BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 180, No. 1, 1991
Pages 197-203
October 15, 1991
Isolation and sequence of a cDNA encoding human platelet phosphofructokinase ] ,2 Craig J. Simpson and Linda A. FothergilI-Gilmore Department of Biochemist~ University of Edinburgh George Square, Edinburgh EH8 9XD, Scotland Received August 26, 1991
A cDNA encoding human platelet 6-phosphofructokinase (PFK; EC 2.7.1.11) has been isolated from a human lymphocyte Raji cell line cDNA library using a cDNA for human muscle PFK as a probe. The platelet cDNA contains 900bp of carboxy terminal coding sequence and 238bp of downstream untranslated region. The deduced amino acid sequence shows 71% identity to the amino acid sequence for the human muscle isoenzyme and 63% identity to the human liver isoenzyme. Almost all of the amino acid residues contributing to catalytic and effector sites in the three isoenzymes are conserved. The platelet gene has been assigned to chromosome 10p15.2-p15.3 by using the cDNA clone as a biotinylated probe against human chromosome spreads (Morrison et al. 1991, submitted to Human Genetics). © 1991 Academic Press, I n c . Mammalian 6-phosphofructokinase (PFK) (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) is a key regulatory enzyme in glycolysis, (Bloxham & Lardy, 1973; Uyeda, 1979). It is a tetrameric enzyme of Mr ~ 340,000 (Dunaway, 1983) which catalyses the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate. There are three human isoenzymes of PFK encoded by the muscle, liver and platelet genes (Vora& Francke, 1981 ; Vora, 1982). The gene for the muscle enzyme has been sequenced (Nakajima eta/., 1987; Sharma eta/., 1989) and has been assigned to chromosome lp32-q32 (Vora, 1982) The gene encoding the liver enzyme has also been sequenced (Levanon et el., 1989; Elson et el., 1990) and has been assigned to chromosome 21q22.3 (van Keuren et el., 1986). The platelet gene has been assigned to chromosome 10p using immunological techniques (Vora et el., 1983) Another putative isoenzyme designated PFKX has been described and Iocalised to chromosome 12 (Ashley et al., 1987). The muscle, liver and platelet isoenzymes are differentially expressed during development and display distinct tissue specificities (Vote, 1982). ] This work was supported by a grant from the World Health Organisation, grant number 08/181/230. 2The nucleotide sequence presented here has been submitted to the EMBL/GENBANK database under the accession number M64784.
197
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc, All rights of reproduction in any form reserved.
Vol. 180, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Materials and Methods The cDNA clones for muscle and platelet PFK were isolated from a human lymphocyte cDNA library constructed from a Raji cell line. This library was a kind gift from Dr. Nigel Spurr (ICRF, Clare Hall, London). The cDNA for the muscle isoenzyme was isolated using a genomic fragment encoding human muscle PFK isolated in this laboratory. The cDNA for the muscle isoenzyme was then used as a probe to isolate the cDNA encoding platelet PFK. Approximately 5x10 s plaques from the library were screened by plaque hybridisation as described in Sambrook et al. (1989). Probe DNA was labelled by random priming (Feinberg & Vogelstein, 1984). The cDNA representing platelet PFK was subcloned into the plasmid pK19 and designated pCS11. Sau3AI restriction fragments derived from the insert in pCS11 were subcloned into the BamHI site of M13mp18 and M13mp19 for DNA sequencing. The insert was also digested with Smal to give two fragments which were subcloned into the Smal site of pK19 and Bluescript KS+ (Stratagene~). The various inserts were sequenced by the dideoxy chain termination technique (Sanger eta/., 1977). Single stranded M13 phage DNA was isolated by standard procedures (Sambrook et al., 1989), while plasmid DNA for double stranded DNA sequencing was isolated by the alkaline lysis method and purified by CsCI equilibrium density centrifugation (Sambrook et al., 1989). The chromosomal Iocalisation of pCS11 is described elsewhere (Morrison et al., 1991). The sequencing strategy for the platelet clone is shown in Figure 1.The first sixty bases of the clone were sequenced on one strand only. The remaining bases of coding region were sequenced on both strands. Results and Discussion The DNA sequence and inferred amino acid sequence of the platelet PFK clone is shown in Figure 2. The identification of this DNA as that encoding platelet PFK has been inferred from sequence similarities to other PFK sequences, and from its chromosomal location. An alignment of the three human sequences is shown in Figure 3. Eukaryotic PFK is proposed to have arisen as a result of gene duplication and fusion (Poorman et al., 1984) and shows similarities between the amino and carboxyl halves. The amino half is thought to retain catalytic and effector sites similar to the prokaryotic enzymes, while the carboxyl half has evolved to form novel effector sites not found in the smaller prokaryotic enzymes. Residues important for ligand binding in Bacillus stearothermophilus PFK and by implication in the human isoenzymes are shown in Table 1.These residues
E:'~'S'
:200
:400
:600
S" :800
:1000
'E:1200
Figure I . Sequencing strategy for the human platelet clone: E=EcoRI, S=Smal. The coding region extends from 1 to gOObp. The f i r s t 60bp were sequenced on one strand only.
