Cloning and Expression of a cDNA Encoding Bovine Lipoyltransferase*

Lipoyltransferase catalyzes the transfer of the lipoyl group from lipoyl-AMP to the specific lysine residue of the lipoate-dependent enzymes. We have isolated lipoyltransferase I (LipTI) and II (LipTII) from bovine liver (Fujiwara, K., Okamura-Ikeda, K., and Motokawa, Y. (1994) J. Biol. Chem. 269, 16605–16609). N-terminal amino acid sequences of LipTI and LipTII were identical except that LipTI had an additional Asn residue on the N terminus. We cloned LipTII cDNA from a bovine liver cDNA library. The cDNA insert contained a 1119-base pair open reading frame encoding a precursor peptide of 373 amino acids including a mitochondrial targeting signal of 26 amino acids. The calculated molecular mass of the mature protein is 39,137 Da. The predicted amino acid sequence showed 35% identity with that ofEscherichia coli lipoate-protein ligase A. Northern and Southern blot analyses showed a single band, and a single species of mRNA for lipoyltransferase was found by reverse transcription-polymerase chain reaction. Recombinant LipTII was expressed in E. coli and purified to apparent homogeneity. The K m app values of the recombinant enzyme for lipoyl-AMP and apoH-protein were comparable with those of native LipTII. An antibody raised against recombinant enzyme cross-reacted with LipTI and LipTII in a similar manner. The results suggest that LipTI and LipTII are derived from the same translated product but processed differently.

Lipoic acid is a prosthetic group of H-proteins of the glycine cleavage system and the acyltransferase components of the pyruvate, ␣-ketoglutarate, and branched chain ␣-ketoacid dehydrogenase complexes (1)(2)(3)(4). Lipoate attaches to the ⑀-amino group of the specific lysine residue of the proteins via an amide linkage. Lipoylated proteins use this prosthetic group as a carrier of intermediates and reducing equivalents during enzymic reactions. The covalent attachment of lipoic acid occurs in two distinct steps as follows. In mammals, Reaction 1 is catalyzed by lipoate-activating enzyme (5), and Reaction 2 is catalyzed by lipoyl-AMP:N ⑀lysine lipoyltransferase (lipoyltransferase) (6). We have isolated two isoforms of lipoyltransferase, lipoyltransferase I (LipTI) 1 and II (LipTII), from bovine liver mitochondria employing apoH-protein as an acceptor of lipoic acid. We have purified LipTII to homogeneity, but homogeneous LipTI has not been obtained yet. Although the isoforms were separated by the chromatography on a hydroxylapatite column, they showed similar molecular mass (about 40 kDa), catalytic properties, and behavior on column chromatographies except on hydroxylapatite. They also catalyze the transfer of lipoyl group from lipoyl-AMP to apolipoyl domains of acyltransferase components of the pyruvate, ␣-ketoglutarate, and branched chain ␣-ketoacid dehydrogenase complexes (7). In contrast, lipoate-protein ligase of Escherichia coli catalyzes both Reactions 1 and 2 (8).
To understand the structure and function of LipTII and its relationship to LipTI, we isolated and sequenced cDNA encoding bovine liver LipTII and expressed the protein in E. coli. Deduced amino acid sequence of LipTII contained the N-terminal amino acid sequence of LipTI determined by Edman degradation. An antibody raised against the recombinant protein cross-reacted with LipTI and LipTII in a similar manner. Only one mRNA species was obtained from bovine live by RT-PCR reaction. The evidence reported here suggests the possibility that LipTI and LipTII are derived from the same translated product but processed differently.
Isolation and Sequencing of cDNA Clone-DNA manipulations were accomplished by standard techniques (9). Two oligonucleotides encoding for two different regions of LipTII were used for screening. Probe 1 (5Ј-AA(T/C)GA(T/C)GT(T/C/A/G)TA(T/C)CA(T/C)AA-3Ј) encoded for residues 13-18, and probe 2 (5Ј-CAT(A/G)TG(A/G)TC(A/G)TG(A/T/ G)ATCCA-3Ј) encoded for residues 24 -29 (see Fig. 1B). They were end-labeled with [␥-32 P]ATP by T4 polynucleotide kinase and used to screen a gt10 bovine liver cDNA library (CLONTECH). The hybridization and washing of Hybond N ϩ membranes (Amersham) were carried out as described previously except that the final washings were carried out at 41 and 44°C with probe 1 and probe 2, respectively (10). The insert cDNA prepared from a positive clone was subcloned into pTZ18U and fully sequenced in both orientations employing a 373A DNA sequencing system (Applied Biosystems).
