Cloning and sequencing of human complementary DNA for the phosphoribosylpyrophosphate synthetase-associated protein 39

BiochimicaL et Biophysics Acta

ELSEVIER

Biochimica

et Biophysics

Acta 1306 (1996) 27-30

Short sequence-paper

Cloning and sequencing of human complementary DNA for the phosphoribosylpyrophosphate synthetase-associated protein 39 ’ Toshiharu Ishizuka, Kazuko Kita, Tomoko Sonoda, Sumio Ishijima, Kunio Sawa, Nobuo Suzuki, Masamiti Tatibana * Department of Biochemistry, Chiba University School of Medicine, l-8-1 Inohana, Chuo-ku, Chiba 260, Japan Received 24 November

1995; accepted 21 January

1996

Abstract A human cDNA encoding a human homologue of the rat phosphoribosylpyrophosphate synthetase-associated protein of 39 kDa was isolated. The deduced protein contains 356 amino acids and has a calculated molecular mass of 38561. The amino acid sequence is 98% identical to that of the rat. The corresponding mRNA is present in all human tissues examined. Keywords: Nucleotide

synthesis;

l’hosphoribosylpyrophosphate

synthetase;

Phosphoribosylpyrophosphate (PRPP) synthetase (EC 2.7.6.1) catalyzes the formation of PRPP from ATP and ribose 5-phosphate. PRPP is a primary substrate for the synthesis of almost all nucleotides [ 1,2] and a critical control factor for de novo synthesis of purines 13-51 and pyrimidines [6-81. The rat liver enzyme exists as a complex aggregate of 34, 39-, and 41-kDa components, the 34-kDa species being the catalytic subunit [9,10]. In the rat liver the 34-kDa subunit is a mixture of two highly homologous isoforms, PRS I and PRS II [lO,ll]. These two isoforms differ in heat stability and sensitivity to nucleotide inhibition [12-141. The PRS I and PRS II mRNAs are encoded in the two distinct genes, designated PRPSl and PRPS2, respectively [15,16]. We termed the 39- a:nd 41-kDa components PRPP synthetase-associated proteins (PAPS). Since the removal of the PAPS from the rat liver enzyme complex results in

Abbreviations: PRPP, S-phosphoribosyl 1-pyrophosphate; PRS I and PRS II, PRPP synthetase subunit I and II, respectively; PAP, PRPP syntbetase-associated protein: PAP39, PAP of 39 kDa; PCR, polymerase chain reaction; bp, base pail(s); EST, expressed sequence tag; kb, kilo base(s); SSC, 150 n-&l NaCl/lS mM sodium citrate. * Corresponding author. Fax: -t 81 43 2262041. ’The nucleotide sequence reported in this paper has been submitted the GSDB/DDBJ/EMBL/NCBI databases under the accession number D61391. 0167-4781/96/$1.5.00 0 1996 Elsevier Science B.V. All rights reserved PI1 SO167-4781(96)00030-9

cDNA cloning; (Human)

an increased enzymatic activity of the remaining catalytic subunits [ 171, we proposed that PAPS play a negative regulatory role in PRPP synthesis. We previously reported the cloning of the cDNA for the 39-kDa component (PAP39), the major component of the PAPS, from rat liver [17]. The deduced amino acid sequence turned out to be very similar to the amino acid sequences of the 34-kDa subunits. Here, we report the cloning and sequencing of a human cDNA encoding a protein homologous to the rat PAP39. To clone the human homologue of PAP39, we employed the polymerase chain reaction (PCR) and degenerate primers. We used S-ATHTAYGTNATGGCNACNCA-3’ as the primer (H = A, C, or T; Y = C or T; N = A, G, C, or T) and 5’-ATNGGNGAYTCYTCDAT-3’ as the antisense primer (D = A, G, or T) based on the amino acid sequence of rat PAP39 (residues 280-286 and 297-302, respectively, see Fig. 2). From the cDNA pool of HepG2 cells (human hepatocarcinoma, derived from Riken, Ref. [18]) we obtained an amplification product of 68 base pairs (bp). This product was cloned in pBluescript II (Stratagene), sequenced, and expected to code for a protein fragment that was 100% identical to the rat PAP39 fragment (nucleotide position 887-954, see Fig. 1). To clone a more 5’ extending cDNA, we performed a second PCR using an internal sequence of the 68-bp product, 5’-GAGGACTCCTCAATCAGGCGAGGGGCCTCT-3’ (nucleotide position 922-9511, as the antisense primer and a