198
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60 ATTCCr~TACTTGGAAGAGATCGCCAC~}ATGCGCACCAGCATCAACGCGCTGCTG I P K Y L E E I A T Q M R T S I N A L L 120 ATCATCGGT~TTCC~TACCTGGGACTCCTGG~ICTGT~ I I G G F E A Y L G L L E L S A A R E K 180 ~.GGAGTTC~CATGGTCATGGTT~ACTGTGTC~TGTGCCG H E E F C V P M V M V P A T V S N N V P 240 G
S
D
F
S
I
G
A
D
T
A
L
N
T
I
T
D
T
C
D
3O0 CGCATCAAGC~GTCCGCCAGCGGAACCA~GTGT R I K Q S A S G T K R R
TCATCATCGAC~CATGGGC V F I I E T M G 360 GGCTACTGTGGCT~CTGGCC~ATGGG~TCC~GCTGATGCCGCAT~ G Y C G Y L A N M G G L A A G A D A A Y 420 ATT T T C G ~ C C T TCGACATCAGGGATCTGCAGTCCA~X~GTGGAGCACCTGACGC~ I F E E P F D I R D L Q S N V E H L T E 480 ARAAT GA~.~C C~J~CATCCAGAGAGGCC T TGT GCT C~I~%ATGAGAGCT GC~JGTGAAAAC K M K T T I Q R G L V L R N E S C S E N 540 TACACCACCGACTTCATTTACC~CTGTA~TGTTTGACTGC Y T T D F I Y Q L Y S E E G K G V F D C 600 AGGA~TGGGTCACAT~CCTCTCCATTTGATAGAAAC R K N V L G H M O Q G G A P S P F D R N 660 TTTGGAACCAAAATCTCTGCCAGAGCTATGG~TGGATC~TGAA~TCA~C F G' T K I S A R A M E W I T E K L K E A 720 CGGGGCAGAGGA~TT TACCACCGATGAT TCCATTTGTGTGCTGGGAATAAGCAAA R G R G K K F T T D D S I C V L G I S K 780 AGAAACGTTAT T T T T C A R C C T G T G G C ~ J G A G C T G A A G ~ C G G A T T TTGAGCACAGG R N V I F Q P V A E L K K Q T D F E H R 840 ATTCCCAA~L%GTGGTGGCTCAAGCTACGGCCCCTCATGAAAATCCTGGCCAAGTAC I P K E Q W W L K L R P L M K I L A K Y 900 A~)GCC~CTATC~a~.~TGTCGGACTCAGGC~GGAACATGTGC~CCTGGAGTGTC K A S Y D V S D S G Q L E H V Q P W S V 960 tgaccoagtcccgcctgcatgtgcctgcagccaccgtggaotgtgtctgtttgtaacact taagttatt tt atcagcact t tatgcacgtat t attgacat tgaataoctaatcggcgag tguccat ctgccccaccagct cccagtgcgtgctgtctgtggagtgtgtotcatgcttt c agatgtgat atgagcagaatt aati~aaacatttgcct atgAn 2. Partia] D N A sequenced and i n f e r r e d a m i n o acid sequence of human platelet phosphofructokinase. The f i r s t residue of the sequence corresponds to residue 74 o f Bacillus stearothermophilus PFK. Figure
have been identified from crystal structure data for PFK from E. coil
stearothermophi/us
and B.
(Shirakihara & Evans, 1988; Rypniewski & Evans, 1989;
Schirmer & Evans, 1990), The platelet and muscle isoenzymes are more closely related to each other than to the liver isoenzyme, and can be considered to be equally distant from the liver form. The platelet amino acid residues retain 71% and 63% identity to the muscle and liver isoenzymes respectively, while the muscle and liver isoenzymes retain 64% identity. The platelet clone has been Iocalised to human chromosome 10p15.2-p15.3 in work presented elsewhere (Morrison et al., 1991). Human platelet PFK has previously been assigned to 10pter-p11.1 (Vora et al., 1983). The availability of this sequence should allow the isolation of a full length cDNA clone for the platelet isoenzyme and permit the detailed comparison of all three human isoenzymes. 199
V o l . 180, No. 1, 1991
Hb~FKN HLPFKN Hb~FKC HLPFKC
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
M T H E E H H A A K T L G I G K A I A V L T S G G O A Q G Z ~ A A V R A V V R V G IF T G A R V F F V H E G Y Q G L V D G M A A V D LEKLRAS G ~ K A I G V L T S G G D R Q G ~ N A A V R A V T R M G IYVGAKVF L I Y B G Y E G L V E G A V ~ V G A P A A G ~ e ~ A A V R S TVRI GLIQGNRVLVVHDGF~.GLAKG AI LNVGAPA~AAVRSAVRTG IS HGHTVYVVHDGTEGLAKG .°°, *. ,*******,,°* *° * * .°°*.°**,°* 46
HP~FKN HLPFKN Hb~FKC HLPFKC HPPFKC
