Isolation and partial characterisation of a Chinese hamster C

© 7992 Oxford University Press Nucleic Acids Research, Vol. 20, No. 8 1891-1895

Isolation and partial characterisation of a Chinese hamsterC^-alkylguanine-DNA alkyltransferase cDNA

Joseph A.Rafferty*, Rhoderick H.Elder, Amanda J.Watson, Lynn Cawkwell, Philip M.Potter*and Geoffrey P.MargisonCRC Department of Chemical Carcinogenesis, Paterson Institute for Cancer Research, ChristieHospital, Wilmslow Road, Manchester M20 9BX, UK

Received January 30, 1992; Revised and Accepted March 23, 1992 EMBL accession no. X65081

ABSTRACT

The cDNA encoding Chinese hamster C^-alkylguanine-DNA-alkyltransferase (ATase) has been isolated froma library prepared from RNA isolated from V79 lungflbroblasts which had an upregulated level of this repairactivity following stepwlse selection with achloroethylating agent (1,2). Expression of the cDNAin E.coll produced functionally active ATase at levelsof 2.5% of total cellular protein as determined by Invitro assay. The recombinant hamster protein has amolecular weight of 28 kDa as estimated by SDS-PA-GE and fluorography and this was identical to that inthe upregulated cells. The characteristic PCHRVpentapeptide of the alkyl acceptor site has beenIdentified and there is a 68 amino add residue regionwhich Is 90% conserved across all the mammalianproteins so far analysed: In contrast, the N- and C-termlnal domains diverge by as much as 50% betweenspecies. Polyclonal antibodies to the human and ratATases hybridised to the hamster protein on westernanalysis suggesting at least one common epitopeshared across species. However, in antibody inhibitionexperiments neither of the antisera cross reacted withthe hamster ATase in a way which interfered withfunctional activity whereas the anti-human antibodiesinhibited the human ATase and the anti-rat antibodiesinhibited the rat and mouse ATases. There maytherefore be significant tertiary structural differencesbetween the hamster protein and the other mammalianATases.

INTRODUCTION

N-nitroso compounds and related alkylating agents exhibit tissuespecific and dose dependent carcinogenicity in a number ofexperimental animal systems (3). Although these agents producea variety of alkylation products in DNA, it is now widely acceptedthat C^-alkylguanine and 04-alkylthymine may be the principallesions of biological importance (4,5,6,7,8).

Repair of C^-alkG was first demonstrated in bacteria (9) andin rat liver (10) and since then there have been numerous reportsof this activity in normal mammalian cells and tissues (11,12,13).O'-alkG is repaired by C^-alkylguanine-DNA-alkyltransferase(ATase) which in an autoinactivating reaction transfers the alkylgroup from DNA to a cysteine residue within the protein,restoring an intact guanine moiety without involving an excisionrepair process. The human (14,15,16,17), rat (18,19,20) andmouse (21) ATase cDNAs have been isolated and the encodedrepair proteins partially characterised.

Generally, tissues with lower levels of ATase are moresusceptible to tumour induction by alkylating agents than thosethat express high levels (22,23). However, normal adult Chineseand Syrian hamsters but not rats, are sensitive to the inductionof liver tumours after a single dose (20mg/kg) of NDMA despiteall species metabolising the agent to equivalent extents (24,25)and having essentially similar levels of hepatic ATase. WhileNDMA induces the expression of ATase only in rat liver, thiseffect is only detectable at relatively low doses (0.2 or 2.0mg/kg),and at 20mg/kg any increased levels of repair protein are rapidlyconsumed by the much higher levels of substrate produced inDNA at this dose (26). Consequently other factors must be evokedto explain the extreme hepatocarcinogenicity of NDMA inhamsters. In order to address the involvement of ATasetranscription and translation in this process we have isolated theChinese hamster ATase cDNA from a library prepared fromcultured lung fibroblasts that were upregulated for this activityfollowing stepwise selection with a chloroethylating agent (1,2).In the present report, we show functional expression of high levelsof the recombinant protein in E.coli and demonstrate that the purerecombinant protein is immunologically distiguishable from otherrodent and human ATases.