Southern Blot Analysis-Bovine genomic DNA (3 g) isolated from blood leukocytes (11) was digested with EcoRV, HindIII, KpnI, PstI, or * This investigation was supported in part by grants from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AB006441.
SacI. DNA fragments were resolved on a 0.7% agarose gel, treated with 0.25 M HCl for 20 min, and transferred to Hybond N ϩ under alkaline condition. The membranes were hybridized with a 1.1-kilobase pair SphI/HincII cDNA fragment (see Fig. 1A) labeled with [␣-32 P]dCTP using Multiprime DNA labeling system (Amersham). Hybridization and the final washing with 0.1 ϫ SSPE/0.1% SDS (1 ϫ SSPE ϭ 0.15 M NaCl, 10 mM NaH 2 PO 4 , 1 mM EDTA, pH 7.4) at 60°C for 5 min were carried out according to the protocol provided by Amersham.
Northern Blot Analysis-Total RNA was prepared from bovine liver using TRIzol Reagent (Life Technologies, Inc.) according to the manufacturer's protocol. Poly(A) ϩ RNA was purified from the total RNA employing Message Maker Reagent kit (Life Technologies, Inc.). 5 g of poly(A) ϩ RNA was electrophoresed on a formaldehyde/agarose gel with 0.24 -9.5-kilobase RNA ladder markers (Life Technologies, Inc.) and transferred to Hybond N ϩ (9). Conditions of hybridization with the 32 P-labeled SphI/HincII cDNA fragment and washing of the membrane were as described (10).
RT-PCR-RT-PCR reaction was performed to isolate a variant cDNA for lipoyltransferase employing poly(A) ϩ RNA prepared as above. A mixture of 500 ng of poly(A) ϩ RNA and 2.5 pmol of gene-specific reverse primer, 5Ј-CATCAAAACTGTCAACATTAAG-3Ј (nucleotides 1247-1226; see PCR amplification was performed for 30 cycles of 94°C for 1 min, 51°C for 2 min, and 72°C for 3 min. After agarose gel electrophoresis, an amplified fragment was isolated with QIAEX (QIAGEN), digested with XbaI, and cloned into pTZ18U.
Expression of Recombinant Lipoyltransferase-For expression of the recombinant mature form of LipTII (designated LipT(T)) an SphI/XbaI cDNA fragment encoding the full-length mature LipTII was subcloned into phagemid vector pTZ19U. Creation of an NdeI site adjacent to the codon for the N-terminal Thr residue of the mature protein and a BamHI site 53 bases downstream from the stop codon was carried out by site-directed mutagenesis according to the method of Kunkel et al. (12) using a Bio-Rad kit. Antisense primer 1 (5Ј-TCCACTTTTAACTGT-CATATGAAAGCCAGCTGCT-3Ј; nucleotides 182-149 (see Fig. 1B); the NdeI site is underlined, and modified bases are shown in boldface letters) and antisense primer 2 (5Ј-TTTAAAAATGCACTGGATC-CATTTTAATTTTCTCA-3Ј; nucleotides 1280 -1246; the BamHI site is underlined and modified bases are shown in boldface letters) were employed, respectively. NdeI and BamHI sites originally present in the coding region of LipTII cDNA (see Fig. 1A) were deleted simultaneously with antisense primer 3 (5Ј-CCTTCTAAATTCATGTGGTCGTGTATC-CAGTCTTCTAC-3Ј; nucleotides 266 -228; modified bases are shown in boldface letters) without altering the amino acid sequences. The NdeI/ BamHI fragment was isolated from the mutant phagemid and ligated to the expression plasmid pET-3a (13) digested with NdeI and BamHI. The nucleotide sequence of the resultant expression vector, pLipT(T), was confirmed by DNA sequencing. The expression vector for LipT(N), which has an additional Asn residue at the N-terminal end of LipT(T), was constructed using antisense primers 2, 3, and 4 (5Ј-ACTTTTAACT-GTGTTCATATGGCCAGCTGCTGG-3Ј; nucleotides 179 -147; the NdeI site is underlined and modified bases are shown in boldface letters) as described for the construction of pLipT(T). The resulting plasmid was designated pLipT(N).
pLipT(T) and pLipT(N) were introduced separately into E. coli BL21(DE3)pLysS (13). Cells were grown in 400 ml of LB medium (9) containing 30 g/ml ampicillin and 25 g/ml chloramphenicol. Expression was induced by the addition of 25 M isopropyl-␤-D-thiogalactopyranoside at the start of the culture. After incubation at 30°C for 22 h, cells were harvested by centrifugation.