G D H I ~Y~ATWE S V S ~ T V I GSARCEDFREREGRLRAAYNLVKRG ITNLCVlGGDGS L G ~ N I K Q A N W L S V S N I IQLGGTI IGSARS K A F T T R E G R R A A A Y N L V Q H G I T N L C V I G G D G S L --QIEEAGWSYVGGWTGQGGSKLGTKRT-- -LPKKSFEOI SANITKFNIQGLVI IGGFEAY - - Q V Q E V G W H D V A G W L G R G G S M L G T K R T - - - L P K G Q L E S IVEN I R I Y G I H A L L V V G G ~ E A Y I P K - Y L E E I A T Q M R T - S INALLI I G G F E A Y °.....* *° **, °** * °° ,* °* ,°** .. 106
H~FKN HLPFKN HP~FKC HLPFKC HPPFKC
TGADTFRSEWSDLLSDLQK~J3KITDEEATKS S Y L N I V G L V G S I D N D F C G T D M T I G T D S A L H TGANIFRSEWGS LLEELVAEGKI SETTAWTYSHLNI~LVGS ID~FCGTDMTIGTDSALH TGGLELMEGRKQF ................ DELCIPFVVIPATVSNNVPGSDFSVGADTALN E G V L Q L V E A R G R Y ................ E E L C I V M C V I P A T IS N V P G T D F SLGSDTAVN L G L L E L S A A R E K H . . . . . . . . . . . . . . . . E E F C V P M V M V P A T V S N N V P G S D F S IG A D T A L N • • , ° °.,.*. *.*°°.*o*,*,, 144
H~FKN HLPFKN HMPFKC HLPFKC HPPFKC
RIME IVDAIT-TTAQSHQRTFVLEVMGRHCGYLALVTSLSCGADWVF IPECPPDDDWEEHL R I M E V I D A I T -T T A Q S H Q R T F V L E V M G R H C G Y L A L V S A L A S G A D W L F I PEAP P E D G W E N F M T ICTTCDRIKQSAAGTKRRVF IIETMGGYCGYLATMAGLAAGADAAYIFEEPFT IRDLQAN A A M E S C D R I K Q S A S G T K R R V F I V E T M G G Y C G Y L A T V T G I A V G A D A A Y V F E D P FN I H D L K V N T I T D T C D R I K Q S A S G T K R R V F I I E T M G G Y C G Y L A N M G G L A ~ = A D A A Y I FEEP FD I R D L Q S N • *, °., ,..*.*.,*°** °***** ...,, *** .. * *
Hb~PFKN HLPFKN H~FKC HLPFKC HPPFKC
C R R L S~.TRT~S R L N I I I V A E G A I D K N G ~ I T SED I K N L V V K - - R L G Y D T R V T V L G H V Q R G C E R L G E T R S R G S R L N I II I A E G A I D R N G E P I S S S Y V E D L V V Q - - R L G F D T R V T V L G H V Q ~ V E H L V Q K M K T T V E R G L V L R N E K C N ~ N .... YT TDF I F N L Y SEEGKG I F D S RKNVLGFR4QQG V E H M T E K M K T D I Q R G L V L R N E K C H D Y .... Y T T E F L Y N L Y S S E G K G V F D C R T N V L G H L Q Q G V E H L T E E M K T T I Q R G L V L R N E S C S ~ .... YT TDF I Y Q L Y S E E G K G V F D C R K N V L G ~ 4 Q Q G
HMPFKN HLPFKN HP~FKC HLPFKC HPPFKC
GTP SAFDRI LG S R M G V E A V M A L L E . . . . . . . . . . . . . G T P D T P A C V V S LS G N Q A V R R L P L M GTP S A F D R I L S S K~F.44EAVMALLE . . . . . . . . . . . . . A T P D T P A C V V T L S G N Q S V R R L P L M GSP T P F D R N F A T E M ~ S G K I K E SYRNGRIFANTPDS - ~ V F F Q P V A GAP TPFD R N Y G T K L G V K A M L W L S E K L R E V Y R K G R V F A N A P D S - A C V I G L K K K A V A F F S P V T GAP S P F D R N F G T K I SARAMEWI T E ~ G R G K K F T - T D D S - ICVLG ISKRNVIFFQPVA
253
• "*''*** HbPFKN HLPFKN Hb~FKC HLPFKC HPPFKC
.....
*"
" "
*"
** . . . . . . .
;92
ECVQVTKDVTKA~DEK---KFDEALKLP~3RSFbSqNWEVYKL--LAHVRPPVSKSGSHTV ECVQMTKEVQKAb~)DK- - - R F D E A T Q L R G G S FEI~NWN I Y K L - - L T H Q K P P K E K S N F S L ELKDQTDFEHRIPKEQWWLKLRP ILKILAK-YE TDLDTSDHAHLEH IT R K R S G E A A V ELKKDTDFEHRMPREQWWLS LRLML~ -YRI S M A A Y V S G E L E H V T R R T LSMDGG~ E L K K Q T D F E H R I P K E Q W W L K L R P T ~4KILAK-YKASYDVSD S G Q L E H V Q P W S V 319
Figure 3. Alignment o f the human phosphofructokinases. • - identity - similarity N - AMINO HALF
~FI( - BE~EN MUSCLE PFK HIJPFK -- H E ~ % N L I V E R P F K H P P F K - HEI4AN P L A T E L E T P F K Nu~Ei ~ is a ¢ ¢ o r d l n g to B.stearothezatophilus
PFK.
