Molecular cloning of murine folylpoly-γ-glutamate synthetase

BB.

ELSEVIER

Biochi~ic~a et BiophysicaA~ta

Biochimicaet BiophysicaActa 1305 (1996) 11-14

Short sequence-paper

Molecular cloning of murine folylpoly-y-glutamate synthetase 1 Michael J. Spinella, Kevin E. Brigle,

I.

David Goldman

*

Departments of Medicine and Pharmacologyand the Massey Cancer Center, Virginia Commonwealth University, Medical College of Virginia, Richmond, VA 23298, USA

Received 18 July 1995; revised 11 September1995; accepted20 September 1995

Abstract

Folylpoly-y-glutamate synthetase (FPGS) is essential for mammalian cell survival and is a major determinant of cytotoxicity and selectivity for folate antimetabolites. Here we describe the cloning of a cDNA encoding murine FPGS isolated from L1210 leukemia cells. The amino acid sequence of murine FPGS is 82% identical to human FPGS [1] with identical discrete regions of up to 41 residues. Murine FPGS contains two AUG initiation codons, shown to be responsible for mitochondrial and cytosolic forms of the enzyme in human cells [2]. Previous studies indicated species, tissue, and tumor specific differences in mammalian FPGS. The availability of murine FPGS expands the knowledge and understanding of the spectrum of these variations. Keywords: Folylpolyglutamatesynthetase; cDNA (murine); L1210

Folylpoly-y-glutamate synthetase (FPGS) catalyzes the ATP-dependent addition of glutamyl residues to naturally occurring folates and antifolate agents. This modification results in increased cellular retention and increased affinity and activity for intracellular enzyme sites. FPGS is essential for the survival of proliferating mammalian cells and there is a clear correlation between antifolate sensitivity/selectivity and FPGS expression [3-7]. Studies on FPGS isolated from a number of mammalian sources have reported differences in activity spectra of folate substrates. This include differences between murine and human liver [8], and among murine L1210 leukemia cells, murine liver, and normal mouse intestine [9,10]. The sequences of FPGS from E. coli and L. casei have been previously reported [11,12]. These bacterial enzymes have markedly different physical properties and substrate preferences for folates compared to mammalian enzymes [13], which is reflected in their low sequence identity with the cloned human enzyme. Thus, the bacterial enzymes are not good models to study how folates interact with mammalian enzymes. In order to study the regulation of polyglutamylation in an LI210 transport model [14], it was necessary

* Corresponding author. Fax: + 1 (804) 8288079. nThe sequence data in this paper have been submitted to the EMBL/GenBank Data Libraries under the accession number U32197. 0167-4781/96/$15.00 © 1996 Elsevier Science B.V. All fights reserved SSDI 01 67-478 1(95)00193-X

to isolate a cDNA clone encoding murine FPGS. We report here the sequence relationship between human FPGS and a second mammalian FPGS isolated from murine L1210 leukemia cells. Murine FPGS was isolated from an L1210 derived lambda phage h g t l l cDNA library [15] screened with the full length human FPGS cDNA. Hybridizations were performed as previously described [15]. From a screen of 5 • l0 s plaques, two positive clones were obtained and the nucleotide sequence of the longest one (1.7 kb) was determined. As this clone was not full length, the 5' terminus of the message was determined by primer extension utilizing a murine FPGS-specific antisense oligonucleotide (Fig. 1A) and 5'RACE (Gibco/BRL). Fig. 1A shows the nucleotide sequence and deduced amino acid sequence of murine FPGS, a 587 amino acid protein with a predicted molecular mass of 65 kDa. The protein contained a 42 residue N-terminal mitochondrial leader sequence that shared 67% identity with this region in the human protein and 83% identity with the N-terminal 72 resides reported from hamster [2]. The amino acid comparisons of FPGS homologs between mouse, human, E. coli and L. casei (Fig. 1B) revealed marked (82%) amino acid identity between the mouse and human sequence. However, two 14 residue regions in the center of the proteins display only 33% and 36% identity. The limited sequence identity of the bacterial proteins compared to the mammalian proteins

12

M.J. Spinella et al. / Biochimica et Biophysica Acta 1305 (1996) 11-14

centered about the two putative ATP binding sites [I 1-13] and at the C-terminus. The percent identity of the FPGS proteins are summarized in Table 1. At the nucleotide level

a 83% identity was noted in the coding region between murine and human FPGS; in contrast sequences in the 3'-UTR showed only 52% identity.