T. Ishizuka et al. /Biochimica

28

et Biophysics Acta 1306 (1996) 27-30

TCGCAATCATCGTGGATGAC-3’ (nucleotide position 741-760) as the sense primer in combination with a primer, 5’-CCATCCTAATACGACTCACTATAGGGC-3’, positioned in the adapters ligated to the cDNA at both ends. Two PCR products were obtained, each spanning a region from the adapters on either side into the 902-bp fragment. These two products were cloned and sequenced. Both clones overlapped the sequence of the 902-bp product and we were able to link all three sequences (Fig. 1). The resulting cDNA of 1760 bp contains a single open reading frame initiating with an ATG codon at nucleotide position 50-53 and terminating with a TAG stop codon at

degenerate primer, S-ATGAAYGCHGCHMGVACHGG3’ (M = A or C; V = A, G, or C), corresponding to amino acid residues l-7 in the rat sequence as the sense primer. The PCR product of 902 bp was cloned and sequenced. The sequence contained the original 68-bp sequence and coded for a protein fragment that was 99% identical to the rat PAP39 (nucleotide position 50-951). To clone the 5’ and 3’ ends of the cDNA, an anchored PCR approach was taken utilizing the Marathon’” cDNA Amplification kit (Clonetech). We used the oligomer 5’-TGAGTTAAACCTGCTTTCG-3’ which is nucleotide position 134- 153 of the 902-bp product as the antisense primer and 5’-

1 ggtgcgcaag~cac~acctcggagctctccccgttcccccgccggcc AX 1

AAC WC

GCT CGC ACC GGC

71TAccGAGTcTpccpCGccAAc~AcGGccGccn;cAn;GAG~GcCAAGcGcATcpd3A

130

SYRVFLANSTAACTELAICRIT

27

131GAG CGC CIT ffiTGCT GAA TX

GQZAAG

TCT dp

GTATAT

CAAGAGACCAAT

GZAGAAACA

28ERLGAELGKSVVYQETNGET AIT ATA CAG ACA ATA CCC

4SRVEIKEFVRGQDIFIIQTIP Tpo CTC A‘ICA'IGGCT TAC GCAcn;

AAGA"'GCC

310

AX! CCC TAC TI'CCCC TACXC

AAG CAGAGCAAGA'IG

370

I

KQ

68RDVNTAVMELLIMAYALKTA

87

311TQT GCC AGGAACATTA?TCXGc)?c 8SC

ARN

I

IGV

PYF

PY

S

SKM

107

TCCAlTGTGTGCAAGClG~AGcAXC!ATGCXCXGAAAGCAoDI

lO8RKRGS

TPA

I

I

TMDLHQKE

491GIGGACAACClTAGAGCC

I

TCXCCTTX

l48VDNLRAS

490

GCAGI'C ATT GTAGCT

l6SYRNAV

AAG

ClGCTl'CAGTATATC I

P

147

CAGGAAGAA

AITCCA

AAT

550

Q

I

TAT

610

EE

TCT C?CTGATGcTGCAAAGAM:GCC

KS

IVA

SF

QGFF

PFLLQY

551 TAC AGAAAT

A

PDA

PN

CAGTCC AQ

KR

167

SY

187

611GcGGAGAGAcn:cCrcrC,GGTTn;oCCG~A~cAcGGGGAADcTcAGTocAcooAAcn:

670 207

188AERLRLGLAVIHGEAQCTEL 671GACATG.GAcGATGQTCX3l'CAC

TZXCCGCCPATGGTCAAAAAT~ACTGTGCAC

208DMDDGRHS

22SLEL

M

PLM

IA

I

IVDD

851 GCC GCGGAGATC

CTGAAAGAGAGAQX

26SAAE

LKERGAY

I

911AW

CXTCT'XAGAGWCCCTCGC

2881

L

3281

790

dc

247

I

850

I

267

CpGATl'GAGGAGWC IEES

CCTCAT

GAG @IT CAGAAG

L

TAT Gl’l’ ATGOCC M

FVA ACC CAC GGC

910

HG

207

AT

TCCDTAGACGAGGTGGTGGTG

970

SVDEVVV

307

PK

SEAI

S

IYV

CTGCAATGTCCC

AlTCITTcTGAAGccATTca:AGAATC I

DDVE

Occ TATAAGA'X

NTVPHEVQKLQC

SL

GATWlT

227 UI’AGT’PDGA

AlTGACGATCXGGAGAQl'TITGlTGCT

K

SAEAPRL

971ACGAATACT 1031 AlTAdl’l%

730

AKEKPPITVVGDV G’lGGATGACATl’

CGCAWWAAlCATC

248GGR

308T

CCAGGC

PPMVKNATVHPG

731en;GAGTpGccATpoAn:A~ocCAAAGpGAAGccAccGATAAcp

791 OOAQX

430 127

IVCKLLASMLAKAGL

431AcI~AATTATcAcTATGGAT~cATcAA~GAAATAcAADGc~TK:AM:~~ l28TIi

250 67

DPGAATACAGCTGTGATGGAO

371AGGAAGAGGm

190 47

191 AGA GlT G?A ATA AAA GAA TPT GTT CGT GGC CAA GAT ATT TX

251AGAGAT

70 7

MNAARTG

AAGATAAAGmW I

GAT

CACAATOOAGAGTCCATGGCC

RRIHNGESMAY

1030 327

KTVD TAC

1090 347

1091 ClT lTC CGA AAC ATC ACT GTG GAT GAC TAG ctttcacgagggtctcgaccct~acctcctgagggaaac 1160 348LFRNI

357

TVDD*

1161 atgg~gcagtgccatga~gatacagtgtttccttg~~gagg~ctcg~~gcct~~t~gatatcttct

1240

1241 tttgcccggattgat~gga~~gatt~g~gtca~~g~g~cagagct~tggat~~t~~cat~cc

1320

1321 ttacatgtct~tgtcatcagccctgttcct~gttc~gctgctttct~at~tct~tcttat~ta

1400

1401 c~gaggagtt~ggcacataMgtcttaactt~ctc~taatgttc~ttta~t~ttca~tc~g~tg~~

1480

1461 ttgatgttgaacctg~taggg~ctgagcgcctgtsgcc~

1560

1561 gtcttttatagagaatcgtatttttctttca~ttgctatgcctacagc~ttg~

tgaagcattcatgttgtta 1640

1641 catcttccaa~atgtcagat~g-tagcatcccacctct~~tctgagt~ctctg~gttgc~~t~

1720

1721 tttgttgtaaaaaaw

1760

Fig. 1. Nucleotide sequence and the deduced amino acid sequence of the human PAP39 cDNA. The nucleotides the left and right margins.

and amino acids are numbered

5’ to 3’ on

29

T. Ishizuka et al. / Biochimica et Biophysics Acta 1306 (1996) 27-30 Human: lMNMR!l-GYRVFLAN.STAAePELAKRI

Rat:

TF.RLGAELGKWVYQETNGETRVEIKEFURGQDI

llllllIIIII.lll11llllllIlIlllllllllllIIlIlIIIIIIlIIII~IIIIII

1MNMR'IGY-RVFSANSTNiCTELTiKRI TERLGAEI.GKSWYQETI-YXWRVEIKESVRGQDI 61 FIIQTIPRDVNTAVMEtLLIMAYALKTACARNIIGVIPYFPYSKQS

KMRKRGSIVCKLLAS

llllllllllllllIiIIlIIIIIllIIIIIIIIIlIIIIIIlIlllllllllllIiIll 61 FIIQTIPRDVWAVMRLLIMAYALKTACARNIIGVIPYFPYSKQSKMNC+GSIVCF.LIAS 121 MLAKAGLTHIITMDLHQKEIQGFFSFPVDNLRASPFLLQYIQEEIPNYFNAVIVAKSPDA