Variable expression of the liver, muscle and platelet genes leads to the formation of homo-and heterotetrameric forms of the enzyme with different catalytic and regulatory properties. The least variation in subunit composition occurs in postnatal skeletal muscle which only contains muscle-type subunits. Placenta has predominantly liver-type subunits whereas platelets, brain and fibroblasts have a higher proportion of platelet type subunits (Dunaway et al., 1988; Dunaway & Kasten, 1989). 200
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Table 1. Ligand binding residues from Bacillus PFK (Schirmer & Evans, 1990) compared to equivalent residues from human PFK Residue number
BS
HMN
HLN
HMC
HLC
HPC
A. catalytic site binding F6P and ATP (probably not in carboxyl half) 11 Gly Gly Gly Gly Gly 72 Arg Arg Arg Arg Arg 73 Cys Cys Set Thr Thr 103 Asp Asp Asp Phe Phe Phe 104 Gly Gly Gly Glu Glu Giu 105 Ser Ser Set Ala Ala Ala 125 Thr Ser Ser Thr Thr Thr 127 Asp Asp Asp Set Ser Ser 162 Arg Arg Arg Arg Arg Arg 243 Arg Arg Arg Arg Arg Arg 252 Arg Arg Arg Gin Gin Gin ADP effector site (probably ATP inhibition site in carboxyl half of mammalian PFK) 21 Arg Arg Arg Arg Arg Arg 25 Arg Arg Arg Arg Arg Arg 57 Val Val Val Val Val Val 154 Arg Thr Thr Lys Lys Lys 187 Glu Asp Asp Asp Asp Asp 211 Arg Thr Thr Lys Lys Lys 213 Lys Lys Lys Lys Lys Lys 214 Lys Lys Lys Lys Lys Lys 215 His His His His His His Key.N = amino half. C = carboxyl half. BS-Bacillus stearothermophilus PFK. HM = human muscle PFK HL = human liver PFK HP = human platelet PFK
The muscle homotetramer has a higher affinity for fructose 6-phosphate than liver rich isoenzymes from the placenta, kidneys and liver. The presence of high levels of platelet-type subunits in fibroblasts promotes even lower affinities for fructose 6-phosphate. Similarly, muscle homotetramers are less susceptible to inhibition by ATP than isoenzymes rich in liver-type subunits and ATP inhibition becomes more pronounced as the contribution of platelet-type subunit increases in an isoenzyme pool (Dunaway et al., 1988). It has been suggested that the muscletype subunit will predominate in tissues such as skeletal muscle, heart or brain with 201
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a high rate of glycolysis, whereas the liver-type will predominate in tissues such as liver and kidney where gluconeogenesis is active (Vora, 1982). Dunaway et al. (1988) suggest that the platelet-type isoenzyme has an intrinsic enhanced sensitivity to inhibition by ATP. Coupled with a decreased affinity for fructose 6phosphate, the presence of this isoenzyme could exert a dampening influence on glycolysis and so protect a tissue from excessive lactate accumulation or allow other tissues which are more immediately important for survival to compete more effectively for available glucose.
Acknowledgments: The authors thank Dr. Nigel Spurr and Mr. Paul Hunt for providing materials that made this study possible.
References
Ashley, P.L., Price, E.R., Lebo, R.V. & Cox, D.R. (1987) Cytogenet. Cell. Genet. HGM9. Bloxham, D.R & Lardy, H.A. (1973) in The Enzymes (Boyer, P.D. ed.) 3rd. edition, vol. 13, pp. 239-278. Academic Press, London. Dunaway, G:A. (1983) Mol. Cell. Biochem. 52, 75-91. Dunaway, G.A., Kasten, T.R, Sebo, T. & Trapp, R. (1988) Biochem. J. 251,677-683. Dunaway, G.A. & Kasten,T.R (1989) Mol. Cell. Biochem. 87, 71-77. Elson, A., Levanon, D., Brandeis, M., Dafni, N., Bernstein, Y., Danciger, E. & Groner, Y. (1990) Genomics 7, 47-56. Feinberg, A.P. & Vogelstein, B. (1984)Anal. Biochem. 137, 266-267. Levanon, D., Danciger, E., Dafni, N., Bernstein, Y., Elson, A., Moens, W., Brandeis, M. & Groner, Y (1989) DNA 8, 733-743. Morrison, N., Simpson, C.J., FothergilI-Gilmore, L.A., Boyd, E. & Connor, J.M. (1991) Human Genetics, submitted for publication. Nakajima, H., Noguchi, T., Yamasaki, T., Kono, N., Tanaka, T. & Tarui, S. (1987) FEBS Lett. 223, 113-116. Poorman, R.A., Randolph, A., Kemp, R.G. & Heinrikson, R.L. (1984) Nature 309, 467-469. Rypniewski, W.R. & Evans, RR. (1989) J. Mol. Biol. 207, 805-821. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: A laboratory manual, 2nd edition, Cold Spring Harbour Press. 2O2
Vol. 18o, No. 1, 1 9 9 1
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Sanger, F.,Nicklen, S. & Coulson, A.R. (1977) Proc. Natl. Acad. Sci. 74, 5463-5467. Schirmer, T. & Evans, P.R. (1990) Nature 343, 140-145. Sharma, P.M., Reddy, G.R., Vora, S., Babior, B.B. & McLachlan, A. (1989) Gene 77, 177-183. Shirakihara, Y. & Evans, P.R. (1988) J. Mol. Biol. 204, 973-994. Uyeda, K. (1979) Adv. Enzymol. 48, 193-244. van Keuren, M., Drabkin, H., Hart, I., Harker, D., Patterson, D. & Vora, S. (1986) Human Genetics, 74, 34-40. Vora, S. & Francke, U. (1981) Proc. Natl. Acad. Sci. 78, 3738-3742. Vora, S. (1982) in Isozymes: Current topics in Biological and Medical Research, 6, pp. 119-167, (Alan R. Liss, Inc., New York). Vora, S., Miranda, A. & Francke, U. (1983) Hum. Genet.. 63, 374-379
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Vol. 180, No. 1, 1991
Pages 197-203
October 15, 1991
Isolation and sequence of a cDNA encoding human platelet phosphofructokinase ] ,2 Craig J. Simpson and Linda A. FothergilI-Gilmore Department of Biochemist~ University of Edinburgh George Square, Edinburgh EH8 9XD, Scotland Received August 26, 1991
