Vol. 183, No. 3, 1992
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March 31, 1992
NUCLEOTIDE AND PREDICTED AMINO ACID SEQUENCE OF A cDNA CLONE ENCODING PART OF HUMAN T R A N S K E T O L A S E
Mahin Abedinia, Roy Layfield, Sheelagh M. Jones, Peter F. Nixon* and John S. Mattick Centre for Molecular Biology & Biotechnology, Department of Biochemistry, University of Queensland, Brisbane, QLD 4072 Australia Received February 12, 1992
Transketolase is a key enzyme in the pentose-phosphate pathway which has been implicated in the latent human genetic disease, Wemicke-Korsakoff syndrome. Here we report the cloning and partial characterisation of the coding sequences encoding human transketolase from a human brain cDNA library. The library was screened with oligonucleotide probes based on the amino acid sequence of proteolytic fragments of the purified protein. Northern blots showed that the transketolase mRNA is approximately 2.2 kb, close to the minimum expected, of which approximately 60% was represented in the largest cDNA clone. Sequence analysis of the transketolase coding sequences reveals a number of homologies with related enzymes from other species.
® 1992
Academic Press, Inc.
Transketolase (EC 2.2.1.1,TK) catalyses the transfer of a two-carbon ketol unit from xylulose 5-phosphate to an aldose acceptor, either ribose 5-phosphate or erythrose 5-phosphate. Along with transaldolase, TK provides the link between the pentose-phosphate pathway and the glycolytic pathway, enabling the recycling of pentose sugars under conditions where NADPH production is required for reductive biosynthesis. TK is also one of the few major enzymes which requires thiamin diphosphate (ThDP) as a cofactor, others being pyruvate dehydrogenase, 2oxoglutarate dehydrogenase and branched chain 2-oxoacid dehydrogenase. TK assays provide a sensitive measure of thiamin deficiency. There is evidence of heterogeneity of human erythrocyte, leucocyte and fibroblast TK by isoelectric focussing [1-4], as well as by variation in its affinity for ThDP [5, 6]. In Alzheimers disease, TK appears to be modified [4, 7] and a variant form of the enzyme, having decreased affinity for ThDP, has been implicated in the specific brain damage characterised in man as the Wernicke-Korsakoff (WK) syndrome [5, 6]. However, others [2, 8] have reported an identical affinity of TK for ThDP in this condition, and constancy of isoelectric focussing patterns [9,10]. *To whom correspondence should be addressed.
Abbreviations: BCKDH (branched chain ketoacid dehydrogenase); DHAS (dihydroxyacetone synthase); ThDP (thiamin diphosphate); TK (transketolase) and WK (Wernicke-Korsakoff). 0006-291X/92 $1.50 1159
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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The WK syndrome, which encompasses a characteristic acute encephalopathy followed by a chronic amnesia, is only observed following an episode of severe thiamin deficiency. However, not all thiamin-deficient individuals develop the WK syndrome, and on this basis the disorder is widely quoted as a latent genetic disease that is only revealed under particular environmental conditions, ie. malnutrition [11]. TK has been well characterised with respect to catalytic and chemical properties from yeast [for review see 12] but little is known about the mammalian enzyme other than that, as in yeast, it is a homodimer of subunits of approximately 70,000 molecular weight [13]. The gene encoding dihydroxyacetone synthase (DHAS), a novel TK from the formaldehyde-utilising yeast Hansenula
polymorpha [14] and the TK gene of Rhodobacter sphaeroides [15] have been cloned and sequenced. Here we present the cloning and partial sequence of the human TK cDNA.
Materials and Methods
Purification of human erythrocyte transketolase. TK was purified from blood supplied by the Brisbane Red Cross Blood Transfusion Service and collected from polycythemic or hemochromatotic patients. Erythrocytes were washed twice in 154 mM saline, filtered (Sepacel) to remove any residual leucocytes, suspended in an equal volume of water and lyzed by freezing and thawing. The hemolysate (500 ml) was dialyzed against 25 mM Tris-HC1 pH 8.5 (buffer A) until its conductivity matched that of the buffer, loaded onto a column of Blue Trisacryl M (I.B.F., France), 5 cm x 70 cm, equilibrated with buffer A. The matrix was washed with the same buffer to remove all unbound protein, then eluted by a linear gradient of sodium chloride from 0-500 mM in buffer A. TK was eluted at 70-100 mM NaC1, ahead of the major protein peak, and was dialyzed against 5 mM sodium phosphate pH 6.8 (buffer B) then adsorbed to hydroxylapatite (Biogel HT, Biorad) in a column 1 cm x 2 cm. Proteins were eluted by successive gradients of sodium chloride, first 5-300 mM then 300-750 mM in buffer B. TK was eluted early in the second gradient. Gel filtration in Biogel P4 (Biorad) was used to exchange the buffer to 10 mM Tris-HC1 pH 7.5. TK activities were assayed [16] by use of a Centrifichem 400 centrifugal analyzer.
Generation and purification of peptides from transketolase. Trypsin (Sigma) and endoproteinase Lys C (Boehringer Mannheim) were used to generate suitable peptides for sequencing. Peptides 1 and 2 were generated by incubation of TK in 10 mM Tris-HC1 pH 7.5 with trypsin at a ratio of 50:1 for either 15 rain at 25°C in the presence of 0.1% SDS or for 2hr at 25°C in the presence of 3M urea respectively. Peptide 3 was generated by incubation of TK in 10 mM Tris-HC1 pH 7.5, 0.1% SDS with endoproteinase Lys C at a ratio of 40:1 for 15 min at 25°C. The three peptides were analysed by 12.5% SDS/PAGE and transferred by electroblotting onto Immobilon membrane (Millipore) in 10 mM CAPS, 10% methanol pH 10.5 at 30 v for 16 h using a BioRad Mini Protean system. After staining with Coomassie Blue the relevant peptides were cut out of the membrane and directly sequenced. Amino acid sequences were analyzed by use of an Applied Biosystems 470A gas phase sequencer.
cDNA library screening. A human brain cDNA library constructed in kgtl 1 (Clontech) was screened for TK sequences using oligonucleotide probes that were designed by reference to the amino acid sequence of purified TK peptides 1 and 3, and biased towards human codon usage [17]. Oligonucleotide sequences were antisense, ie. complementary to the mRNA sequence. These were: Oligo 1 (corresponding to peptide 1) 5'-TGT GGC CAC GCC ATC AGA GGG GTA GAA GAC TGT AGA GGT GGG CAC AGA-3' and Oligo 3 (corresponding to peptide 3) 5'-GAT GAA GCA GAT GCC CTT GGT GTT GGC AGC CAG CTC CAC AGC-3'. All oligonucleotides were prepared on a Milligen 7500 DNA synthesizer. For hybridisations, oligonucleotides were labelled to a specific activity of 108 cprn/~g using T4 polynucleotide kinase and [U32p]-ATP [18]. A total of 500,000 pfu were screened and recombinant phage were transferred onto Hybond N, according to the suppliers instructions 1160
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(Amersham). Prehybridiization was carried out in 6 X SSC [18], 0.1% SDS, 5 X Denhardts solution [18] and 100 ~tg/ml denatured salmon sperm DNA at 42°C for 2 h. Hybridization, for 2 h at 50°C, was in the same buffer but contained 20 ~tg/ml yeast tRNA instead of cartier DNA and 0.5 pmol/ml end-labelled oligonucleotide probe. The stringency of washing was 6 X SSC, 0.1% SDS at 55°C. Recombinant phage giving positive signals were plaque purified and the cDNA insert size estimated using the polymerase chain reaction.