MATERIALS & METHODS

Bacteria and cloning vectors

The \g/10 arms and host C600 Hfl E.coli were obtained fromAmersham International pic (AI). The plasmids pUC8.0 and

* To whom correspondence should be addressed

+ Present address: Department of Pharmacology, St Jude Childrens Research Hospital, Memphis, TN, USA

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1892 Nucleic Acids Research, Vol. 20, No. 8

pUC8.1 (27) were obtained from Dr.J. Brennand. The plasmidpchAT consists of the 654bp protein coding sequence of thehamster ATase (produced by PCR mediated site directedmutagenesis using oligonucleotides which create 5' and 3'flanking BamHI sites) inserted into the BamHI site of pUC8.0.

Cell cultureThe growth conditions for the Chinese hamster lung fibroblastline (RJKO) and the 120/ig/ml mitozolomide selected population(GM + 120) have been reported elsewhere (2).

cDNA library preparationTotal RNA was prepared from mitozolomide selected Chinesehamster lung fibroblasts (1) and poly A+ mRNA was isolated byoligo-dT cellulose chromatography. cDNA was synthesised usingan AI cDNA kit and using EcoRI linkers. Following packagingthe XgflO phage were transfected into E.coli C600 Hfl.

Library screeningApproximately 8X105 plaques were plated, lifted in duplicateonto Hybond N+ (AT) and probed with a [32P]-labelled 65Obphuman ATase cDNA (17) in the usual manner (28). The filterswere washed twice in 2 xSSC/ 0.1 % SDS at 62°C and were thenexposed to X-ray film at — 70 °C for 48h. Positive plaques werepicked and phage eluted overnight in 100/iL of SM buffer (28).Subsequent screenings were carried out as described above.

DNA sequencingInsert DNA in plasmids or M13 was sequenced using an AIMultiwell sequencing kit and the dideoxy chain terminationmethod.

Nucleic acids analysisBacteriophage Xg/10 DNA was isolated using the Qiagen> Lambda Kit< (Qiagen, Germany). Northern and Southernanalysis and associated protocols were followed as previouslydescribed (Potter et al, 1991).

PCR analysis of recombinant clones and site directedmutagenesis of the hamster ATase cDNAPlate eluates of plaque pure clones were used directly for PCRanalysis of insert size. Aliquots (1/iL) of eluate were incubatedin 50/iL of Taq DNA polymerase buffer containing 150/tM ofeach dNTP, 0.5units Taq polymerase (IBI Ltd) and witholigonucleotides (20pmol each) that flanked the 5' and 3' endsof the cloning site in \gtlO. After 30 cycles (95°C, 30s; 50°C,60s; 72°C, 90s) the amplification products were examined afterelectrophoresis in 1 % agarose gels. Site directed mutagenesis ofthe hamster cDNA was achieved using Vent DNA Polymerase(New England Biolabs) and oligonucleotides previously described(18,21) which hybridise 5' to the translation initation codon and3' to the stop codon. BamHI sites were created immediately 5'and 3' to the respective codons following PCR amplification(95°C, 30s; 55°C, 45s; 72°C, 60s; 20 cycles). Amplificationproducts of the expected size were cloned into the appropriatesite of pUC8.0 following isolation from LMP agarose.

Computer analysis of nucleic acid and protein sequence dataAll sequence analysis was carried out on a MicroVax 3600computer using the University of Wisconsin Genetics ComputerGroup (GCG) molecular analysis software (29). All programswere run using the default parameters.

Alkyltransferase assay

Protein extracts of pelleted bacterial cultures were prepared bysonication and assayed using [3H]-methylated calf thymus DNAas substrate as previously described (1).