A cDNA encoding human platelet 6-phosphofructokinase (PFK; EC 2.7.1.11) has been isolated from a human lymphocyte Raji cell line cDNA library using a cDNA for human muscle PFK as a probe. The platelet cDNA contains 900bp of carboxy terminal coding sequence and 238bp of downstream untranslated region. The deduced amino acid sequence shows 71% identity to the amino acid sequence for the human muscle isoenzyme and 63% identity to the human liver isoenzyme. Almost all of the amino acid residues contributing to catalytic and effector sites in the three isoenzymes are conserved. The platelet gene has been assigned to chromosome 10p15.2-p15.3 by using the cDNA clone as a biotinylated probe against human chromosome spreads (Morrison et al. 1991, submitted to Human Genetics). © 1991 Academic Press, I n c . Mammalian 6-phosphofructokinase (PFK) (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) is a key regulatory enzyme in glycolysis, (Bloxham & Lardy, 1973; Uyeda, 1979). It is a tetrameric enzyme of Mr ~ 340,000 (Dunaway, 1983) which catalyses the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate. There are three human isoenzymes of PFK encoded by the muscle, liver and platelet genes (Vora& Francke, 1981 ; Vora, 1982). The gene for the muscle enzyme has been sequenced (Nakajima eta/., 1987; Sharma eta/., 1989) and has been assigned to chromosome lp32-q32 (Vora, 1982) The gene encoding the liver enzyme has also been sequenced (Levanon et el., 1989; Elson et el., 1990) and has been assigned to chromosome 21q22.3 (van Keuren et el., 1986). The platelet gene has been assigned to chromosome 10p using immunological techniques (Vora et el., 1983) Another putative isoenzyme designated PFKX has been described and Iocalised to chromosome 12 (Ashley et al., 1987). The muscle, liver and platelet isoenzymes are differentially expressed during development and display distinct tissue specificities (Vote, 1982). ] This work was supported by a grant from the World Health Organisation, grant number 08/181/230. 2The nucleotide sequence presented here has been submitted to the EMBL/GENBANK database under the accession number M64784.
197
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc, All rights of reproduction in any form reserved.
Vol. 180, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Materials and Methods The cDNA clones for muscle and platelet PFK were isolated from a human lymphocyte cDNA library constructed from a Raji cell line. This library was a kind gift from Dr. Nigel Spurr (ICRF, Clare Hall, London). The cDNA for the muscle isoenzyme was isolated using a genomic fragment encoding human muscle PFK isolated in this laboratory. The cDNA for the muscle isoenzyme was then used as a probe to isolate the cDNA encoding platelet PFK. Approximately 5x10 s plaques from the library were screened by plaque hybridisation as described in Sambrook et al. (1989). Probe DNA was labelled by random priming (Feinberg & Vogelstein, 1984). The cDNA representing platelet PFK was subcloned into the plasmid pK19 and designated pCS11. Sau3AI restriction fragments derived from the insert in pCS11 were subcloned into the BamHI site of M13mp18 and M13mp19 for DNA sequencing. The insert was also digested with Smal to give two fragments which were subcloned into the Smal site of pK19 and Bluescript KS+ (Stratagene~). The various inserts were sequenced by the dideoxy chain termination technique (Sanger eta/., 1977). Single stranded M13 phage DNA was isolated by standard procedures (Sambrook et al., 1989), while plasmid DNA for double stranded DNA sequencing was isolated by the alkaline lysis method and purified by CsCI equilibrium density centrifugation (Sambrook et al., 1989). The chromosomal Iocalisation of pCS11 is described elsewhere (Morrison et al., 1991). The sequencing strategy for the platelet clone is shown in Figure 1.The first sixty bases of the clone were sequenced on one strand only. The remaining bases of coding region were sequenced on both strands. Results and Discussion The DNA sequence and inferred amino acid sequence of the platelet PFK clone is shown in Figure 2. The identification of this DNA as that encoding platelet PFK has been inferred from sequence similarities to other PFK sequences, and from its chromosomal location. An alignment of the three human sequences is shown in Figure 3. Eukaryotic PFK is proposed to have arisen as a result of gene duplication and fusion (Poorman et al., 1984) and shows similarities between the amino and carboxyl halves. The amino half is thought to retain catalytic and effector sites similar to the prokaryotic enzymes, while the carboxyl half has evolved to form novel effector sites not found in the smaller prokaryotic enzymes. Residues important for ligand binding in Bacillus stearothermophilus PFK and by implication in the human isoenzymes are shown in Table 1.These residues
E:'~'S'
:200
:400
:600
S" :800
:1000
'E:1200
Figure I . Sequencing strategy for the human platelet clone: E=EcoRI, S=Smal. The coding region extends from 1 to gOObp. The f i r s t 60bp were sequenced on one strand only.