A -6

1 AAGACTATGTCGTGGGCGCGGAGCCGACTGTGCTCGACTCTGTCCCTGGCAGCTGTTTCTGCGCGTGGT M S W A R S R L C S T L S L A A V S A R G

21

63

GCAACGACGGAGGGCGCGGCGCC-GCGGGGGATGAGCGCGTGGCCAGCGCCACAGGAGCCGGGCATGGAG A T T E G A A R R G M S A W P A P Q E P G M E

44

132

T A T C A G G A T G CT G T G C G C A C G C T C A A C A C C C T ~ C C A A T G C Y Q D A V R T L N T L Q T N

67

201

C A A C G G A G T G A C CC C C A G G CG C A C G T G G A G G C T A T ~ T G T A C C T G G C A C G G A G T G G A C T G C A G G T G Q R S D P Q A H V E A M E M Y L A R S G L Q

A

CAGCTACCTGGAGCAGGTAAAGCGC S Y L E Q V K R V

270

GAGGACTTGAAC CGG CTAAACATTATTCATGTCACTGGG~C E D L N R L N I I H V T G T

339

AC CGAAC GGAT CCTACGG~TTACGGC T E R I L R N Y G

CTGAAGAC CC-C~TCTTTAGCTCTCCT CACATGGTGCAGGTG L K T G F F S S P H M V Q V

136

408

CGGGAGCGGATTCGAATCAACGGGAA R E R I R I N G K

CCAATCAGCCCCGAGCTCTTCACCAAGCACTTCTGGTGCCTC P I S P E L F T K H F W C L

159

477

TATAACCAGCTGGAGGAGTTCAAGGACGACAGCCATGTCTCCATGCCCTCTTACTTCCGCTTCCTCACA Y N Q L E E F K D D S H V S M P S Y F R F L T

182

546

CTCATGGC CTTCCATGTCTTCCTCCAAGAGAAGGTGGACCTGGCAGTGGTGGAGGTGGGCATTGGCGGG L M A F H V F L Q E K V D L A V V E V G I G G

205

615

GCTTTTGACTGCACCAACATCATCAGAAAGCCAGTGGTGTGTGGAGTCTCCTCTCTTGGCATTGACCAC A F D C T N I I R K P V V C G V S S L G I D H

228

684

AC CAGTCTAC TAGGAGATACAGTGGAGA~TAGCATGGCAGA~GGGGGCATCTTTAAGC T S L L G D T V E K I A W Q K G G I F K

251

753

CCTGCCTTCACTGTGGTGCAGCCAGAAGGTCCCCTGGCTGTGCTGAGGGATCGAGCCCAGCAGATTGGA P A F T V V Q P E G P L A V L R D R A Q Q I G

274

822

TGCCCGTTGTACCTGTGTCCGC CATTGGAAGC CCTGGAGGAGGTTGGACTGCCATTGAGCCTGGGTCTG C P L Y L C P P L E A L E E V G L P L S L G L

297

891

GAGGGAGCACACCAGCGGTCTAATGCTGCCTTGGCCTTGCAGCTGGCCCACTGTTGGCTGGAGCGGCAG E G A H Q R S N A A L A L Q L A H C W L E R Q

320

960

GACCAC CAAGACATCCAGGAGCTGAAGGTATCCAGGCCAAGCATACGGTGGCAGCTGCCCCTGGCACCT D H Q D I Q E L K V S R P S I R W Q L P L A P

343

1029

GTGTTCCGCC CTACCCCTCACATGAGGCGTGGGCTTCGGGACACAGTGTGGCCTGGCCGGACACAGATA V F R P T P H M R R G L R D T V W P G R T Q I

366

1098

CTC CAGC~ACCCCTTAC L Q R G P L

389

T

C A A A G ~ T CCAC CTGTGCCTT C K G K G S T C A F

90

CTGGTGT C P G V

CTGGTACCTGGATGGCGCCCATACCACCAGCAGTGTGCAGGCCTGTGTG W Y L D G A H T T S S V Q A C V

113

1167

CACTGGTAC CGC CAGTCATTGGAGC GCAGCA~C G CACCGATGGAGGGTCCGAAGTACACATCTTGCTC H W Y R Q S L E R S K R T D G G S E V H I L L

