Carnitine Biosynthesis: Identification of the cDNA Encoding Human γ-Butyrobetaine Hydroxylase

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.

250, 506 –510 (1998)

RC989343

Carnitine Biosynthesis: Identification of the cDNA Encoding Human g-Butyrobetaine Hydroxylase Fre´de´ric M. Vaz, Sandy van Gool, Rob Ofman, Lodewijk Ijlst, and Ronald J. A. Wanders Department of Clinical Chemistry and Pediatrics, Academic Medical Center, University of Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands

Received August 14, 1998

g-Butyrobetaine hydroxylase (EC 1.14.11.1) is the last enzyme in the biosynthetic pathway of L-carnitine and catalyzes the formation of L-carnitine from g-butyrobetaine, a reaction dependent on a-ketoglutarate, Fe21, and oxygen. We report the purification of the protein from rat liver to apparent homogeneity, which allowed N-terminal sequencing using Edman degradation. The obtained amino acid sequence was used to screen the expressed sequence tag database and led to the identification of a human cDNA containing an open reading frame of 1161 base pairs encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44.7 kDa. Heterologous expression of the open reading frame in the yeast Saccharomyces cerevisiae confirmed that the cDNA encodes the human g-butyrobetaine hydroxylase. Northern blot analysis showed g-butyrobetaine hydroxylase expression in kidney (high), liver (moderate), and brain (very low), while no expression could be detected in the other investigated tissues. © 1998 Academic Press

Carnitine (3-hydroxy-4-N-trimethylaminobutyrate) is best known for its function in mitochondrial b-oxidation since it plays an indispensable role in the transport of activated fatty acids across the mitochondrial membrane (1, 2). Many organisms, ranging from bacteria to mammals, are able to synthesize carnitine (3–5). In man, carnitine is synthesized in kidney, liver and, presumably, in brain from the essential amino acids lysine and methionine (3, 6). The lysine becomes available as e-N-trimethyllysine after degradation of proteins of which lysine residues have been trimethylated by a protein-dependent methyl transferase using S-adenosylmethionine as the methyl donor (3). As the first step in carnitine biosynthesis, the e-N-trimethyllysine is hydroxylated at the 3-position by e-N-trimethyllysine hydroxylase (3, 6). Subsequently, the resulting b-hydroxy-e-N-trimethyllysine is cleaved into g-trimethylaminobutyraldehyde and glycine by b-hydroxy0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

e-N-trimethyllysine aldolase (3, 6), after which the aldehyde is oxidized by g-trimethylaminobutyraldehyde dehydrogenase to yield g-butyrobetaine (3, 6, 7). The final step involves the hydroxylation of g-butyrobetaine at the 3-position by g-butyrobetaine hydroxylase (g-BBH)1 to yield L-carnitine (3, 6, 8). Of all the enzymes of the carnitine biosynthetic pathway, g-BBH is the best-studied enzyme. Like e-N-trimethyllysine hydroxylase, g-BBH is a non-heme ferrousiron dioxygenase that requires a-ketoglutarate, Fe21 and molecular oxygen as cofactors (9). In this class of enzymes, the hydroxylation of the substrate is linked to the oxidative decarboxylation of a-ketoglutarate. g-BBH has been isolated from various sources including human kidney (10, 11), calf (12) and rat liver (13, 14) and the bacterium Pseudomonas AK1 (15). The complete amino acid sequence of the Pseudomonas AK1 enzyme has been determined by Edman degradation (16). Although the different enzymatic steps and the intermediates of the carnitine biosynthesis are well documented, thus far none of the mammalian enzymes has been characterized at the molecular level. We now report the identification of a human cDNA encoding g-BBH as demonstrated by heterologous expression in yeast. Our results show that g-BBH is expressed in only a few human tissues.