198
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60 ATTCCr~TACTTGGAAGAGATCGCCAC~}ATGCGCACCAGCATCAACGCGCTGCTG I P K Y L E E I A T Q M R T S I N A L L 120 ATCATCGGT~TTCC~TACCTGGGACTCCTGG~ICTGT~ I I G G F E A Y L G L L E L S A A R E K 180 ~.GGAGTTC~CATGGTCATGGTT~ACTGTGTC~TGTGCCG H E E F C V P M V M V P A T V S N N V P 240 G
S
D
F
S
I
G
A
D
T
A
L
N
T
I
T
D
T
C
D
3O0 CGCATCAAGC~GTCCGCCAGCGGAACCA~GTGT R I K Q S A S G T K R R
TCATCATCGAC~CATGGGC V F I I E T M G 360 GGCTACTGTGGCT~CTGGCC~ATGGG~TCC~GCTGATGCCGCAT~ G Y C G Y L A N M G G L A A G A D A A Y 420 ATT T T C G ~ C C T TCGACATCAGGGATCTGCAGTCCA~X~GTGGAGCACCTGACGC~ I F E E P F D I R D L Q S N V E H L T E 480 ARAAT GA~.~C C~J~CATCCAGAGAGGCC T TGT GCT C~I~%ATGAGAGCT GC~JGTGAAAAC K M K T T I Q R G L V L R N E S C S E N 540 TACACCACCGACTTCATTTACC~CTGTA~TGTTTGACTGC Y T T D F I Y Q L Y S E E G K G V F D C 600 AGGA~TGGGTCACAT~CCTCTCCATTTGATAGAAAC R K N V L G H M O Q G G A P S P F D R N 660 TTTGGAACCAAAATCTCTGCCAGAGCTATGG~TGGATC~TGAA~TCA~C F G' T K I S A R A M E W I T E K L K E A 720 CGGGGCAGAGGA~TT TACCACCGATGAT TCCATTTGTGTGCTGGGAATAAGCAAA R G R G K K F T T D D S I C V L G I S K 780 AGAAACGTTAT T T T T C A R C C T G T G G C ~ J G A G C T G A A G ~ C G G A T T TTGAGCACAGG R N V I F Q P V A E L K K Q T D F E H R 840 ATTCCCAA~L%GTGGTGGCTCAAGCTACGGCCCCTCATGAAAATCCTGGCCAAGTAC I P K E Q W W L K L R P L M K I L A K Y 900 A~)GCC~CTATC~a~.~TGTCGGACTCAGGC~GGAACATGTGC~CCTGGAGTGTC K A S Y D V S D S G Q L E H V Q P W S V 960 tgaccoagtcccgcctgcatgtgcctgcagccaccgtggaotgtgtctgtttgtaacact taagttatt tt atcagcact t tatgcacgtat t attgacat tgaataoctaatcggcgag tguccat ctgccccaccagct cccagtgcgtgctgtctgtggagtgtgtotcatgcttt c agatgtgat atgagcagaatt aati~aaacatttgcct atgAn 2. Partia] D N A sequenced and i n f e r r e d a m i n o acid sequence of human platelet phosphofructokinase. The f i r s t residue of the sequence corresponds to residue 74 o f Bacillus stearothermophilus PFK. Figure
have been identified from crystal structure data for PFK from E. coil
stearothermophi/us
and B.
(Shirakihara & Evans, 1988; Rypniewski & Evans, 1989;
Schirmer & Evans, 1990), The platelet and muscle isoenzymes are more closely related to each other than to the liver isoenzyme, and can be considered to be equally distant from the liver form. The platelet amino acid residues retain 71% and 63% identity to the muscle and liver isoenzymes respectively, while the muscle and liver isoenzymes retain 64% identity. The platelet clone has been Iocalised to human chromosome 10p15.2-p15.3 in work presented elsewhere (Morrison et al., 1991). Human platelet PFK has previously been assigned to 10pter-p11.1 (Vora et al., 1983). The availability of this sequence should allow the isolation of a full length cDNA clone for the platelet isoenzyme and permit the detailed comparison of all three human isoenzymes. 199
V o l . 180, No. 1, 1991
Hb~FKN HLPFKN Hb~FKC HLPFKC
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
M T H E E H H A A K T L G I G K A I A V L T S G G O A Q G Z ~ A A V R A V V R V G IF T G A R V F F V H E G Y Q G L V D G M A A V D LEKLRAS G ~ K A I G V L T S G G D R Q G ~ N A A V R A V T R M G IYVGAKVF L I Y B G Y E G L V E G A V ~ V G A P A A G ~ e ~ A A V R S TVRI GLIQGNRVLVVHDGF~.GLAKG AI LNVGAPA~AAVRSAVRTG IS HGHTVYVVHDGTEGLAKG .°°, *. ,*******,,°* *° * * .°°*.°**,°* 46
HP~FKN HLPFKN Hb~FKC HLPFKC HPPFKC