Purification of LipT(T)-All purification steps were performed at 4°C unless indicated. The cell pellet was frozen and thawed in 40 ml of 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 30 M p-amidinophenylmethanesulfonyl fluoride, 10 g/ml leupeptin and sonicated twice for 1 min with a duty cycle of 50% (Branson Sonifier 250). The inclusion bodies were pelleted by centrifugation at 15,000 ϫ g for 30 min. The cell extract was obtained by centrifugation of the supernatant fraction at 105,000 ϫ g for 1 h. LipT(T) was purified from the extract by the chromatography on hydroxylapatite (2.5 ϫ 6 cm), DEAE-Sepharose (1 ϫ 3 cm), and apoH-protein-Sepharose affinity columns (3 ml) as described for the purification of lipoyltransferase from bovine liver (6). Enzyme activity and steady state kinetic constants were determined as described previously (6). One unit of lipoyltransferase activity is defined as that catalyzing the formation of 1 nmol of lipoylated H-protein/min.
Preparation of Antibody-Purified LipT(T) (0.2 mg) was emulsified with an equal volume of Freund's complete adjuvant and subcutaneously injected into a rabbit. 2 and 4 weeks later, boosters containing 0.15 mg of LipT(T) in Freund's incomplete adjuvant were injected. The blood sample was taken 10 days after the second booster injection. IgG fraction was obtained by precipitation with ammonium sulfate at 40% saturation.
Western Blot Analysis and Immunoprecipitation-10 ng of LipT(T), LipTI, and LipTII were separated by 12.5% SDS-PAGE, electroblotted onto Immobilon-P (Millipore), and incubated with anti-LipT(T) antibody diluted 2000-fold with 0.05% Tween 20, 20 mM Tris-HCl, pH 7.5, 0.15 M NaCl. The antigens were located with ProtoBlot AP system (Promega) as described (2). Immunoprecipitation was carried out in a mixture of 14-l phosphate-buffered saline containing the indicated amount of IgG and 21.6 or 24.1 ng of LipTI or LipTII, respectively. The amount of protein was kept constant by the addition of bovine serum albumin. The control experiment was carried out with nonimmune IgG. After incubation at 4°C for 16 h, the supernatant fractions were obtained by centrifugation at 15,000 ϫ g for 20 min, and a quarter of each supernatant fraction was employed for the assay of lipoyltransferase activity.
Amino Acid Sequence Analysis-The N-terminal amino acid sequences of LipTI, LipTII, and lysylpeptides were determined using an Applied Biosystems 477A protein sequencer and a Hewlet Packard G100A protein sequencer. The lysylpeptides were obtained by digestion of carboxymethylated LipTII with lysylendopeptidase and separation of the peptides by high performance liquid chromatography on an ODS column as described previously (2).
Other Methods-SDS-PAGE were carried out as described (14). Protein concentrations were determined by the method of Bradford (15) with bovine serum albumin as a standard.

RESULTS
Cloning and Sequencing of Bovine Liver Lipoyltransferase II-The N-terminal amino acid sequence of LipTII and amino acid sequences of several lysylpeptides derived from LipTII were examined (Fig. 1B). To isolate cDNA encoding LipTII, two degenerated oligonucleotide probes were synthesized based on the amino acid sequence of the N-terminal region of LipTII. Approximately 2.4 ϫ 10 6 independent clones in a gt10 bovine liver cDNA library were screened, and only one positive clone that hybridized with both probes was obtained. It may reflect a low abundance mRNA of lipoyltransferase as expected from the yield of the purified enzyme (6).
The insert cDNA of the clone is 1326-bp long and consists of an 89-bp 5Ј-untranslated region, a 1119-bp open reading frame, and a 118-bp 3Ј-untranslated region (Fig. 1). The sequence AGCATGC surrounding the first inflame ATG codon is in good agreement with the optimum translation initiation sequence (ACCATGG) described by Kozak (16). The open reading frame encodes a 373-amino acid protein. The predicted amino acid sequence completely matches the N-terminal amino acid sequence of purified LipTII and amino acid sequences of lysylpeptides (Fig. 1B). Thus, a mitochondrial targeting signal of 26 amino acids is predicted. The predicted size of the mature protein (347 amino acids with a calculated molecular mass of 39,137 Da) is in good agreement with the size previously determined by SDS-PAGE (40 kDa) (6). A putative polyadenylation signal, ATTAAA, is located 20 bp upstream from the poly(A) tail. The N-terminal amino acid sequence of LipTI was determined to be NTVKSGLILQSISNDVYHNL-. This sequence was identical to the residues Ϫ1 to 19 of LipTII (Fig. 1B), suggesting that LipTI and LipTII are produced from the same translated product by alternative processing in mitochondria.