MATERIALS AND METHODS Materials. Q-Sepharose, chromatofocusing PBE 94, phenyl Sepharose HP, and 32P-ATP were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden), g-butyrobetaine and carnitine from Sigma (St. Louis, MO), and the Econo-Pac CHT-II hydroxylapatite column, from Bio-Rad (Hercules, CA). All other reagents were of

Abbreviations used: g-BBH, g-butyrobetaine hydroxylase; DTT, dithiothreitol; ORF, open reading frame; EST, expressed sequence tag; PCR, polymerase chain reaction; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1

Purification of g-BBH from Rat Liver Purification step

Protein (mg)

Specific activity (nmol/min/mg)

Activity (nmol/min)

Yield (%)

Purification (-fold)

20.000g supernatant Q-Sepharose Hydroxylapatite CHT-II Chromatofocusing PBE 94 Phenyl Sepharose HP

4403 240 33 4 0.8

0.4 3.6 8.6 34.3 111.9

1606 873 281 137 90

100 54.3 17.5 8.5 5.6

— 9 22 86 280

analytical grade. The pGEM-T vector was purchased from Promega (Madison, WI). The S. cerevisiae strain INVSC2 and the pYES2 expression vector were both purchased from Invitrogen (Carlsbad, CA).

USA) as described by the manufacturer of the transfer unit. Proteins were visualized with Coomassie Brilliant Blue. N-terminal amino acid sequencing was performed using a Procise 494 protein sequencer.

Purification of g-BBH from rat liver. The purification procedure was according to the one described by Lindstedt et al. (15) with major modifications. All protein isolation steps were carried out at 4°C, except for the phenyl Sepharose column-step, which was performed at room temperature. Livers of five Wistar rats were homogenized in a 50 mM potassium phosphate buffer, pH 6.8, containing 20 mM KCl and 2 mM dithiothreitol (DTT), by five passes of a Teflon pestle in a Potter-Elvehjem glass homogenizer at 500 rpm. The crude homogenate was centrifuged for 60 min at 20.000g. To remove floating fat particles, the supernatant was passed through a column of glass pearls. The filtrate was applied onto a Q-Sepharose column (140 ml), which was pre-equilibrated with a 20 mM sodium phosphate buffer, pH 6.8, containing 75 mM KCl. After washing of the column with equilibration buffer, bound proteins were eluted with a linear gradient from 75 to 350 mM KCl in the same buffer. Fractions containing g-BBH activity were pooled and concentrated with an Amicon Ultrafiltration unit using a YM10 membrane. The concentrate was dialyzed against a 25 mM sodium phosphate buffer, pH 7.0, containing 100 g/liter glycerol, 2 mM DTT and loaded onto an Econo-Pac CHT-II hydroxylapatite column (5 ml) equilibrated with the same buffer. Bound proteins were eluted with a linear gradient from 25 to 150 mM sodium phosphate. Fractions with g-BBH activity were pooled and dialyzed against a 10 mM sodium phosphate buffer, pH 7.0, containing 100 g/liter glycerol and 2 mM DTT. This dialysate was applied onto a PBE 94 chromatofocusing column (15 ml) preequilibrated with a 25 mM imidazole/HCl buffer, pH 6.5, and bound proteins eluted with 125 ml/liter Polybuffer 74, pH 4.6. The pH of the fractions was measured and subsequently adjusted to 7.4 by the addition of 1 M sodium phosphate buffer, pH 7.4. Fractions containing g-BBH activity were pooled and dialyzed against a 20 mM sodium phosphate buffer, pH 7.4, containing 100 g/liter glycerol and 2 mM DTT. An equal volume of the dialysis buffer supplemented with 1 M ammonium sulphate was slowly added to the dialysate. Precipitated proteins were removed by centrifugation and the supernatant was applied onto a phenyl Sepharose HP column (2 ml) preequilibrated with a 20 mM sodium phosphate buffer, pH 7.4, containing 100 g/liter glycerol, 2 mM DTT and 0.5 M ammonium sulphate. Proteins were eluted with a linear gradient from 0.5 to 0.15 M ammonium sulphate in the same buffer. Fractions were tested for g-BBH activity and analysed by SDS–PAGE followed by silverstaining.