DNA sequence analysis. Lambda DNA for subcloning was prepared using the CTAB method [19]. cDNA inserts were subcloned into the phagemid pBS+/- (Stratagene) for restriction analysis and generation of smaller, overlapping clones. Sequence determination of both strands was performed using the Sanger dideoxy chain termination method [20] with modifications for the direct sequencing of doublestranded DNA [21]. Preparation of RNA and northern hybridization. RNA was prepared fl'om the human Burkitt's lymphoma cell line, B JAB, using Nonidet P-40 buffer (BDH) and protein~LseK/SDS according to Favaloro et al [22]. For Northern hybridisation, polyA+ RNA (1 ixg/lane) and total RNA (30 txg/lane) were denatured and electrophoresed through a formaldehyde denaturing gel (1.2% agarose) in MOPS buffer [16] at 5 V/cm for 3 h. After electrophoresis, RNA samples were transferred to Hybond N (Amersham), and prehybridised for 4 hrs at 42°C in 5 X SSPE [16], 50% formamide, 0.1% SDS, 5 X Denhardts solution and 100 ~tg/ml denatured salmon sperm DNA. Hybridisation, for 16 hrs at 42°C, was in the same buffer with a human TK cDNA probe l~Lbelledaccording to the method of Feinberg & Vogelstein [23], added to 106 cpm/ml. The stringency of washing was 2 X SSC, 0.1% SDS at 65°C.
Results and Discussion
Purification and partial amino acid sequence of Human Transketolase. TK purified from human erythrocytes migrated as a single band with a molecular weight of 69,000 in SDS-polyacrylamide gels (10 ~tg protein, silver stained, data not shown). Since attempts to obtain amino-terminal sequence were unsuccessful, suggesting that it is blocked, enzymatic proteolysis of the protein was carried out to generate peptides suitable for amino acid sequencing. The resultant sequences were: Peptide 1 SVPTSTVFYPSDGVAT, Peptide 2 ILATPPQE and
Peptide 3 AVELAANTKGICFI. Isolation and partial chanlcterisation of human TK cDNA A human brain cDNA library was screened with oligonucleotide probes (oligos 1 and 3) derived from the amino acid sequence of TK peptides 1 and 3 respectively, each biased toward human codon usage. Clonted sequences hybridizing to both probes were selected for further study, and the largest of these was subcloned into pBS+/-, restriction mapped and sequenced. The complete nucleotide and deduced amino acid sequence of this cDNA (clone pTK2), is given in Figure 1, with regions corresponding to the TK peptides 1, 2 and 3 underlined. Clone pTK2 is 1318 bp in length and has a translation stop codon at 1177 bp, leaving 141 nts of 3' untranslated sequence in which a polyA addition site occurs 20 nts upstream of the polyA tail. Homology of the nucleotide sequences enccxting peptides 1 and 3 to the corresponding probes used for the library screening was 77% for olilgo 1 and 81% for oligo 3. Northern hybridisation of mRNA purified from BJAB (human lymphocyte) cell lines with the cDNA insert of clone pTK2 as probe identified a strong band at ~2.2 kb (Figure 2), suggesting 1161
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GCTGT GCAAGGCCT T TGCGAGGCCAAGCACCAGCCAACAGCCATCAT A V Q G L C E A K H Q P T A GGC CGAGGGATCACGGGGGTAGAAGATAAGGAGT G R G I T G V E D K A TGGCT GAGCAGA M A E Q
E
I
I
TGCCAAGACCT A K T
C T TGGCAT GGGAAGCCCC S W H G K P
TCAAG F K
TCCCCAAAAAC L P K N
TCATCCAGGAGAT C TACAGCCAGAT C CAGAGCAAAAAGAAGATCC I I Q E I Y S Q I Q S K K K .I
T GGCA L A
63
126
189
ACCCCTCCACAGGAGGACGCACCCTCAGTGGACATTGCCAACATCCGCATGCCCAGCCTGCCC T P P Q E D A P S V D I A N I R M P S L P PEPTIDE 2 A GCTACAAAGT T GGGGACAAGATAGCCACCCGCAAGGCC TAC GGGCAGGCAC TGGCCAAGCTG S Y K V G D K I A T R K A Y G Q A L A K L
252
GGCCATGCCAGTGACCGCATCATCGCCCTGGATGGGGACACCAAAAATTCCACCTTCTCGGAG G H A S D R I I A L D G D T K N S T
378 F
S
315
E
ATCTTCAAAAAGGAGCACCCGGACCGCTTCATCGAGTGCTACAT I F K K E H P D R F I E C
I
TGCCGAGCAGAACATGGTG A E Q N M V
441
Y
AGCATCGCGGTGGGCTGTGCCACCCGCAACAGGACGGTGCCCT S I A V G C A T R N R T V
TCTGCAGCACTTT TGCAGCC F C S T F A A
504
P
T TCTTCACGCGGGCCT F F T R A
TTGACCAGAT TCGCATGGCCGCCATCTCCGAGAGCAACATCAACCTC F D Q I R M A A I S E S N I N
TGCGGCTCCCACTGCGGCGT C G S H C G CTGGCTATGT L A M
V
TTCCATCGGGGAAGACGGGCCCTCCCAGATGGCCCTAGAAGAT S I G E D G P S Q M A L
567 L 630
E
TTCGGTCAGTCCCCACATCAACCGTC F R S V P T S T
TTTTACCCAAGTGATGGCGTTGCTACA V F Y P S D G V A PEPTIDE 1 GAGAAGGCAGTGGAACTAGCCGCCAATACAAAGGGTATCTGT TTCATCCGGACCAGCCGCCCA E K A V E L A A N T K G I C F I R T S R PEPTIDE 3 GAAAATGCCATCATCTATAACAACAATGAGGACT TCCAGGTCGGACAAGCCAAGGTGGTCCTG E N A I I Y N N N E D F Q V G Q A K V V
D 693 T 756 P 819 L
AAGAGCAAGGATGACCAGGTGACCGTTATCGGGGCTGGGGTGACCCTGCACGAGGCCTTGGCC K S K D D Q V T V I G A G V T L H E
A
L
A
GCTGCCGAACTGCTGAAGAAAGAAAAGATCAACATCCGCGTGCTGGACCCCTTCACCATCAAG A A E L L K K E K I N I R V L D P F
T
I
K
TCTCGACAGCGCTCGTGCCACCAAGGGCAGGATCCTCACCGTG L D S A R A T K G R I L
T
V
TGGTGAGGCTGTGTCCAGTGCAGTAGTGGGCGAGCCT G E A V S S A V V G E
P
CCCCTGGACAGAAAACTCAT P L D R K L GAGGACCAT E D H
I
TATTATGAAGGTGGCAT Y Y E G G
I
882
945
GGCATCAC TGTCACCCACCTGGCAGTTAACCGGGTACCAAGAAGTGGGAAGCCAGCTGAGCTG G I T V T H L A V N R V P R S G K P A C TGAAGATGT L K M
T TGGTATCACAGGGATGCCATTGCACAAGCTGTGAGGGGCCTCATCACCAAGG F G I T G M P L H K L *
1008
1071
1134 E
L 1197
CCTAGGGCGGGTATGAAGTGTGGGGCGGGGGTCTCATACATTCCTGAGATTCTGGGAAAGGTG
1260
C T T CAAAGATG
1318
TAC T GAGAGGAGGGGTAAATATA TG T T T TGAGAAAAAAAAAAAAAAA polyA signal
Fil~ore 1. Nu¢leotide and deduced amino acid seauence of the human TK cDNA done. nTK2. The sequence represents the C-terminal 60% of the complete TK mRNA (see text) and is numbered relative to the first nucleotide (+1). The peptide sequences which are underlined have a perfect match with TK peptides 1, 2 and 3 which were sequenced from purified human TK. Also underlined in the nucleotide sequence is the polyA addition signal, 20 nts upstream of the polyA tail. The translation stop codon (TGA) is marked by an asterisk (*).