SDS-PAGE and fluorographySeparation of proteins by SDS-PAGE has been describedelsewhere (18). Protein samples for fluorography were separatedin 16% polyacrylamide gels prior to electroblotting onto HybondC (AI) or immobilon (Millipore). Radiolabelled proteins weredetected by wetting the filter in Optiphase Hi-safe (LKB)scintillation fluid, wrapping the filter in Saran and exposure toX-ray film at -80°C.

Overexpression in E.coli and purification of recombinantChinese hamster ATaseE.coli DH5a harbouring the plasmid pchAT was expanded toa 1L culture in LB medium and the bacteria were harvested.Extracts of such cultures were shown to contain the hamsterATase at ca. 2.5% of total protein as determined by in vitroactivity assay. Double stranded DNA cellulose (Sigma) affinitypurification of the ATase from bacterial extracts was carried outas described (21).

Western analysis and antibody inhibition assayATase antisera were raised in Half-lop rabbits by repeatedinjection of recombinant proteins purified to apparenthomogeneity by DNA cellulose chromatography. Aliquots ofATase were subjected to SDS-PAGE, electroblotted to HybondC (AI) membrane and probed with the rabbit anti-human (Lee,SM., Rafferty, JA., Fan, CY., Potter, PM., Elder, RH. &Margison, GP., in preparation) and anti-rat (Rafferty, JA., Elder,RH., Cawkwell, L. & Margison, GP., in preparation) ATasepolyclonal antibodies (pAb). The antibody reaction was visualisedusing an alkaline phosphatase method (30). The ability of these

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residue conHnvd in all aequencescoaservatire aubatitutiona

Figure 1. Alignment of the Chinese hamster, human, mouse and ratCP-alkylguanine-DNA-alkyltransferase amino acid sequences as determined bythe Ousts] computer programme. Line X shows the pattern of conserved residues0 and conserved substitutions (+) between the four mammalian proteins. LineY represents the effect on conservation if the E.coli ogt and ada, the B.subrilisdas and the yeast ATases are included in the line up. A ( - ) symbol denotes agap in a sequence relative to the hamster protein.


Nucleic Acids Research, Vol. 20, No. 8 1893

antisera to functionally inhibit chAT was determined bypreincubation of the pure recombinant proteins with the pAhisfollowed by residual ATase activity measurements. Fifty toseventy-five fmoles of ATase was incubated on ice for 4h withvarious amounts of either anti-human or anti-rat antisera in a totalvolume of 200/xL of 50mM Tris-HCl (pH 8.3), lmM EDTA,3mM dithiothreitol, 50/ig/mL calf thymus DNA and lmg/mLBSA. Residual ATase activity was then determined by incubationat 37°C with excess substrate as described (1). Samples wereroutinely assayed in duplicate.

RESULTSIsolation of the Chinese hamster cDNAPlaque hybridisation of a Chinese hamster V79 lung fibroblastcDNA library in XgrlO gave 15 putative positive clones followinga primary screening with a I32!5]-labelled 650bp human ATasecDNA. Three of these were picked and eluted into SM bufferovernight and one of the three was consistently strongly positivethrough secondary and tertiary screenings. Plate lysates from thisclone were used to analyse insert size by PCR using \gt 10 primers.The approximately 400bp PCR product obtained by this approachhybridised at high stringency to the human probe indicatingextensive sequence homology. The recombinant vector wasrecovered from the host bacteria and the 400bp insert isolated andligated into the EcoRI site of pUC19. DNA sequencing of the 5'and 3' ends of this clone demonstrated it had homology with 3'

rCh kDa H R +

- 46.0-

- 30.0 •

- 21.5 •

- 14.3 -

Figure 2. Fluorography of an extract of E.coli harbouring the 1.1kb Chinesehamster ATase fragment ligated into the Kpnl site of pUC19 (rCh) and of cellextracts of the chloroethylaling agent selected Chinese hamster lung fibroblastcells, RJKO (R) and GM + 120 ( + ) . A 28 kDa protein is expressed in E.coliand this is the same size as the endogenous hamster ATase. The human ATase(H) is included for size comparison.

kDa

46.0-

30.0-

21 5

14.3

Hx O Mr H Ch R M H

coding sequence of the rat and human ATase cDNAs (83% and61 % respectively) and that the clone also had a 141bp 3'-UTR.