g-BBH activity measurement. g-BBH activity was determined in a two step procedure in which the produced carnitine is measured in a radioisotopic assay. The standard assay medium for g-BBH was composed of a 20 mM potassium phosphate buffer, pH 7.0, containing 20 mM KCl, 3 mM a-ketoglutarate, 10 mM sodium ascorbate, 2 g/liter Triton X-100, 0.25 mM (NH4)2Fe(SO4)2 and 0.2 mM g-butyrobetaine. Ascorbate is needed as a reducing agent to maintain the reduced state of the iron atom. Samples (for tissue homogenate: 0.1–1 mg total protein) were added to the reaction mixture and incubated at 37°C for 60 min. The reactions were terminated by boiling for 3 min. Precipitates were pelleted by centrifugation in an Eppendorf centrifuge at maximum speed for 5 min and the amount of free carnitine was determined in the supernatant as described by Barth et al. (19). Protein concentrations were determined by the method of Bradford (20), using bovine serum albumin as standard.

SDS–PAGE, Western blotting, and automated Edman degradation. SDS–PAGE was performed as described by Laemmli (17). Gels were stained using the silver-staining procedure described by Rabilloud et al. (18). A Multiphor II Nova Blot electrophoretic transfer unit (Pharmacia Biotech) was used to transfer proteins onto a PVDF (polyvinylidene di-fluoride) membrane (Millipore, Bedford,

Cloning and expression of the human g-BBH in yeast. Expressed sequence tag (EST) clone 150-I19 (IMAGE ID: 30861) was obtained from the UK HGMP Resource Center in Cambridge (UK). The complete open reading frame (ORF) of g-BBH was amplified in a polymerase chain reaction (PCR) with this clone as template using the following primers: a BamHI-tagged forward primer 59-aaaggatccaaaATGGCTTGTACCATCCAAAAG-39 and an XhoI-tagged reverse primer 59-aaaactcgagTCAGTTTCCATTCTCCACCC-39. The PCR

FIG. 1. g-BBH purification overview by silver-stained 10% SDS– PAGE, lane 1: 20.000g rat liver supernatant, lanes 2, 3, 4, and 5 contain pools of active fractions of Q-Sepharose, hydroxylapatite CHT-II, chromatofocusing PBE 94, and phenyl Sepharose HP eluates, respectively.

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FIG. 2. cDNA and deduced amino acid sequence of human g-BBH (GenBank Accession No. AF082868). The C/T and A/T polymorphism are indicated by #. The positions of the two putative polyadenylation signals (AATAAA) are underlined.

product was cloned downstream of the galactose-inducible GAL1 promoter into the BamHI and XhoI sites of the yeast expression vector pYES2. To assess the integrity of the PCR process the ORF was sequenced. The construct was transformed to the S. cerevisiae strain INVSC2 using the lithium acetate procedure (21). Transformed yeast cells were grown on minimal glucose medium (6.7 g/liter yeast nitrogen base, 3 g/liter glucose) to fully repress transcription of the GAL1 promoter. Cells were transferred to minimal lactate medium (6.7 g/liter yeast nitrogen base, 20 g/liter lactate) and galactose was added to a final concentration of 4 g/liter to induce

protein expression. After overnight induction, spheroplasts were prepared using zymolyase according to Franzusoff et al. (22) and lyzed in a 10 mM sodium phosphate buffer, pH 7.4, containing 140 mM NaCl, 1 mM DTT and 1 g/liter Triton X-100. Northern blot analysis. A human 12-lane multiple tissue Northern blot containing poly(A)1 RNA was purchased from Clontech (Palo Alto, CA). Probes were labeled with 32P-ATP using the random priming technique and hybridization was performed according to standard procedures (23).