that the human TK gene transcribes a mRNA of ~2200 nt, consistent with the protein molecular weight of 69,000 (about 600 codons). This would indicate that clone pTK2 represents approximately 60% of the complete coding sequence, and since three peptides sequenced from purified human TK have a complete match with the translated peptide sequence of pTK2, occuring 1162
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I
2
3
4
--28S --18S
Figure 2. Northern blot analvsis of human TK mRNA. Total RNA samples (30 ~g each in lanes 1, 2 and 3) and polyA+ RNA (1 ~tg in lane 4), isolated from the human Burkitt's lymphoma cell line (BJAB), were probed with the human TK cDNA, pTK2. Hybridisation revealed a strong band at N2.2 kb.
in the same open reading frame as the stop codon indicated (Fig 1), this clone appears to represent the C-terminal portion of the complete TK sequence. A search of the GenBank database with the peptide sequence of pTK2 revealed extensive homology to two other sequences, dihydroxyacetone synthase (DHAS), a transketolase of unusual substrate specificity from the formaldehyde utilising yeast Hansenula polymorpha [14], and the recently cloned and sequenced transketolase of Rhodobacter sphaeroides, TKLB [15]. Dot plots of the amino acid sequences for DHAS or TKLB with that of pTK2 revealed a number of homologous regions. A more detailed alignment of the three sequences showed that there was 24% identity with 40% similarity between pTK2 and DHAS, and 19% identity with 41% similarity between pTK2 and TKLB (Figure 3). This correlates with the grouping of ThDP-dependent enzymes reported by Zhang et al [25] in which the yeast DHAS, the human TK and the Ell3 subunit of both human and P. putida BCKDH can be grouped into a class within which positional identities are found at 20-25% of residues and similarities at 40-45%. It is interesting to note that whilst the level of similarity between all three sequences is virtually the same, the highest level of positional identity with the human sequence is not with the TK of the photosynthetic prokaryote R. sphaeroides (TKLB), despite the identity of the many reactions catalysed by these two proteins. Rather, the greater positional identity is between the human sequence and that of the DHAS ofH. polymorpha, consistent with the eukaryotic origins of both but perhaps unexpected in view of the unique substrate specificity of DHAS, which catalyses the transfer of a glycoaldehyde group from xylulose 5-phosphate to formaldehyde [14], in addition to the more common TK reactions. The highly conserved region reported as a putative ThDP-binding motif, present in all thiamin-dependent enzymes sequenced so far [26], is not found in the sequence encoded by pTK2. In DHAS however, the proposed motif is present in the N-terminal portion of the sequence at residues 158 to 208 [26] and, the comparable region of TKLB (residues 147 to 197) has the same motif. Anticipating that the ThDP-binding motif of the human TK will also be present in the Nterminal -200 residues (based on the comparative alignment in Fig 3), it is not surprising that this 1163
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DHAS
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RELKIKYGMNPAQKFYIPQDVYDFFKEKPAEGDKLVAEWKS-LLVAKYVKAYPEEG : : : : : ::: : : :
[338] :
:
::
:
:
pTK2 AVQGLCEAKHQPTAI-IAKTFK---GRGIT-GVEDKESWHGKPL-PKNM-AEQIITKLB
AAARERLGWDHPP-FEIPADLYEAWGRIAARGADARAAWETRLLQASPLRA
.....
DHAS
QEFLARMRGELPKNWKSFLPQQEFTGDAPTRAAARELVRALGQNCK-SV-IA-GC:: : : : : :: :::: : : ::
pTK2
QE-IYSQIQSKKKILAT--PPQE---DAPSVDIANIRMPSL-PSYKVGDKIA-TRK :: : :: : :: :: ......
[48] [320]
[390] :
:
:
::
::
:
:
::
[96]
TKLB
AFETAEAADT
SALPPAIAAYKARL--SAE---APKVATRK-ASEMALGVV
DHAS
ADLSVSVNLQWPGVKY-FMDP-SL-STQCGL-SGDYSGRYIE-YGIREHAMCAIAN : :: :: : : : :: : : ::::::
[364]
[441] :
:
::
:
:::
pTK2 AYGQALAKLGHASDRIIALDGDTKNSTFSEIFKKEHPDRFIECY-IAEQNNVSIAV [151] TKLB
: ::1 : : : :: : :: : : 1 NEALPFAVGGSADLTGSNLTR-SKGM-VSVA-PGAFAGSYIH-YGIREHGM-AAAM
DHAS
-GLAAYNKGTFL-PITSTFFMFYLYAAPAIRMAGLQELKAIHIGTHDSINEGENGP
pTK2
-GCATRN-RT-V-PFCSTFAAFFTRAFDQIRMAAISESNINLCGSHCGVSIGEDGP : : : : :: : : : :: :::::: :
TKLB
NGIALHGG---LRPYGGTFMAFADYCRPSIRLSALMGVPVTYV~THDSIGLGEDGP
pTK2
SQMALEDLAMFRSVPTSTVFYPSDGVAT :: :::::::::::: :::
TKLB
THQPVEHLASLRAIPNLAVIRPAD~VETAEAWEIAMTATSTPTLLVLSRQNLPTVR
DHAS
y
y
--
loop
--
::
:::::::::
~yy
G G
[468]
G
.... :::::
:
[5501 :
:
:
:
EKAVELAANTKGICFIRTSRPENA : :: :: : : :
: [255] :
: [524]
zI
y
[603] ::
IIYNNNEDFQVGQAKVVLKSKDDQVTVIGAGVTLHEALAAAELLKKEKINIRVL-D ::: : : : : :::::::: :: : : : : : : : :
TKLB
TEHRDENLTARGAYLLRD-PGERQVTLIATGSELELALAAADLLAAEGIAAAWSA
DHAS
PCTRLFDEQSIGYRRSVL-RKD-GRQVPTVVVDGHVAFGWER-YATASYCMNTYGK : : :: : : : :: : : : :
pTK2
PFTIKPLDRKLILD-SARARATKGRILTVE--D-HYYEGGIG-EAVSSAVVGEPGI : : : ::: :: : : :
TKLB
PCFELFAAQPADYRATVLGRA--PRV-GCEAALRQGWDLFLGPQ-DGFVGMTGFGA
DHAS
S LPP -EVI
pTK2
;VT.LAVNRVPRS;K-;
E-LL
::::
[203]
pTK2
-GYNPAT
:
[415]
ASRAQRRRNAAGYILEDAENAEVQI--IGVGAEMEFADKAAKIL-GRKFRTRVLSI : : : : :: :: : :: : ::
YEYF
::
[495]
THQPVESPALFRAYANIYYMRPVDSAEVFGLFQKAVELP-FSSILSLSRNEVLQYL :: ::: ::::: : : :: ::::::
•
:
,]
.