412

1236

TTCAACTCTACTGGTGACAC-GGACTCTGCTGCCCTGCTGAAGCTGCTGCAGCCCTGCCAGTTTGACTAC F N S T G D R D S A A L L K L L Q P C Q F D Y

435

1305

GC TGT CT T CTG C C CCAACGTGACAGAGGTTT CATC CATAGGAAATGCAGACCAGCAGAACTTCAC A V F C P N V T E V S S I G N A D Q Q N F T

458

1374

ACTCTGGACCAGGTGCTGCTCCGCTGCCTCCAACACCAGCAGCATTGGAACGGCCTGGCTGAGAAACAG T L D Q V L L R C L Q H Q Q H W N G L A E K Q

481

1443

GCTAGCTCCAAC CTCTGGAGCAGCTGCAGCCCAGACCCTGCTGGGCCAGGCTCCCTGCTGCTGGCCCCG A S S N L W S S C S P D P A G P G S L L L A P

504

1512

CACC CACCTCAGCCTACTAGGACGAGCTCCCTCGTTTTCAGCTGCATCTCCCACGCCTTGCTGTGGATC H P P Q P T R T S S L V F S C I S H A L L W I

527

1581

AGCCAAGGCCGGGATCCCATCTTTCAGCCCCAGAGCCTTCCAAGGAATCTTCTCAACCACCCCACAGCC S Q G R D P I F Q P Q S L P R N L L N H P T A

550

1650

AACAGCGGGGC CAGCATTCTCCGTGAGGCTGCTGCCATCCATGTACTGGTTACAGGAAGCCTGCACCTG N S G A S I L R E A A A I H V L V T G S L H L

573

TGTG V

1719

GTGGGCGGGGTTCTGAAACTGCTGGATCCCTCTATGTCCCAGTAGCCAAGGACCATCCTACATCGGTCT V G G V L K L L D P S M S Q * 587

1788 1857 1926 1995 2064 2133 2202

GC CTTTCCACAGACTCTTATACTCAGTGCCTTGTGATTTCTGCTCTCAGATTTTTTCGGACTGCGCAGG GTCCTGGGCTCTTGGTAGAGTGTAC~TGGGAGAGGCTCTCTGTACCTCGGCCTCTCCTTCCTCTGGC AGAGACAG CAGGGTGCTTC C CAGAGTC CC CACCATCGTAGAGCTT~TGGCC CATCTCC CT~CTGC CTC CAGGCTCAGGTCCAGCTTACTGTTGCACGTGCCTC~CAGC C~TCCTGCCTGAGGTTAGAC CA GAGAC CTC CTCCTC C CTC CCAATGC~ CTG~G/~CCTTGCCTGGCATT~C"I'GT GTTG CTGCaAACTAATTG~GC T'FrTA~CCCT~FFrATTT~AT~TA~TA~TGACA~CTT T T G A T T G A ~

Fig. 1. (A) Nucleotide and deduced amino acid sequence of murine FPGS isolated form L1210 cells. Initiating codons and the polyadenylation signal are in bold. The region in which the RACE primer was designed is underscored. Nucleotides derived from RACE are from - 6 to 500; nucleotides derived from the cDNA are from 452 to 2224. (B) Amino acid sequence comparison of FPGS from mouse, human [1,2], E. coli [11], and L. casei [12]. Mitoehondrial leader sequences are in bold. Putative ATP binding sites are underscored [13]. Sequence identity is indicated by a vertical line. Identity between a single bacterial enzyme and one of the mammalian enzymes is indicated by a dot. The PILEUP program from the Wisconsin Genetics Group, Madison, WI, USA was used for the sequence alignments and comparisons.