G D H I ~Y~ATWE S V S ~ T V I GSARCEDFREREGRLRAAYNLVKRG ITNLCVlGGDGS L G ~ N I K Q A N W L S V S N I IQLGGTI IGSARS K A F T T R E G R R A A A Y N L V Q H G I T N L C V I G G D G S L --QIEEAGWSYVGGWTGQGGSKLGTKRT-- -LPKKSFEOI SANITKFNIQGLVI IGGFEAY - - Q V Q E V G W H D V A G W L G R G G S M L G T K R T - - - L P K G Q L E S IVEN I R I Y G I H A L L V V G G ~ E A Y I P K - Y L E E I A T Q M R T - S INALLI I G G F E A Y °.....* *° **, °** * °° ,* °* ,°** .. 106
H~FKN HLPFKN HP~FKC HLPFKC HPPFKC
TGADTFRSEWSDLLSDLQK~J3KITDEEATKS S Y L N I V G L V G S I D N D F C G T D M T I G T D S A L H TGANIFRSEWGS LLEELVAEGKI SETTAWTYSHLNI~LVGS ID~FCGTDMTIGTDSALH TGGLELMEGRKQF ................ DELCIPFVVIPATVSNNVPGSDFSVGADTALN E G V L Q L V E A R G R Y ................ E E L C I V M C V I P A T IS N V P G T D F SLGSDTAVN L G L L E L S A A R E K H . . . . . . . . . . . . . . . . E E F C V P M V M V P A T V S N N V P G S D F S IG A D T A L N • • , ° °.,.*. *.*°°.*o*,*,, 144
H~FKN HLPFKN HMPFKC HLPFKC HPPFKC
RIME IVDAIT-TTAQSHQRTFVLEVMGRHCGYLALVTSLSCGADWVF IPECPPDDDWEEHL R I M E V I D A I T -T T A Q S H Q R T F V L E V M G R H C G Y L A L V S A L A S G A D W L F I PEAP P E D G W E N F M T ICTTCDRIKQSAAGTKRRVF IIETMGGYCGYLATMAGLAAGADAAYIFEEPFT IRDLQAN A A M E S C D R I K Q S A S G T K R R V F I V E T M G G Y C G Y L A T V T G I A V G A D A A Y V F E D P FN I H D L K V N T I T D T C D R I K Q S A S G T K R R V F I I E T M G G Y C G Y L A N M G G L A ~ = A D A A Y I FEEP FD I R D L Q S N • *, °., ,..*.*.,*°** °***** ...,, *** .. * *
Hb~PFKN HLPFKN H~FKC HLPFKC HPPFKC
C R R L S~.TRT~S R L N I I I V A E G A I D K N G ~ I T SED I K N L V V K - - R L G Y D T R V T V L G H V Q R G C E R L G E T R S R G S R L N I II I A E G A I D R N G E P I S S S Y V E D L V V Q - - R L G F D T R V T V L G H V Q ~ V E H L V Q K M K T T V E R G L V L R N E K C N ~ N .... YT TDF I F N L Y SEEGKG I F D S RKNVLGFR4QQG V E H M T E K M K T D I Q R G L V L R N E K C H D Y .... Y T T E F L Y N L Y S S E G K G V F D C R T N V L G H L Q Q G V E H L T E E M K T T I Q R G L V L R N E S C S ~ .... YT TDF I Y Q L Y S E E G K G V F D C R K N V L G ~ 4 Q Q G
HMPFKN HLPFKN HP~FKC HLPFKC HPPFKC
GTP SAFDRI LG S R M G V E A V M A L L E . . . . . . . . . . . . . G T P D T P A C V V S LS G N Q A V R R L P L M GTP S A F D R I L S S K~F.44EAVMALLE . . . . . . . . . . . . . A T P D T P A C V V T L S G N Q S V R R L P L M GSP T P F D R N F A T E M ~ S G K I K E SYRNGRIFANTPDS - ~ V F F Q P V A GAP TPFD R N Y G T K L G V K A M L W L S E K L R E V Y R K G R V F A N A P D S - A C V I G L K K K A V A F F S P V T GAP S P F D R N F G T K I SARAMEWI T E ~ G R G K K F T - T D D S - ICVLG ISKRNVIFFQPVA
253
• "*''*** HbPFKN HLPFKN Hb~FKC HLPFKC HPPFKC
.....
*"
" "
*"
** . . . . . . .
;92
ECVQVTKDVTKA~DEK---KFDEALKLP~3RSFbSqNWEVYKL--LAHVRPPVSKSGSHTV ECVQMTKEVQKAb~)DK- - - R F D E A T Q L R G G S FEI~NWN I Y K L - - L T H Q K P P K E K S N F S L ELKDQTDFEHRIPKEQWWLKLRP ILKILAK-YE TDLDTSDHAHLEH IT R K R S G E A A V ELKKDTDFEHRMPREQWWLS LRLML~ -YRI S M A A Y V S G E L E H V T R R T LSMDGG~ E L K K Q T D F E H R I P K E Q W W L K L R P T ~4KILAK-YKASYDVSD S G Q L E H V Q P W S V 319
Figure 3. Alignment o f the human phosphofructokinases. • - identity - similarity N - AMINO HALF
~FI( - BE~EN MUSCLE PFK HIJPFK -- H E ~ % N L I V E R P F K H P P F K - HEI4AN P L A T E L E T P F K Nu~Ei ~ is a ¢ ¢ o r d l n g to B.stearothezatophilus
PFK.