DHAS
:
IAKKVEAYVRACQRDP
;
T
LLLHRLP
......
. . . . . . . . . . . .
::
::: [310]
:
:
:: [579]
[656] :
::
:
:
:
:
:
[359] ::
:
[631]
GPEGKA
[702]
....... . . . . . . .
[ 6 5 7 ]
Figure 3. Alignment 0[ nentide seeuenees encoded bv the D H A S and T K L B ~enes with the derived amino acid seouence of clone nTK2. The complete sequence of pTK2 is aligned with the C-terminal 60% of the yeast DHAS sequence [14] and the TKLB sequence of R. sphaeroides [15]. Conserved residues or identities with a score of +1 or more on the Dayhoff similarity matrix [24] are indicated by a double-dot (:). The encoded pTK2 peptide has 24% identity with 40% similarity to the DHAS sequence of yeast, and 19% identity with 41% similarity to the TKLB sequence of R. sphaeroides. A highly conserved region at residues 483-511 in DHAS, 192-227 in pTK2 and 455-483 in TKLB (boxed) has a match with 6 of the 11 amino acids identified for the "~c~-fold fingerprint" of NAD-binding enzymes [27]. The consensus for each of the 11 essential amino acids is given below the alignment. Asterisks above the sequence indicate agreement with the consensus, where x may be K, R, H, S, T, Q or N; y may be A, I, L, V, M or C, and z is D or E. Numbers given in parentheses refer to the amino acid residues of each sequence relative to either the initiator methionine (for DHAS and TKLB) or the first residue in the available sequence (pTK2). 1164
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motif has not been identified in the sequence we publish here. However, there are several stretches that do in fact display a high level of similarity or positional identity between all three sequences. A highly conserved region at residues 483-511 in DHAS, 192-227 in pTK2 and 455-483 in TKLB, is indicated in Fig 3. This region contains residues at some of the key positions reported for the "~t~13-fold fingerprint" observed in NAD-binding enzymes [27]. The fingerprint describes the positions of 11 amino acids which are essential for nucleotide binding at the 13cry-fold. The aligned TK region given in Fig 3 has a match with 6 of the 11 required amino acids as shown and, although speculative, it should be considered that this highly conserved region may have some functional significance, such as nucleotide binding. This is interesting since TK actually has no requirement for nucleotide binding, and the conserved domain illustrated here may represent some evolutionary oversight. To our knowledge this is the first report of any mammalian TK sequence and should contribute to the search for putative TK variants associated with the WK syndrome.
Acknowledgments We are grateful to Jenny L. Cassidy for assistance in the RNA isolation. This work was supported by the National Health and Medical Research Council. M.A. is supported by a University of Queensland Postdoctoral fellowship.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19]
Kaczmarek, M. J. & Nixon, P. F. (1983). Clin. Chim. Acta. 130, 349-356. Nixon, P. F., Kaczmarek, M. J., Tate, J., Kerr, R. A. & Price, J. (1984). Europ. J. Clin. Invest. 14, 278-281. Paoletti, F., Mocali, A., Marchi, M., & Truschi, F. (1989). Biochem. Biophys. Res. Comm. 161, 150-155. Paoletti, F., Mocali, A., Marchi, M., Sorbi, S. & Piacentini, S. (1990). Biochem. Biophys. Res. Comm. 172, 396-401. Blass, J. P. & Gibson, G. E. (1977). N. England J. Med. 197, 1367-1370. Mukherjee, A. B., Svoronos, S., Ghazanfari, A., Martin, P. R., Fisher, A., Roecklein, B., Rodbard, D., Staton, R., Behar, D., Berg, C. J. & Manjunath, R. (1987). J. Clin. Invest. 79, 1039-1043. Sheu, K-F. R., Clarke, D. D., Kim, Y-T., Blass, J. P., Harding, B. J. & DeCicco, S. (1988). Arch. Neurol. 45, 841-845. Tate, J. R. & Nixon, P. F. (1987). Anal. Biochem. 160, 78-87, Blansjaar, B. A., Zwang, R. & Blijenberg, B. G. (1991). J. Neurol. Sciences. 106, 88-90. Kaufmann, A., Uhlhaas, S., Friedl, W. & Propping, P. (1987). Clin. Chim. Acta. 162, 215-219. Stryer, L. (1988). Biochemistry. (W.H. Freeman & Co., N. Y. Third edition ). pp.435. Kochetov, G. A. (1986). Biokhimiya. 51, 2020-2029. Takeuchi, T., Nishino, K. & Itokawa, Y. (1986). Biochim. Biophys. Acta. 872, 24-32. Janowicz, Z. A., Eckart, M. R., Drewke, C., Roggenkamp, R. O. & Hollenberg, C. P. (1985). Nucleic Acids Res. 13, 3043-3062. Chen, J-H., Gibson, J. L., McCue, L. A. & Tabita, F. R. (1991). J. Biol. Chem. 266, 20447-20452. Smeets, E. H. J., Muller, H. & De Wael, J. (1971). Clin. Chim. Acta. 33, 379-386. Lathe, R. (1985). J. Mol. Biol. 183, 1-12. Sambrook J., Fritsch E.F. and Maniatis T. (1989). Molecular Cloning, a Laboratory Manual. Cold Spring Harbour Laboratory (Second Edition). Manfioletti, G. & Schneider, C. (1988). Nucl. Acid Res. 16, 2873-2884. 1165
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[20] Sanger, F., Nicklen, S. & Coulson, A. R. (1977). Proc. Natl. Acad. Sci. USA. 74, 54635467. [21] Haltiner, M., Kempe, T. & Tjian, R. (1985). Nucl. Acids Res. 13, 1015. [22] Favaloro, J. ,Treisman, R. & Kamen, R. (1980). Methods Enzymol. 65, 718-749. [23] Feinberg, A. P. & Vogelstein, B. (1983). Anal. Biochem. 132, 6-13. [24] Dayhoff, M.O., Barker, W.C. & Hunt, L.T. (1983). Methods in Enzymology. 91, 524-545. [25] Zhang, B., Zhao, Y., Harris, R. A. & Crabb, D. W. (1991). Mol. Biol. Med. 8, 39-47. [26] Hawkins, C. F., Borges, A. & Perham, R. N. (1989). FEBS 255, 77-82. [27] Wierenga, R. K., Terpstra, P. & Hol, W. G. J. (1986). J. Mol. Biol. 187, 101-107.