M.J. Spinella et al. / Biochimica et Biophysica Acta 1305 (1996) 11-14

13

B Mouse

MSWARSRLCSTLSLAAVSAII

PGMEYQDAVRTLNTLQTNAS

GATTEGARRRGNSAWPAPQE

Human E. L.

60

coli casel

..........................................

~1~irarp~spf~swn~

..........................................

MNYTETVAYIHS F - PRLA

YLEQVKRQRSDPQAHVEAME

Mouse

MYLARSGLQVEDLNRLNI IH VTGTKGKGSTCAFTERILRN I

Human LVA~--LG~"LKPAbF-gFT

~rGO--aRR- I,¢r.-I~--~

I,Gm, O . . . . . .

Mouse

YGLKTGFFSSPHMVQVRERI

RINGKPISPELFTKHFWCLY

Human

~.~~LV~l~.~i

E. c o l i L . casel

A~/~/YS~HLVRY~V SGLTVGLYTSPFIMRFNERI

Mouse

LTLMAFHVF II

coli case~

'

Human E. L.

73 63

NQLEEFKDDSHVSMPSYFRF ' II I' HR~.~.T~G~CV.~M~PY~

180

AEI~.S---ARGDISLTY~EY AALERLQQQQADFNVTEFEF

126 123

I~AVVE VGIGGAFDCTNI I R K P W C G VSSLGIDHTSLLGDTVEKIA I ~ II

240

I

0~Ga~-I-a

'

I

~IN~Q~.~~R~.Y

I

RVQ~ELPESA--HTASF-MIDHEPIPDAALVNAVAFVR

I

I

180

240

G~ LS ALWL ~ K ~ A Q ~ V V I ~~'TS IAL~TDW~.~PDRES ~'6 185 ITALGYWYFRQRQVDVAVIE VGIGGDTDSTNVI -TPVVSV LTEVALDHQKLLGHTITAIA 182 WQKGGI FKPGVPAFTV- VQP EGPLAVLRDRAQQIGCP-LY

MOUSe

120 120

'

I I

coli casel

18 17

VTGTNGKGSAANAIAHVLEA

II

E

'

I

~'L-HSi-TIDLGLiRVS

E. L.

60

LCPPLEALEE--VGLPLSLG

296

A ~ L A ~ - - G ~ A ~

296

/11

Human

IIII/I

I

II

II

RE~FRS~.KIAIQG,'-~ EMP-iTIADVAQEKGALLQR KHKAG I I KRG I PVVTGNLVP DAA -A W A A K V A T T G S Q W L R

RGVEWNYSVTD- HDWA- - FS 239 FDRDFSVPKAKLHGWGQRFT 241

Human

LEGAHQRSNAALALQLAHCW !~G!~iT~p~p~v~_I!!

LERQDHQDIQELKVSRPSIR ~RHGAG~.P~IGLL

WQLPLAPVFRPTPHMRRGLR 356 W~.~V~Q~S~LI~.~IrlIilIII//ill I I / 356

E. L.

PQPNAAT~GLE--

YEDQDGRI-SDLEVPLVGDY

QQRNMAIAIQTAKVYAKQTE

: - - -'(rS~. . . . . . NAI~D6iA 282 W--PLTP ..... QNIRQGLA 293

DTVWPGRTQILQRGPLTWYL

DGAHTTSSVQACVHWYRQSL

ERSKRTDGGSEVHILLFNST

E. L.

coli casel

Mouse

coli casel

Mouse

'I

Human E. L.

coli casel

Mouse

'II

416

SAIAiaiFQiVSESPiVIFASHWPARLEKISDTPLIVI - DGAHNPDGINGLITALKQLF

KNGR ....... MLAVI-GML 333 SQP ........ ITVIA-GIL 343

GDRDSAALLKLLQPCQFDYA

TVT

VFCPNVTEVSSIGNADQQNF

VLLRCLQHQ HWNG 476 I

•

I

I

Human E. L.

