxisomes. In thiamine deficient rats, however, phytol administration results in death . Presently nothing is known about the transport of thiamine or its phosphate esters across the peroxisomal membrane. Hence, we analyzed the presence of this vitamin in peroxisomes and verified whether peroxisomes are able to synthesize it starting from thiamine.
Results and discussion
Studies on the distribution of thiamine and its phosphate esters in animal tissues appeared in the literature a few decades ago. In rat, the distribution of this vitamin has been shown to be tissue specific [6, 7]. So far, most consistent data are available for rat brain [7, 8], while less is known about the subcellular localization in rat liver. With regard to TPP, its content in rat brain is reported to be highest in mitochondrial and synaptosomal fractions , whereas in skeletal muscle  and rat liver  most TPP is reported as cytosolic.
Since the discovery of the TPP-dependent HACL1, it became clear that TPP, and in general the thiamine status of the cell , plays an important role in peroxisomal α-oxidation. Until now, TPP has never been measured in peroxisomes, nor has its transport over the peroxisomal membrane been considered. This study was undertaken to address these issues.
The presence of TPP in peroxisomes raises the question whether thiamine or TPP is transported across the peroxisomal membrane. Transport of thiamine over the plasma membrane is performed by the high affinity carriers ThTR1 and ThTR2, encoded by the SLC19A2 and SLC19A3 gene, respectively. This transport system would also be present in the mitochondrial membrane  but nothing is known about a peroxisomal transporter. Uptake of thiamine would require a peroxisomal TPK to convert the vitamin into active TPP. To investigate the presence of such a kinase, we analyzed whether TPP could be formed from thiamine intraperoxisomally. The TPK activity profile overlapped with the distribution of lactate dehydrogenase (LDH) activity (Figure 2B–C). As LDH is a marker enzyme for the cytosol and TPK activity was absent or too low to be measured reliably in the Nycodenz purified peroxisomes (data not shown), we can conclude that TPK is exclusively cytosolic. This is in accordance with previous results [19, 20, 30] and with computer-based prediction studies, which show no peroxisome targeting signal in the primary amino acid sequence of mammalian TPK. Thus, we can conclude that peroxisomal TPP is not the product of an in situ pyrophosphorylation of thiamine, suggesting that TPP has to enter the peroxisome as such.
Whether TPP is transported across the peroxisomal membrane via a specific carrier has not been discovered yet. In human mitochondria, Song and Singleton  detected a saturable TPP transport system and the yeast mitochondrial counterpart has been functionally characterized . More recently, mitochondrial TPP transport in mammals has been linked to the deoxynucleotide carrier, a protein encoded by the SLC25A19 gene, mutations in which cause Amish lethal microcephaly .
In order to better understand peroxisomal metabolism, it would certainly be useful to know more about the translocation mechanism of metabolites and cofactors across the peroxisomal membrane. So far, evidence for functional transporters in mammalian peroxisomes is limited to ATP  and phosphate carriers . With regard to TPP, a carrier has not yet been identified, but one can also envision that the uptake of this vitamin in peroxisomes is linked with the import/tetramerization of HACL1.
Using HPLC coupled with fluorimetry, we detected and measured, for the first time, the presence of TPP in purified rat liver peroxisomes. In addition, our results show that peroxisomes are devoid of thiamine pyrophosphokinase activity, which implies that vitamin B1 is entering the peroxisome in its diphosphorylated form. The TPP transport may be due to the existence of a specific peroxisomal TPP carrier or it may be linked, as a cofactor-protein complex, to the import of the peroxisomal TPP-dependent enzyme HACL1.
Thiamine hydrochloride was purchased from Janssen Chimica, TMP from Fluka and TPP from Sigma. Potassium hexacyanoferrate [K3Fe(CN)6] was purchased from Merck.
Animal studies were approved by the University Ethics committee. Male Wistar rats, weighing approximately 200 g, and Swiss Webster mice, weighing approximately 30 g, were maintained on a constant light-dark cycle and a standard laboratory diet. Rats were fasted overnight before sacrifice.
Preparation of homogenates and subcellular fractions
Homogenates of rat liver were prepared in 0.25 M sucrose containing 5 mM Mops-NaOH, pH 7.2, 1 mM dithiothreitol and 0.1% (v/v) ethanol (homogenization medium). Protease inhibitors were added to the homogenization medium just before use. This medium was also supplemented with phosphatase inhibitors (5 mM NaF and 50 μM orthovanadate) to prevent degradation of TPP and TMP. Subcellular fractionation into a nuclear (N), heavy mitochondrial (M), light mitochondrial (L), microsomal (P) and soluble (S) fraction was performed as described previously . Fraction L was subfractionated over a Nycodenz gradient in order to obtain purified peroxisomes . Marker enzymes and protein [25, 27] and HACL1  were measured as described previously.
Determination of thiamine and its phosphate esters
To investigate the stability of TPP in homogenates, mouse liver was homogenized in sucrose medium and stored at -20°C for 5 days. When prepared in the absence of phosphatase inhibitors, TPP levels dropped by approximately 42%, compared to the use of medium with inhibitors. The amount of TPP measured in perchloric liver extracts or homogenates made in the presence of inhibitors was comparable.
When analyzing samples containing Nycodenz, the baseline was less stable and aberrant peaks were noticed that interfered with the accurate determination of low levels of TPP (< 1 pmol/sample). Apparently, these peaks originated from the tri-iodinated benzoate compound, but their exact nature was unclear (Figure 1B). When required, data were corrected by analyzing the corresponding fractions collected from blank gradients.
Thiamine pyrophosphokinase activity measurement
Thiamine pyrophosphokinase activity was measured in rat liver fractions. Samples (50 μl) were incubated at 37°C with 8 mM thiamine, 24 mM ATP, 8 mM MgSO4 and 40 mM Na-phosphate buffer, pH 7.4 containing 5 mM NaF and 0.1 mM orthovanadate as phosphatase inhibitors (adapted from ). Final volume was 200 μl. After 1 h incubation, 100 μl sample was treated with 100 μl perchloric acid and further processed as described above to establish the amount of TPP formed. Values were corrected for endogenous TPP by omitting thiamine from the reaction mixture and used to calculate TPK activity, which was expressed as nmol TPP produced/mg protein/h at 37°C.
This work was supported by grants from the 'Geconcerteerde onderzoeksacties van de Vlaamse Gemeenschap' (GOA 2004/08), the 'Fonds voor Wetenschappelijk Onderzoek-Vlaanderen' (G.0115.02) and the FP6 European Union Project 'Peroxisome' (LSHG-CT-2004-512018). PF and MS were supported by a fellowship from the 'Fonds voor Wetenschappelijk Onderzoek-Vlaanderen'. The authors would like to thank Chantal Brees, Wendy Geens and Luc Govaert for technical help.
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