Protein sequence similarity between LipTII and known proteins was analyzed by searching the data library of the DNA Data Bank of Japan using the FASTA program (version 3.0). LipTII showed 35, 32, and 28% identity with E. coli lipoateprotein ligase A (8), yeast hypothetical protein (accession number, Swiss-Prot P47051), and Mycoplasma genitalium probable lipoate-protein ligase A (P47512), respectively (Fig. 2). In particular, amino acids 8 -86 of LipTII shares high homology with residues 6 -85 of E. coli enzyme (59% identity), suggesting that the N-terminal domain of these proteins may be responsible for the lipoate-transferring activity. In contrast, the C-terminal part of these proteins is less homologous. Presumably, the C-terminal half of E. coli lipoate-protein ligase A may be involved in the lipoate activating activity that bovine lipoyltransferase lacks.
Southern and Northern Blot Analyses-Because LipTI and LipTII share the identical amino acid sequence around the N-terminal region, Southern blot analysis was carried out to examine whether more than one gene encoding lipoyltransferase was present employing the LipTII cDNA as a probe. A single band was detected in each enzyme digest (Fig. 3A). The result suggests the presence of a single copy gene encoding lipoyltransferase.
A Northern blot that contains size fractionated bovine liver poly(A) ϩ RNA was probed with the 32 P-labeled SphI/HincII cDNA fragment. A single mRNA species of about 1.5 kilobases long was detected (Fig. 3B). The size of the message corresponds to that of the cloned cDNA.
RT-PCR Analysis-Although Northern blot analysis showed the presence of a single mRNA species for lipoyltransferase, a possibility exists that the mRNA includes a variant for LipTI with minor differences at the internal region of the sequence produced by alternative splicing. To examine the possibility, RT-PCR was utilized with primers that were synthesized based on the findings that LipTI has an N-terminal amino acid sequence identical with that of LipTII and a molecular mass similar to LipTII. If mRNA for LipTI co-exists, two kinds of cDNA should be isolated with identical ratio by the cloning of the RT-PCR products, because the levels of LipTI and LipTII in bovine liver are nearly equal (6). The PCR products showed only a single band of about 1100 bp on agarose gel electrophoresis (data not shown). Nucleotide sequences of the cDNAs from 24 independent clones were identical to that of LipTII (Fig. 1B), except that a few of them showed a single base replacement at a different site caused by misreading by polymerase in the PCR reaction. The result suggests that LipTI and LipTII are products from a single mRNA species.
Expression and Purification of Recombinant LipTII and Kinetic Analysis-LipT(T) was expressed in E. coli as described under "Experimental Procedures." The induction of BL21(DE3)-pLysS cells harboring pLipT(T) with isopropyl-␤-D-thiogalactopyranoside resulted in high level expression of a 40-kDa protein (Fig. 4A). Although most of the expressed protein was sequestered in inclusion bodies, the supernatant fraction obtained from the cell extracts exhibited about 100-fold higher lipoyltransferase activity (5.97 units/mg protein) than that from control cells harboring pET-3a (0.04 unit/mg protein). The protein from inclusion bodies solubilized with 6 M urea had a low lipoyltransferase activity. Attempts to recover fully active LipT(T) from the inclusion bodies through the use of urea, followed by dialysis for 2 days during which urea was incrementally diluted, were unsuccessful. We purified LipT(T) from the supernatant fraction to homogeneity by the successive chromatographies on hydroxylapatite, DEAE-Sepharose, and apoH-protein-Sepharose affinity columns (Fig. 4B). Table I summarizes a typical purification of LipT(T) from a 400-ml culture. LipT(T) emerged from the hydroxylapatite column at about 230 mM phosphate, nearly the same concentration at which LipTII was eluted from the column (6). The N-terminal amino acid sequence of LipT(T) showed that the initiation Met residue was cleaved off by an E. coli methionyl-aminopeptidase in agreement with the rule for the methionine removal from protein in E. coli (17). Steady state kinetic studies were carried out by varying the concentration of one substrate while keeping the concentration of the other substrate constant. The K m app values for lipoyl-AMP and apoH-protein of LipT(T) were com- parable with those of LipTII, but the V max app value was more than 3-fold that of LipTII (Table II). The difference may be due in part to the inactivation of LipTII during the purification because the purification of LipTII required another two steps of column chromatography (6). LipT(T) could not lipoylate apoHprotein with lipoate plus MgATP, confirming the previous observation that bovine lipoyltransferase has no ability to activate lipoate to lipoyl-AMP (6).