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NUCLEOTIDE AND PREDICTED AMINO ACID SEQUENCE OF A cDNA CLONE ENCODING PART OF HUMAN T R A N S K E T O L A S E
Mahin Abedinia, Roy Layfield, Sheelagh M. Jones, Peter F. Nixon* and John S. Mattick Centre for Molecular Biology & Biotechnology, Department of Biochemistry, University of Queensland, Brisbane, QLD 4072 Australia Received February 12, 1992
Transketolase is a key enzyme in the pentose-phosphate pathway which has been implicated in the latent human genetic disease, Wemicke-Korsakoff syndrome. Here we report the cloning and partial characterisation of the coding sequences encoding human transketolase from a human brain cDNA library. The library was screened with oligonucleotide probes based on the amino acid sequence of proteolytic fragments of the purified protein. Northern blots showed that the transketolase mRNA is approximately 2.2 kb, close to the minimum expected, of which approximately 60% was represented in the largest cDNA clone. Sequence analysis of the transketolase coding sequences reveals a number of homologies with related enzymes from other species.
® 1992
Academic Press, Inc.
Transketolase (EC 2.2.1.1,TK) catalyses the transfer of a two-carbon ketol unit from xylulose 5-phosphate to an aldose acceptor, either ribose 5-phosphate or erythrose 5-phosphate. Along with transaldolase, TK provides the link between the pentose-phosphate pathway and the glycolytic pathway, enabling the recycling of pentose sugars under conditions where NADPH production is required for reductive biosynthesis. TK is also one of the few major enzymes which requires thiamin diphosphate (ThDP) as a cofactor, others being pyruvate dehydrogenase, 2oxoglutarate dehydrogenase and branched chain 2-oxoacid dehydrogenase. TK assays provide a sensitive measure of thiamin deficiency. There is evidence of heterogeneity of human erythrocyte, leucocyte and fibroblast TK by isoelectric focussing [1-4], as well as by variation in its affinity for ThDP [5, 6]. In Alzheimers disease, TK appears to be modified [4, 7] and a variant form of the enzyme, having decreased affinity for ThDP, has been implicated in the specific brain damage characterised in man as the Wernicke-Korsakoff (WK) syndrome [5, 6]. However, others [2, 8] have reported an identical affinity of TK for ThDP in this condition, and constancy of isoelectric focussing patterns [9,10]. *To whom correspondence should be addressed.
Abbreviations: BCKDH (branched chain ketoacid dehydrogenase); DHAS (dihydroxyacetone synthase); ThDP (thiamin diphosphate); TK (transketolase) and WK (Wernicke-Korsakoff). 0006-291X/92 $1.50 1159
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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The WK syndrome, which encompasses a characteristic acute encephalopathy followed by a chronic amnesia, is only observed following an episode of severe thiamin deficiency. However, not all thiamin-deficient individuals develop the WK syndrome, and on this basis the disorder is widely quoted as a latent genetic disease that is only revealed under particular environmental conditions, ie. malnutrition [11]. TK has been well characterised with respect to catalytic and chemical properties from yeast [for review see 12] but little is known about the mammalian enzyme other than that, as in yeast, it is a homodimer of subunits of approximately 70,000 molecular weight [13]. The gene encoding dihydroxyacetone synthase (DHAS), a novel TK from the formaldehyde-utilising yeast Hansenula
polymorpha [14] and the TK gene of Rhodobacter sphaeroides [15] have been cloned and sequenced. Here we present the cloning and partial sequence of the human TK cDNA.
Materials and Methods
Purification of human erythrocyte transketolase. TK was purified from blood supplied by the Brisbane Red Cross Blood Transfusion Service and collected from polycythemic or hemochromatotic patients. Erythrocytes were washed twice in 154 mM saline, filtered (Sepacel) to remove any residual leucocytes, suspended in an equal volume of water and lyzed by freezing and thawing. The hemolysate (500 ml) was dialyzed against 25 mM Tris-HC1 pH 8.5 (buffer A) until its conductivity matched that of the buffer, loaded onto a column of Blue Trisacryl M (I.B.F., France), 5 cm x 70 cm, equilibrated with buffer A. The matrix was washed with the same buffer to remove all unbound protein, then eluted by a linear gradient of sodium chloride from 0-500 mM in buffer A. TK was eluted at 70-100 mM NaC1, ahead of the major protein peak, and was dialyzed against 5 mM sodium phosphate pH 6.8 (buffer B) then adsorbed to hydroxylapatite (Biogel HT, Biorad) in a column 1 cm x 2 cm. Proteins were eluted by successive gradients of sodium chloride, first 5-300 mM then 300-750 mM in buffer B. TK was eluted early in the second gradient. Gel filtration in Biogel P4 (Biorad) was used to exchange the buffer to 10 mM Tris-HC1 pH 7.5. TK activities were assayed [16] by use of a Centrifichem 400 centrifugal analyzer.
Generation and purification of peptides from transketolase. Trypsin (Sigma) and endoproteinase Lys C (Boehringer Mannheim) were used to generate suitable peptides for sequencing. Peptides 1 and 2 were generated by incubation of TK in 10 mM Tris-HC1 pH 7.5 with trypsin at a ratio of 50:1 for either 15 rain at 25°C in the presence of 0.1% SDS or for 2hr at 25°C in the presence of 3M urea respectively. Peptide 3 was generated by incubation of TK in 10 mM Tris-HC1 pH 7.5, 0.1% SDS with endoproteinase Lys C at a ratio of 40:1 for 15 min at 25°C. The three peptides were analysed by 12.5% SDS/PAGE and transferred by electroblotting onto Immobilon membrane (Millipore) in 10 mM CAPS, 10% methanol pH 10.5 at 30 v for 16 h using a BioRad Mini Protean system. After staining with Coomassie Blue the relevant peptides were cut out of the membrane and directly sequenced. Amino acid sequences were analyzed by use of an Applied Biosystems 470A gas phase sequencer.
cDNA library screening. A human brain cDNA library constructed in kgtl 1 (Clontech) was screened for TK sequences using oligonucleotide probes that were designed by reference to the amino acid sequence of purified TK peptides 1 and 3, and biased towards human codon usage [17]. Oligonucleotide sequences were antisense, ie. complementary to the mRNA sequence. These were: Oligo 1 (corresponding to peptide 1) 5'-TGT GGC CAC GCC ATC AGA GGG GTA GAA GAC TGT AGA GGT GGG CAC AGA-3' and Oligo 3 (corresponding to peptide 3) 5'-GAT GAA GCA GAT GCC CTT GGT GTT GGC AGC CAG CTC CAC AGC-3'. All oligonucleotides were prepared on a Milligen 7500 DNA synthesizer. For hybridisations, oligonucleotides were labelled to a specific activity of 108 cprn/~g using T4 polynucleotide kinase and [U32p]-ATP [18]. A total of 500,000 pfu were screened and recombinant phage were transferred onto Hybond N, according to the suppliers instructions 1160
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(Amersham). Prehybridiization was carried out in 6 X SSC [18], 0.1% SDS, 5 X Denhardts solution [18] and 100 ~tg/ml denatured salmon sperm DNA at 42°C for 2 h. Hybridization, for 2 h at 50°C, was in the same buffer but contained 20 ~tg/ml yeast tRNA instead of cartier DNA and 0.5 pmol/ml end-labelled oligonucleotide probe. The stringency of washing was 6 X SSC, 0.1% SDS at 55°C. Recombinant phage giving positive signals were plaque purified and the cDNA insert size estimated using the polymerase chain reaction.