Following removal of the polyA+ tail by TaqI digestion, thisfragment was [^PJ-labelled and used to rescreen the remainingtwelve plaques from the primary screening. These were elutedinto SM, PCR analysis carried out to determine insert size andthe amplification products probed with the 400bp fragment. Thelargest product was approximately 1. lkb and this hybridised tothe probe at very high stringency. Insert DNA from the parent\gt\O clone was isolated, cloned into the Kpnl site of pUC19and sequenced completely on both strands using 3 PstI subclonesand the pUC forward and reverse primers. An open reading frame(ORF) of 630bp was found and this was flanked by a 297bp5'-UTR and a 141bp 3'-UTR, including a 30bp polyA+ tail.The ORF encodes a 209 amino acid residue protein with amolecular weight of 22.3kDa as calculated from the predictedamino acid sequence. The Chinese hamster peptide sequence isextensively homologous with the human, rat and mouse ATases(Figure 1) and as expected shows somewhat more similarity tothe other rodents than to the human protein. The trends inhomology and divergence observed for the mammalian proteinsto date (21) are clearly applicable to the hamster sequence. Thereis a 90% conserved central core region of 68 amino acid residuespresent in all four species (amino acid residues 84—152 of thehamster sequence) while the N- (1—83) and C-terminal(153-209) regions show about 50% exact homology. ThePCHRV pentet at the proposed alkyl-acceptor site is conservedin the hamster protein. There is no clear evidence for DNAbinding motifs such as zinc fingers (31), cysteine rich regions(32), leucine zippers (33) or basic zippers (34).

Expression of the Chinese hamster ATase in E.coliIn order to confirm that the Chinese hamster cDNA encoded afunctional protein, cell free sonicates of the host E. coli harbouring

6 8 10 2 4 6

Serum volume (uL)8 10

Figure 3 . Fluorography of Ecoli extracts containing the 647bp hamster codingsequence indicating a 28kDa protein being expressed and confirming the assignmentof the translation initiating codon. The human (H), rat (R), mouse (M), ogi (O)and ada (Hx) proteins are included for comparison. Mr, [l4C]-labelled proteinmolecular weight markers.

Figure 4. The effect on ATase activity of incubation of pure recombinant human(A) or Chinese hamster (B) ATase with increasing amounts of anti-human pAb( • ) or with preimmune serum (O) . Similarly the effect on the activity of purerecombinant rat (C) or Chinese hamster (D) ATase of incubation with anti-ratpAb ( • ) or preimmune serum (O) is also shown. Each point represents the meanof duplicate samples. In all cases the standard error of the mean was < 2 % .


1894 Nucleic Acids Research, Vol. 20, No. 8

the 1.1kb fragment ligated into the Kpnl site of pUC19(pUCchAT) were assayed for ATase activity. Sense orientationclones (with respect to the lacZ promoter) yielded active ATaseat ca. 1.5% of total protein. SDS-PAGE and fluorographyindicated a labeled protein product of 28kDa (Figure 2). Thiswas the same size as the ATase protein in extracts of the Chinesehamster lung fibroblast line from which the library was made(Figure 2). The discrepancy between the predicted and observedsize of the protein is a consistent feature of the ATase family(15,18,21).