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RESULTS AND DISCUSSION Initial experiments in which g-BBH activity was measured in several rat tissues revealed that it was only present in rat liver (results not shown). Therefore, the enzyme was purified from rat liver by liquid chromatography. An overview of the purification is given in Table 1. SDS–PAGE analysis of the individual fractions is shown in Fig. 1. g-BBH activity eluted as a single peak from the phenyl Sepharose column. The purity of the protein was estimated to be greater than 95% as concluded from SDS–PAGE analysis followed by silver staining (Fig. 1, lane 5). After transfer to a PVDF membrane, the protein was N-terminally sequenced by automated Edman degradation, which produced the following sequence: M H C A I L K A E A V D G A R L M Q I F K H D G. This sequence was used to screen the EST database (dbEST) and identified three mouse cDNA sequences with high homology. Using these mouse cDNA sequences, the EST database was rescreened to identify human cDNA sequences. This resulted in a set of 27 human cDNA clones with high homology to the rat and mouse sequences. Since EST clone 150-I19 contained the entire ORF of the candidate cDNA, it was used as a template for further analysis. Based on the EST data, primers were selected to amplify the entire ORF. The amplified fragment was sequenced and contained an ORF of 1161 base pairs, coding for a polypeptide of 387 amino acids (Fig. 2). The cDNA contains a translation initiation signal in the context of a Kozak motif (24). The cDNA sequences revealed two putative polyadenylation signals located at base pairs 1362 and 1492 with the corresponding polyadenylation sites at base pairs 1386 and 1518, respectively. Figure 2 shows the longer transcript. The ORF could also be amplified from human liver cDNA and was identical to the sequence of clone 150-I19 except for two polymorphisms. These include a C/T and an A/G polymorphism at positions 408 and 906, respectively. Both polymorphisms do not result in an amino acid change and were also observed in other EST clones. To confirm that the cDNA identified in the database search encodes the human g-BBH, the encoded protein was expressed under transcriptional control of the galactose-inducible GAL1 promoter in the yeast S. cerevisiae. When grown on either glucose or galactose, no endogenous g-BBH activity could be measured in the yeast INVSC2 (results not shown). However, after overnight induction with galactose of the transformant containing the expression vector with the presumed g-BBH ORF, high g-BBH activity (.1 nmol/min/mg) was measured, while no activity could be measured in a transformant containing the expression vector without insert. Based on the g-BBH activity in the expression system we concluded that the cDNA identified in this study encodes human g-BBH. Furthermore, the

FIG. 3. Distribution of g-BBH expression in human tissues. Northern blot with 1 mg poly(A)1 RNA per lane was probed with 32 P-ATP labeled human g-BBH and exposed for 3 days (top). After stripping, the Northern blot was reprobed with b-actin as a control for equal loading and exposed for 16 h (bottom).

amino acid sequence of the human g-BBH shows considerable homology with the Pseudomonas AK1 g-BBH, which supports our conclusion. However, when comparing the amino acid sequence of human g-BBH with other dioxygenases, like prolyl 4-hydroxylase (25, 26) and phytanoyl-CoA hydroxylase (27, 28), no pattern similarities could be found. Previous studies have shown that g-BBH is located in the cytosol (5, 9, 14). Paul et al. (29) reported that rat liver peroxisomes also contain g-BBH activity, and that the peroxisomal g-BBH has the same cofactor requirements as the cytosolic enzyme. The cDNA identified in this study does not contain either of the peroxisomal targeting sequences [PTS1, PTS2 see for reviews (30, 31)], but this does not rule out the possibility that an isozyme is present in peroxisomes. Studies are underway to resolve this matter. To determine the expression levels of g-BBH in human tissues, the full-length cDNA was used as a probe for Northern blot analysis (Fig. 3). The g-BBH transcript with a molecular weight of approximately 1.7 kb was detected in kidney, liver and brain, but not in heart, skeletal muscle, colon, thymus, spleen, small intestine, placenta, lung, and peripheral blood leukocytes. Highest expression was observed in kidney and moderate levels in liver. Expression in brain was very low and could only be detected in longer exposures of the Northern blot in Fig. 3 (not shown). These data confirm enzyme activity measurements in human tissue homogenates from previous studies, which showed that g-BBH activity is high in kidney, moderate in liver and low in brain (3, 6, 11). ACKNOWLEDGMENTS The authors thank Dr. A. O. Muijsers for the N-terminal amino acid sequencing, C. W. T. van Roermund for help with yeast expression, N. Ponne for operating the ABI automated sequencer, and Dr. H. R. Waterham for critical reading of the manuscript.

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