DNA sequence analysis. Lambda DNA for subcloning was prepared using the CTAB method [19]. cDNA inserts were subcloned into the phagemid pBS+/- (Stratagene) for restriction analysis and generation of smaller, overlapping clones. Sequence determination of both strands was performed using the Sanger dideoxy chain termination method [20] with modifications for the direct sequencing of doublestranded DNA [21]. Preparation of RNA and northern hybridization. RNA was prepared fl'om the human Burkitt's lymphoma cell line, B JAB, using Nonidet P-40 buffer (BDH) and protein~LseK/SDS according to Favaloro et al [22]. For Northern hybridisation, polyA+ RNA (1 ixg/lane) and total RNA (30 txg/lane) were denatured and electrophoresed through a formaldehyde denaturing gel (1.2% agarose) in MOPS buffer [16] at 5 V/cm for 3 h. After electrophoresis, RNA samples were transferred to Hybond N (Amersham), and prehybridised for 4 hrs at 42°C in 5 X SSPE [16], 50% formamide, 0.1% SDS, 5 X Denhardts solution and 100 ~tg/ml denatured salmon sperm DNA. Hybridisation, for 16 hrs at 42°C, was in the same buffer with a human TK cDNA probe l~Lbelledaccording to the method of Feinberg & Vogelstein [23], added to 106 cpm/ml. The stringency of washing was 2 X SSC, 0.1% SDS at 65°C.
Results and Discussion
Purification and partial amino acid sequence of Human Transketolase. TK purified from human erythrocytes migrated as a single band with a molecular weight of 69,000 in SDS-polyacrylamide gels (10 ~tg protein, silver stained, data not shown). Since attempts to obtain amino-terminal sequence were unsuccessful, suggesting that it is blocked, enzymatic proteolysis of the protein was carried out to generate peptides suitable for amino acid sequencing. The resultant sequences were: Peptide 1 SVPTSTVFYPSDGVAT, Peptide 2 ILATPPQE and
Peptide 3 AVELAANTKGICFI. Isolation and partial chanlcterisation of human TK cDNA A human brain cDNA library was screened with oligonucleotide probes (oligos 1 and 3) derived from the amino acid sequence of TK peptides 1 and 3 respectively, each biased toward human codon usage. Clonted sequences hybridizing to both probes were selected for further study, and the largest of these was subcloned into pBS+/-, restriction mapped and sequenced. The complete nucleotide and deduced amino acid sequence of this cDNA (clone pTK2), is given in Figure 1, with regions corresponding to the TK peptides 1, 2 and 3 underlined. Clone pTK2 is 1318 bp in length and has a translation stop codon at 1177 bp, leaving 141 nts of 3' untranslated sequence in which a polyA addition site occurs 20 nts upstream of the polyA tail. Homology of the nucleotide sequences enccxting peptides 1 and 3 to the corresponding probes used for the library screening was 77% for olilgo 1 and 81% for oligo 3. Northern hybridisation of mRNA purified from BJAB (human lymphocyte) cell lines with the cDNA insert of clone pTK2 as probe identified a strong band at ~2.2 kb (Figure 2), suggesting 1161
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GCTGT GCAAGGCCT T TGCGAGGCCAAGCACCAGCCAACAGCCATCAT A V Q G L C E A K H Q P T A GGC CGAGGGATCACGGGGGTAGAAGATAAGGAGT G R G I T G V E D K A TGGCT GAGCAGA M A E Q
E
I
I
TGCCAAGACCT A K T
C T TGGCAT GGGAAGCCCC S W H G K P
TCAAG F K
TCCCCAAAAAC L P K N
TCATCCAGGAGAT C TACAGCCAGAT C CAGAGCAAAAAGAAGATCC I I Q E I Y S Q I Q S K K K .I
T GGCA L A
63
126
189
ACCCCTCCACAGGAGGACGCACCCTCAGTGGACATTGCCAACATCCGCATGCCCAGCCTGCCC T P P Q E D A P S V D I A N I R M P S L P PEPTIDE 2 A GCTACAAAGT T GGGGACAAGATAGCCACCCGCAAGGCC TAC GGGCAGGCAC TGGCCAAGCTG S Y K V G D K I A T R K A Y G Q A L A K L
252
GGCCATGCCAGTGACCGCATCATCGCCCTGGATGGGGACACCAAAAATTCCACCTTCTCGGAG G H A S D R I I A L D G D T K N S T
378 F
S
315
E
ATCTTCAAAAAGGAGCACCCGGACCGCTTCATCGAGTGCTACAT I F K K E H P D R F I E C
I
TGCCGAGCAGAACATGGTG A E Q N M V
441
Y
AGCATCGCGGTGGGCTGTGCCACCCGCAACAGGACGGTGCCCT S I A V G C A T R N R T V
TCTGCAGCACTTT TGCAGCC F C S T F A A
504
P
T TCTTCACGCGGGCCT F F T R A
TTGACCAGAT TCGCATGGCCGCCATCTCCGAGAGCAACATCAACCTC F D Q I R M A A I S E S N I N
TGCGGCTCCCACTGCGGCGT C G S H C G CTGGCTATGT L A M
V
TTCCATCGGGGAAGACGGGCCCTCCCAGATGGCCCTAGAAGAT S I G E D G P S Q M A L
567 L 630
E
TTCGGTCAGTCCCCACATCAACCGTC F R S V P T S T
TTTTACCCAAGTGATGGCGTTGCTACA V F Y P S D G V A PEPTIDE 1 GAGAAGGCAGTGGAACTAGCCGCCAATACAAAGGGTATCTGT TTCATCCGGACCAGCCGCCCA E K A V E L A A N T K G I C F I R T S R PEPTIDE 3 GAAAATGCCATCATCTATAACAACAATGAGGACT TCCAGGTCGGACAAGCCAAGGTGGTCCTG E N A I I Y N N N E D F Q V G Q A K V V
D 693 T 756 P 819 L
AAGAGCAAGGATGACCAGGTGACCGTTATCGGGGCTGGGGTGACCCTGCACGAGGCCTTGGCC K S K D D Q V T V I G A G V T L H E
A
L
A
GCTGCCGAACTGCTGAAGAAAGAAAAGATCAACATCCGCGTGCTGGACCCCTTCACCATCAAG A A E L L K K E K I N I R V L D P F
T
I
K
TCTCGACAGCGCTCGTGCCACCAAGGGCAGGATCCTCACCGTG L D S A R A T K G R I L
T
V
TGGTGAGGCTGTGTCCAGTGCAGTAGTGGGCGAGCCT G E A V S S A V V G E
P
CCCCTGGACAGAAAACTCAT P L D R K L GAGGACCAT E D H
I
TATTATGAAGGTGGCAT Y Y E G G
I
882
945
GGCATCAC TGTCACCCACCTGGCAGTTAACCGGGTACCAAGAAGTGGGAAGCCAGCTGAGCTG G I T V T H L A V N R V P R S G K P A C TGAAGATGT L K M
T TGGTATCACAGGGATGCCATTGCACAAGCTGTGAGGGGCCTCATCACCAAGG F G I T G M P L H K L *
1008
1071
1134 E
L 1197
CCTAGGGCGGGTATGAAGTGTGGGGCGGGGGTCTCATACATTCCTGAGATTCTGGGAAAGGTG
1260
C T T CAAAGATG
1318
TAC T GAGAGGAGGGGTAAATATA TG T T T TGAGAAAAAAAAAAAAAAA polyA signal
Fil~ore 1. Nu¢leotide and deduced amino acid seauence of the human TK cDNA done. nTK2. The sequence represents the C-terminal 60% of the complete TK mRNA (see text) and is numbered relative to the first nucleotide (+1). The peptide sequences which are underlined have a perfect match with TK peptides 1, 2 and 3 which were sequenced from purified human TK. Also underlined in the nucleotide sequence is the polyA addition signal, 20 nts upstream of the polyA tail. The translation stop codon (TGA) is marked by an asterisk (*).