476

coli casel

.

.

.

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.

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ADKDYAAMADRL ................................................ LAEKQAS SNLWS SCS PDPAG PGSLLLAPHPPQPTRTS SLV FSCISHALLWISQGRDPIFQ I I t, •

Mouse

Human E. L.

416

345 356 536 536

coli casel

Mouse

................. .................

I~V T~

VDDWY~LiGPiGATAEQL FSTVYLVPVPGTPRALPEAG

LEHLGNGKSFD~VAQA . . . .

~JU~'mGRL~SWQ~. . . .

PQSLPRNLLNHPTANSGAS I LREAAAIHVLVTGS LHLVGG VLKLLDPSMSQ Illl

!

384 394

587

Human E. L.

coli casel

............... W D ~ ...............

~ L

v-P

plWrGSL

_

sA VRQTLLGGKS

428

Fig. 1 (continued).

Table 1 Comparison of FPGS proteins Percent identity Mouse Mouse

Human

E. coli

L. casei

100

Human

82

100

E. coli L casei

26

26

100

27

26

33

100

In summary FPGS is highly conserved across two mammalian species, but has limited identity to the homologous bacterial enzymes• Additional studies are required to define the critical regions of these proteins responsible for substrate binding and specificity. The authors would like to thank Dr. Richard Moran for FPGS human cDNA and Cathy Leyco for technical assistance in sequencing. This work was supported by National

14

M.J. Spinella et al. / Biochimica et Biophysica Acta 1305 (1996) 11-14

C a n c e r Institute Grants C A - 3 9 8 0 7 (I.D.G. and K.E.B.), and C A - 0 9 3 4 9 (M.J.S.)

References [1] Garrow, T.A., Admon, A. and Shane, B. (1992) Proc. Natl. Acad. Sci. USA 89, 9151-9155. [2] Freemantle, S.J., Taylor, S.M., Krystal, G. and Moran, R.G. (1995) J. Biol. Chem. 270, 9579-9584. [3] Barredo, J.C., Synold, T.W., Laver, J., Relling, M.V., Pui, C.-H., Priest, D.G. and Evans, W.E. (1994) Blood 84, 564-569. [4] Barredo, J. and Moran, R.G. (1992) Mol. Pharmacol. 42, 687-694. [5] McCloskey, D.E., McGuire, J.J., Russell, C.A., Rowan, B.G., Bertino, J.R., Pizzorno, G. and Mini, E. (1991) J. Biol. Chem. 266, 6181-6187.

[6] Fry, D.W., Anderson, L.A., Borst, M. and Goldman, I.D. (1983) Cancer Res. 43, 1087-1092. [7] Fabre, I., Fabre, G. and Goldman, I.D. (1984) Cancer Res. 44, 3190-3195. [8] Clarke, L. and Waxman, D.J. (1987) Arch. Biochem. Biophys. 256, 585-596. [9] Moran, R.G., Colman, P.D., Rosowsky, A., Forsch, R.A. and Chan, K.K. (1984) Mol. Pharmacol. 27, 156-166. [10] Rumberger, B.G., Barrueco, J.R. and Sirotnak, F.M. (1990) Cancer Res. 50, 4639-4643. [11] Bognar, A.L., Osborne, C. and Shane, B. (1987) J. Biol. Chem. 262, 12337-12343. [12] Toy, J. and Bognar, A.L. (1990) J. Biol. Chem. 265, 2492-2499. [13] Shane, B., Garrow, T., Brenner, A., Chen, L., Choi, Y.-J., Hsu, J.-C. and Stover, P. (1993) Adv. Exp. Med. Biol. 338, 629-634. [14] Spinella, M.J., Brigle, K.E., Sierra, E.E. and Goldman, I.D. (1995) J. Biol. Chem. 270, 7842-7849. [15] Brigle, K.E., Spinella, M.J., Sierra, E.E. and Golman, I.D. (1995) J. Biol. Chem., submitted.