IlIIIIIIIIIIIlIIIlIIIIII.IIIIIIIIlIlIIIIlIIIlIIIIIIIIIIIIIII 121 MLAKAGLTHIITMDLHQKEIPGFFCFWTNLRASPFLLQYIQEEIPNYPNAVIVAKSPDA 181 AKRAQSYAEFZU&LAVIHG?XAQCTELDMDEGRHSP

PMVKNATVHPGLELPLJmAKExPP

TRLDMDDxc-ISPPFGLELP-KPP 241 I~AIIVDDIIDDVESJ?VA?AEILKERGAYKIYVMATHGILSAEAPRLIEES

IIlIlIIIIIIIIIlIIIIIlIlIIIIIII.IlIIIIIlllllllllllllllllllIII 241 ITWGDVGGRIAIIVDDIIDDVESFVAAAETL

KERGAYKIYVMATHGILSAEAPFLIEES

301 SVDEWWTMVP~QKLQCPKIKTVDISLILSEAIRRIHNGESMAYLFF.NITVDD

356

..IIIIIIIIIlII.IIIIIIIlIIIIIIIIIlIlIIIIIIIlIIIIIIIIIIIII 301 PID~QKLQCPKIKTVDISLILSRAIRRIHMZESMAYLFRNIWDD

Fig. 2. Alignment of the amino acid sequences of the human and the rat PAP39. The alignment different residues are represented by solid bars and dots, respectively.

position 1118- 1120. The sequence surrounding the initiation ATG codon, GCCATGA, agrees with the consensus established by Kozak [19]. In the 3’-untranslated region, there is a putative polyadenylation signal, AATAAA, located 18 nucleotides upstream of the poly(A) tract. A search for homology with other mammalian genes revealed that the partial sequences of this clone had been submitted to the databases as more than 18 expressed sequence tags (EST& Those ESTs were isolated from fetal brain, fetal liver spleen, and placenta.

12345678 kb 9.5 7.5

2.4 ,1.35

-

Fig. 3. Hybridization of the human PAP39 cDNA probe to a human multiple tissue Northern blot frolm Clontech. The cDNA probe (nucleotide position 50-951) was labeled by random primed labeling method [24]. Each lane contains approx. 2 pg of poly(A)+ RNA isolated from the following human tissues: lane 1, heart; lane 2, brain; lane 3, placenta: lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, pancreas. After a high stringency wash (0.1 X SSC/O.l% SDS at 65°C) the blot was analyzed by autoradiography. The positions of RNA size markers are indicated in the left margin of the blot.

356

was done by the Clustal W [23] program.

Identical

and

The open reading frame accommodates a protein of 356 amino acids with a calculated molecular mass of 38561. Amino acid comparison revealed that this human protein shares 98% identity (7 out of 356 residues differ) with the rat PAP39 (Fig. 2), and 44% identity with human PRS I [20,21] and PRS II [22]. We conclude therefore that we have cloned the cDNA for the human homologue of the rat PAP39. Since the rat PAP39 interacts directly with the catalytic subunits [17], the extremely high degree of identity between the human and the rat PAP39 sequence suggests that the human PAP39 also interacts with the catalytic subunits. Using our cDNA as a probe, Northern blot analysis revealed a major transcript of 2.2 kb in all the human tissues examined (Fig. 3). The results suggest that the human PAP39 mRNA is widely expressed. In fact, we detected a 39-kDa protein in a partially purified PRPP synthetase preparation from human erythrocytes by Westem blot analysis with anti-rat PAP39 antiserum (Tatibana, M. and Kita, K., unpublished data). The physiological role of this protein in human cells remains to be clarified. The cDNA clone for the human PAP39 should provide a powerful tool for studies of this important question. This research was supported by grants from the Ministries of Education, Science and Culture and of Health and Welfare of Japan, and the Gout Research Foundation of Japan. G. Hoschek provided linguistic help in the preparation of this manuscript.

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T. Ishizuka et al. / Biochimica et Biophysics Acta 1306 (1996) 27-30

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