We attempted to express and purify a protein that has an additional Asn residue on the N terminus of LipT(T). The protein designated LipT(N) was expressed in BL21(DE3)pLysS cells. Again the substantial amounts of LipT(N) were segregated in inclusion bodies. The cell extract showed a lipoylation activity of 5.31 units/mg protein comparable with that of LipT(T). LipT(N) was eluted at about 190 mM phosphate from a hydroxylapatite column. The behavior was quite similar to that of LipTI. However, most of the LipT(N) protein passed through the column of DEAE-Sepharose in a condition where LipTI and LipTII activities can be retained on the column. Amino acid sequence analysis of the partially purified LipT(N) revealed that the protein has an initiation Met residue on the N terminus.
Reactivity with Anti-LipT(T) Antibody-To further elucidate  the relationship between LipTI and LipTII, we raised an antibody against the purified LipT(T) and examined the reactivity of LipTI and LipTII with the antibody. Western blotting (Fig.  5A) showed that both LipTI and LipTII were equally recognized by anti-LipT(T) antibody. An immunoprecipitation experiment showed similar inactivation curves of the activities of LipTI and LipTII with the increase of the amount of antibody (Fig.  5B). These results suggest that LipTI and LipTII are structurally related proteins.

DISCUSSION
This report describes the cloning of the full-length cDNA for bovine lipoyltransferase II. The cDNA contained a 1119-bp open reading frame encoding a peptide of 373 amino acids. The protein consists of a mitochondrial targeting signal of 26 amino acids and a mature protein of 347 amino acids. The mitochondrial targeting signal showed characteristic properties such as a high content of basic and hydrophobic amino acid residues, an absence of acidic residues, and amphiphilicity (18). This confirms the localization of lipoyltransferase in mitochondria as concluded previously from the distribution of the activity and translocation experiments of H-protein (2).
Although the cloning of LipTII has not resulted in very high levels of expressed soluble enzyme because the majority of recombinant LipTII (LipT(T)) is produced as inclusion bodies, it has facilitated the isolation and purification of the enzyme to homogeneity in an active form in quantities sufficient for the characterization of the enzyme. LipT(T) expressed in E. coli showed similar properties as compared with the native enzyme but a higher V max app value. LipT(T) was unable to activate lipoic acid in agreement with the previous observation with the native enzyme. Thus it is confirmed that bovine lipoyltransferase catalyzes only lipoate transfer from lipoyl-AMP to apoproteins. Comparison of the primary sequence of LipTII and E. coli lipoateprotein ligase A, which catalyzes both the activation of lipoic acid and the transfer of lipoate, revealed a strong homology in Nterminal region, suggesting that the active site for the transfer of lipoate to proteins is located within the N-terminal half of these proteins. E. coli biotin-protein ligase (BirA) and human holocarboxylase synthetase catalyze the attachment of biotin to biotindependent enzymes by two-step reactions similar to the lipoylation reaction. The enzymes contain the sequence GXGXXG predicted as a consensus sequence associated with ATP binding (19 -21). Inspection of amino acid sequence of bovine LipTII revealed no similar sequence as expected, because LipTII catalyzes no ATP-dependent activation of lipoic acid.
An intriguing finding was that a stretch of the predicted amino acid sequence of LipTII (amino acids Ϫ1 to 19, Fig. 1B) completely matched the N-terminal amino acid sequence of LipTI. Although the internal amino acid sequence of LipTI has not been examined, it seems likely that LipTI and LipTII are derived from the same translated product. Several pieces of evidence favor this possibility: (i) The behaviors of LipT(T) and LipT(N) on a hydroxylapatite column were similar to that of native LipTII and LipTI, respectively. (ii) LipTI and LipTII were equally recognized by anti-LipT(T) antibody. (iii) Southern blot analysis suggested that bovine lipoyltransferase appears to be encoded by a single copy gene. (iv) Northern blot analysis indicated the presence of a single transcript of 1.5 kilobases. (v) A specific mRNA for LipTI could not be detected by RT-PCR analysis.
We cloned and expressed a cDNA encoding mammalian lipoyltransferase for the first time. Purification of lipoyltransferase from animal liver is laborious because of the low content of the enzyme. Here we present a simple purification method to obtain milligram quantities of recombinant bovine lipoyltransferase II. The recombinant enzyme showed the same properties as the native enzyme. The availability of cDNA clone coding for lipoyltransferase should facilitate studies of structural and functional aspects of this enzyme.