that the human TK gene transcribes a mRNA of ~2200 nt, consistent with the protein molecular weight of 69,000 (about 600 codons). This would indicate that clone pTK2 represents approximately 60% of the complete coding sequence, and since three peptides sequenced from purified human TK have a complete match with the translated peptide sequence of pTK2, occuring 1162
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I
2
3
4
--28S --18S
Figure 2. Northern blot analvsis of human TK mRNA. Total RNA samples (30 ~g each in lanes 1, 2 and 3) and polyA+ RNA (1 ~tg in lane 4), isolated from the human Burkitt's lymphoma cell line (BJAB), were probed with the human TK cDNA, pTK2. Hybridisation revealed a strong band at N2.2 kb.
in the same open reading frame as the stop codon indicated (Fig 1), this clone appears to represent the C-terminal portion of the complete TK sequence. A search of the GenBank database with the peptide sequence of pTK2 revealed extensive homology to two other sequences, dihydroxyacetone synthase (DHAS), a transketolase of unusual substrate specificity from the formaldehyde utilising yeast Hansenula polymorpha [14], and the recently cloned and sequenced transketolase of Rhodobacter sphaeroides, TKLB [15]. Dot plots of the amino acid sequences for DHAS or TKLB with that of pTK2 revealed a number of homologous regions. A more detailed alignment of the three sequences showed that there was 24% identity with 40% similarity between pTK2 and DHAS, and 19% identity with 41% similarity between pTK2 and TKLB (Figure 3). This correlates with the grouping of ThDP-dependent enzymes reported by Zhang et al [25] in which the yeast DHAS, the human TK and the Ell3 subunit of both human and P. putida BCKDH can be grouped into a class within which positional identities are found at 20-25% of residues and similarities at 40-45%. It is interesting to note that whilst the level of similarity between all three sequences is virtually the same, the highest level of positional identity with the human sequence is not with the TK of the photosynthetic prokaryote R. sphaeroides (TKLB), despite the identity of the many reactions catalysed by these two proteins. Rather, the greater positional identity is between the human sequence and that of the DHAS ofH. polymorpha, consistent with the eukaryotic origins of both but perhaps unexpected in view of the unique substrate specificity of DHAS, which catalyses the transfer of a glycoaldehyde group from xylulose 5-phosphate to formaldehyde [14], in addition to the more common TK reactions. The highly conserved region reported as a putative ThDP-binding motif, present in all thiamin-dependent enzymes sequenced so far [26], is not found in the sequence encoded by pTK2. In DHAS however, the proposed motif is present in the N-terminal portion of the sequence at residues 158 to 208 [26] and, the comparable region of TKLB (residues 147 to 197) has the same motif. Anticipating that the ThDP-binding motif of the human TK will also be present in the Nterminal -200 residues (based on the comparative alignment in Fig 3), it is not surprising that this 1163
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DHAS
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RELKIKYGMNPAQKFYIPQDVYDFFKEKPAEGDKLVAEWKS-LLVAKYVKAYPEEG : : : : : ::: : : :
[338] :
:
::
:
:
pTK2 AVQGLCEAKHQPTAI-IAKTFK---GRGIT-GVEDKESWHGKPL-PKNM-AEQIITKLB
AAARERLGWDHPP-FEIPADLYEAWGRIAARGADARAAWETRLLQASPLRA
.....
DHAS
QEFLARMRGELPKNWKSFLPQQEFTGDAPTRAAARELVRALGQNCK-SV-IA-GC:: : : : : :: :::: : : ::
pTK2
QE-IYSQIQSKKKILAT--PPQE---DAPSVDIANIRMPSL-PSYKVGDKIA-TRK :: : :: : :: :: ......
[48] [320]
[390] :
:
:
::
::
:
:
::
[96]
TKLB
AFETAEAADT
SALPPAIAAYKARL--SAE---APKVATRK-ASEMALGVV
DHAS
ADLSVSVNLQWPGVKY-FMDP-SL-STQCGL-SGDYSGRYIE-YGIREHAMCAIAN : :: :: : : : :: : : ::::::
[364]
[441] :
:
::
:
:::
pTK2 AYGQALAKLGHASDRIIALDGDTKNSTFSEIFKKEHPDRFIECY-IAEQNNVSIAV [151] TKLB
: ::1 : : : :: : :: : : 1 NEALPFAVGGSADLTGSNLTR-SKGM-VSVA-PGAFAGSYIH-YGIREHGM-AAAM
DHAS
-GLAAYNKGTFL-PITSTFFMFYLYAAPAIRMAGLQELKAIHIGTHDSINEGENGP
pTK2
-GCATRN-RT-V-PFCSTFAAFFTRAFDQIRMAAISESNINLCGSHCGVSIGEDGP : : : : :: : : : :: :::::: :
TKLB
NGIALHGG---LRPYGGTFMAFADYCRPSIRLSALMGVPVTYV~THDSIGLGEDGP
pTK2
SQMALEDLAMFRSVPTSTVFYPSDGVAT :: :::::::::::: :::
TKLB
THQPVEHLASLRAIPNLAVIRPAD~VETAEAWEIAMTATSTPTLLVLSRQNLPTVR
DHAS
y
y
--
loop
--
::
:::::::::
~yy
G G
[468]
G
.... :::::
:
[5501 :
:
:
:
EKAVELAANTKGICFIRTSRPENA : :: :: : : :
: [255] :
: [524]
zI
y
[603] ::
IIYNNNEDFQVGQAKVVLKSKDDQVTVIGAGVTLHEALAAAELLKKEKINIRVL-D ::: : : : : :::::::: :: : : : : : : : :
TKLB
TEHRDENLTARGAYLLRD-PGERQVTLIATGSELELALAAADLLAAEGIAAAWSA
DHAS
PCTRLFDEQSIGYRRSVL-RKD-GRQVPTVVVDGHVAFGWER-YATASYCMNTYGK : : :: : : : :: : : : :
pTK2
PFTIKPLDRKLILD-SARARATKGRILTVE--D-HYYEGGIG-EAVSSAVVGEPGI : : : ::: :: : : :
TKLB
PCFELFAAQPADYRATVLGRA--PRV-GCEAALRQGWDLFLGPQ-DGFVGMTGFGA
DHAS
S LPP -EVI
pTK2
;VT.LAVNRVPRS;K-;
E-LL
::::
[203]
pTK2
-GYNPAT
:
[415]
ASRAQRRRNAAGYILEDAENAEVQI--IGVGAEMEFADKAAKIL-GRKFRTRVLSI : : : : :: :: : :: : ::
YEYF
::
[495]
THQPVESPALFRAYANIYYMRPVDSAEVFGLFQKAVELP-FSSILSLSRNEVLQYL :: ::: ::::: : : :: ::::::
•
:
,]
.