Variable expression of the liver, muscle and platelet genes leads to the formation of homo-and heterotetrameric forms of the enzyme with different catalytic and regulatory properties. The least variation in subunit composition occurs in postnatal skeletal muscle which only contains muscle-type subunits. Placenta has predominantly liver-type subunits whereas platelets, brain and fibroblasts have a higher proportion of platelet type subunits (Dunaway et al., 1988; Dunaway & Kasten, 1989). 200
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Table 1. Ligand binding residues from Bacillus PFK (Schirmer & Evans, 1990) compared to equivalent residues from human PFK Residue number
BS
HMN
HLN
HMC
HLC
HPC
A. catalytic site binding F6P and ATP (probably not in carboxyl half) 11 Gly Gly Gly Gly Gly 72 Arg Arg Arg Arg Arg 73 Cys Cys Set Thr Thr 103 Asp Asp Asp Phe Phe Phe 104 Gly Gly Gly Glu Glu Giu 105 Ser Ser Set Ala Ala Ala 125 Thr Ser Ser Thr Thr Thr 127 Asp Asp Asp Set Ser Ser 162 Arg Arg Arg Arg Arg Arg 243 Arg Arg Arg Arg Arg Arg 252 Arg Arg Arg Gin Gin Gin ADP effector site (probably ATP inhibition site in carboxyl half of mammalian PFK) 21 Arg Arg Arg Arg Arg Arg 25 Arg Arg Arg Arg Arg Arg 57 Val Val Val Val Val Val 154 Arg Thr Thr Lys Lys Lys 187 Glu Asp Asp Asp Asp Asp 211 Arg Thr Thr Lys Lys Lys 213 Lys Lys Lys Lys Lys Lys 214 Lys Lys Lys Lys Lys Lys 215 His His His His His His Key.N = amino half. C = carboxyl half. BS-Bacillus stearothermophilus PFK. HM = human muscle PFK HL = human liver PFK HP = human platelet PFK
The muscle homotetramer has a higher affinity for fructose 6-phosphate than liver rich isoenzymes from the placenta, kidneys and liver. The presence of high levels of platelet-type subunits in fibroblasts promotes even lower affinities for fructose 6-phosphate. Similarly, muscle homotetramers are less susceptible to inhibition by ATP than isoenzymes rich in liver-type subunits and ATP inhibition becomes more pronounced as the contribution of platelet-type subunit increases in an isoenzyme pool (Dunaway et al., 1988). It has been suggested that the muscletype subunit will predominate in tissues such as skeletal muscle, heart or brain with 201
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a high rate of glycolysis, whereas the liver-type will predominate in tissues such as liver and kidney where gluconeogenesis is active (Vora, 1982). Dunaway et al. (1988) suggest that the platelet-type isoenzyme has an intrinsic enhanced sensitivity to inhibition by ATP. Coupled with a decreased affinity for fructose 6phosphate, the presence of this isoenzyme could exert a dampening influence on glycolysis and so protect a tissue from excessive lactate accumulation or allow other tissues which are more immediately important for survival to compete more effectively for available glucose.
Acknowledgments: The authors thank Dr. Nigel Spurr and Mr. Paul Hunt for providing materials that made this study possible.
References
Ashley, P.L., Price, E.R., Lebo, R.V. & Cox, D.R. (1987) Cytogenet. Cell. Genet. HGM9. Bloxham, D.R & Lardy, H.A. (1973) in The Enzymes (Boyer, P.D. ed.) 3rd. edition, vol. 13, pp. 239-278. Academic Press, London. Dunaway, G:A. (1983) Mol. Cell. Biochem. 52, 75-91. Dunaway, G.A., Kasten, T.R, Sebo, T. & Trapp, R. (1988) Biochem. J. 251,677-683. Dunaway, G.A. & Kasten,T.R (1989) Mol. Cell. Biochem. 87, 71-77. Elson, A., Levanon, D., Brandeis, M., Dafni, N., Bernstein, Y., Danciger, E. & Groner, Y. (1990) Genomics 7, 47-56. Feinberg, A.P. & Vogelstein, B. (1984)Anal. Biochem. 137, 266-267. Levanon, D., Danciger, E., Dafni, N., Bernstein, Y., Elson, A., Moens, W., Brandeis, M. & Groner, Y (1989) DNA 8, 733-743. Morrison, N., Simpson, C.J., FothergilI-Gilmore, L.A., Boyd, E. & Connor, J.M. (1991) Human Genetics, submitted for publication. Nakajima, H., Noguchi, T., Yamasaki, T., Kono, N., Tanaka, T. & Tarui, S. (1987) FEBS Lett. 223, 113-116. Poorman, R.A., Randolph, A., Kemp, R.G. & Heinrikson, R.L. (1984) Nature 309, 467-469. Rypniewski, W.R. & Evans, RR. (1989) J. Mol. Biol. 207, 805-821. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: A laboratory manual, 2nd edition, Cold Spring Harbour Press. 2O2
Vol. 18o, No. 1, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
Sanger, F.,Nicklen, S. & Coulson, A.R. (1977) Proc. Natl. Acad. Sci. 74, 5463-5467. Schirmer, T. & Evans, P.R. (1990) Nature 343, 140-145. Sharma, P.M., Reddy, G.R., Vora, S., Babior, B.B. & McLachlan, A. (1989) Gene 77, 177-183. Shirakihara, Y. & Evans, P.R. (1988) J. Mol. Biol. 204, 973-994. Uyeda, K. (1979) Adv. Enzymol. 48, 193-244. van Keuren, M., Drabkin, H., Hart, I., Harker, D., Patterson, D. & Vora, S. (1986) Human Genetics, 74, 34-40. Vora, S. & Francke, U. (1981) Proc. Natl. Acad. Sci. 78, 3738-3742. Vora, S. (1982) in Isozymes: Current topics in Biological and Medical Research, 6, pp. 119-167, (Alan R. Liss, Inc., New York). Vora, S., Miranda, A. & Francke, U. (1983) Hum. Genet.. 63, 374-379
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