DHAS
:
IAKKVEAYVRACQRDP
;
T
LLLHRLP
......
. . . . . . . . . . . .
::
::: [310]
:
:
:: [579]
[656] :
::
:
:
:
:
:
[359] ::
:
[631]
GPEGKA
[702]
....... . . . . . . .
[ 6 5 7 ]
Figure 3. Alignment 0[ nentide seeuenees encoded bv the D H A S and T K L B ~enes with the derived amino acid seouence of clone nTK2. The complete sequence of pTK2 is aligned with the C-terminal 60% of the yeast DHAS sequence [14] and the TKLB sequence of R. sphaeroides [15]. Conserved residues or identities with a score of +1 or more on the Dayhoff similarity matrix [24] are indicated by a double-dot (:). The encoded pTK2 peptide has 24% identity with 40% similarity to the DHAS sequence of yeast, and 19% identity with 41% similarity to the TKLB sequence of R. sphaeroides. A highly conserved region at residues 483-511 in DHAS, 192-227 in pTK2 and 455-483 in TKLB (boxed) has a match with 6 of the 11 amino acids identified for the "~c~-fold fingerprint" of NAD-binding enzymes [27]. The consensus for each of the 11 essential amino acids is given below the alignment. Asterisks above the sequence indicate agreement with the consensus, where x may be K, R, H, S, T, Q or N; y may be A, I, L, V, M or C, and z is D or E. Numbers given in parentheses refer to the amino acid residues of each sequence relative to either the initiator methionine (for DHAS and TKLB) or the first residue in the available sequence (pTK2). 1164
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motif has not been identified in the sequence we publish here. However, there are several stretches that do in fact display a high level of similarity or positional identity between all three sequences. A highly conserved region at residues 483-511 in DHAS, 192-227 in pTK2 and 455-483 in TKLB, is indicated in Fig 3. This region contains residues at some of the key positions reported for the "~t~13-fold fingerprint" observed in NAD-binding enzymes [27]. The fingerprint describes the positions of 11 amino acids which are essential for nucleotide binding at the 13cry-fold. The aligned TK region given in Fig 3 has a match with 6 of the 11 required amino acids as shown and, although speculative, it should be considered that this highly conserved region may have some functional significance, such as nucleotide binding. This is interesting since TK actually has no requirement for nucleotide binding, and the conserved domain illustrated here may represent some evolutionary oversight. To our knowledge this is the first report of any mammalian TK sequence and should contribute to the search for putative TK variants associated with the WK syndrome.
Acknowledgments We are grateful to Jenny L. Cassidy for assistance in the RNA isolation. This work was supported by the National Health and Medical Research Council. M.A. is supported by a University of Queensland Postdoctoral fellowship.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19]
Kaczmarek, M. J. & Nixon, P. F. (1983). Clin. Chim. Acta. 130, 349-356. Nixon, P. F., Kaczmarek, M. J., Tate, J., Kerr, R. A. & Price, J. (1984). Europ. J. Clin. Invest. 14, 278-281. Paoletti, F., Mocali, A., Marchi, M., & Truschi, F. (1989). Biochem. Biophys. Res. Comm. 161, 150-155. Paoletti, F., Mocali, A., Marchi, M., Sorbi, S. & Piacentini, S. (1990). Biochem. Biophys. Res. Comm. 172, 396-401. Blass, J. P. & Gibson, G. E. (1977). N. England J. Med. 197, 1367-1370. Mukherjee, A. B., Svoronos, S., Ghazanfari, A., Martin, P. R., Fisher, A., Roecklein, B., Rodbard, D., Staton, R., Behar, D., Berg, C. J. & Manjunath, R. (1987). J. Clin. Invest. 79, 1039-1043. Sheu, K-F. R., Clarke, D. D., Kim, Y-T., Blass, J. P., Harding, B. J. & DeCicco, S. (1988). Arch. Neurol. 45, 841-845. Tate, J. R. & Nixon, P. F. (1987). Anal. Biochem. 160, 78-87, Blansjaar, B. A., Zwang, R. & Blijenberg, B. G. (1991). J. Neurol. Sciences. 106, 88-90. Kaufmann, A., Uhlhaas, S., Friedl, W. & Propping, P. (1987). Clin. Chim. Acta. 162, 215-219. Stryer, L. (1988). Biochemistry. (W.H. Freeman & Co., N. Y. Third edition ). pp.435. Kochetov, G. A. (1986). Biokhimiya. 51, 2020-2029. Takeuchi, T., Nishino, K. & Itokawa, Y. (1986). Biochim. Biophys. Acta. 872, 24-32. Janowicz, Z. A., Eckart, M. R., Drewke, C., Roggenkamp, R. O. & Hollenberg, C. P. (1985). Nucleic Acids Res. 13, 3043-3062. Chen, J-H., Gibson, J. L., McCue, L. A. & Tabita, F. R. (1991). J. Biol. Chem. 266, 20447-20452. Smeets, E. H. J., Muller, H. & De Wael, J. (1971). Clin. Chim. Acta. 33, 379-386. Lathe, R. (1985). J. Mol. Biol. 183, 1-12. Sambrook J., Fritsch E.F. and Maniatis T. (1989). Molecular Cloning, a Laboratory Manual. Cold Spring Harbour Laboratory (Second Edition). Manfioletti, G. & Schneider, C. (1988). Nucl. Acid Res. 16, 2873-2884. 1165
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[20] Sanger, F., Nicklen, S. & Coulson, A. R. (1977). Proc. Natl. Acad. Sci. USA. 74, 54635467. [21] Haltiner, M., Kempe, T. & Tjian, R. (1985). Nucl. Acids Res. 13, 1015. [22] Favaloro, J. ,Treisman, R. & Kamen, R. (1980). Methods Enzymol. 65, 718-749. [23] Feinberg, A. P. & Vogelstein, B. (1983). Anal. Biochem. 132, 6-13. [24] Dayhoff, M.O., Barker, W.C. & Hunt, L.T. (1983). Methods in Enzymology. 91, 524-545. [25] Zhang, B., Zhao, Y., Harris, R. A. & Crabb, D. W. (1991). Mol. Biol. Med. 8, 39-47. [26] Hawkins, C. F., Borges, A. & Perham, R. N. (1989). FEBS 255, 77-82. [27] Wierenga, R. K., Terpstra, P. & Hol, W. G. J. (1986). J. Mol. Biol. 187, 101-107.
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