In an attempt to increase the levels of expression of the hamsterATase in E.coli, the cDNA was truncated by PCR-mediated sitedirected mutagenesis to produce BamHI sites 5bp upstream ofthe translation initiation codon and 7bp downstream of thetranslation termination codon. The resultant 647bp product wasligated into the BamHI site of pUC8 and bacterial extracts fromclones harbouring sense (pchAT) and antisense constructs wereassayed for ATase activity and subjected to SDS-PAGE andfluorography. Clones containing the cDNA in the correctorientation routinely produced ATase at 2.5% of total cell protein.Crude cell-free sonicates of one of the pchAT clones wassubjected to DNA-cellulose affinity chromatography. ATasecontaining fractions (as determined by SDS-PAGE andCoomassie staining) were pooled and concentrated: aliquots ofthis material gave single bands on SDS-PAGE. Analysis of thisapparently pure recombinant protein by SDS-PAGE fluorographygave a single band of 28kDa confirming that the translationinitiation codon was correctly assigned. Figure 3 shows afluorograph indicating the sizes of all the mammalian ATasesand the E.coli equivalents, ada and ogt.

Structural features of Chinese hamster 06-alkylguanine-DNA-alkyltransferase as determined by cross reaction withanti-human and anti-rat ATase polyclonal antibodiesThe structural relationship between the hamster, human and ratATases has been investigated by examining the effect onfunctional activity of the protein following incubation with eitheranti-human or anti-rat ATase pAbs. Any inhibitory effect of theanti-human antibody is confined to the human ATase and if thereis any cross reaction of this antibody with the Chinese hamsterprotein it does not result in impaired activity (Figure 4A and B).The anti-rat pAb is a potent inhibitor of rat ATase and appearsto suppress this activity more efficiently than the anti-human

hAT pAB

M Ch H R kDa

46(1

14.3

rAT pAB

M Ch H R

Figure 5. Western blot of pure recombinant mouse (M), Chinese hamster (Ch),human (H) and rat (R) ATases incubated with either anti-human (hAT pAb) oranti-rat (rAT pAb) ATase. The approximate positions of the molecular weightmarkers are shown in kDa.

equivalent inhibits the human ATase (Figure 4C). We havepreviously shown that a similar pattern of cross reaction occursif the mouse ATase is preincubated with these two antisera (21).However, the hamster ATase does not appear to interact withthe anti-rat pAb in a way that influences functional activity(Figure 4D) and this distinguishes it from the other recombinantrodent ATases.

Western analyses using these antisera (Figure 5) has shownthat both recognise all four of the mammalian ATases, althougha weaker signal is consistently obtained for the hamster protein.The relative molecular weights of the proteins detected by theantisera are consistent with those determined by fluorography(Figure 3).

DISCUSSION

We have isolated a cDNA for Chinese hamster ATase using acDNA probe for the human homologue: a clone was obtainedwith a 1.1kb insert which contains an open reading frameencoding a protein of 209 amino acids with a predicted molecularweight of 22.3kDa. This was confirmed to express high levelsof ATase by an activity assay. PCR mediated site directedmutagenesis of this to remove most of the 5'- and 3'-UTRsresulted in a 647bp cDNA. Both this and the 1.1kb fragmentencoded proteins of 28kDa confirming that the ATG assignedas the translational start was correct. This rules out translationinitiation from other in frame codons, including one 60nucleotides upstream. The discrepency between the predicted andthe apparent molecular weight of the hamster protein is aconsistent feature of the mammalian ATase family. The highproline content of the mammalian ATases (8—10%) may accountfor their anomalously high molecular weights as determined bySDS-PAGE, a phenomenon which has been described previously(e.g., 35).

The predicted amino acid sequence of the Chinese hamsterATase is highly homologous with rat and mouse and to a lesserdegree with the human protein. When the mammalian proteinsare compared a 68 amino acid residue core region is 90%conserved across all four species (Figure 1, line X). Theimmediate amino (residues 1 —83) and carboxy terminal (residues153 -209) segments are comparatively more divergent than thiscore region, but they still remain well conserved when comparedto the equivalent regions of the bacterial proteins. For amino acidresidues 1-83 and 153-209 the hamster: rat divergence is 17%and 27% respectively while the rat:mouse figures are 8.5% and18% respectively. Consequently these regions may be goodcandidate antigenic sites. Residues 83 — 152 are highly conservedbetween all three species. The human: rodent divergence withinthe N- and C-terminal domains is on average, 44% and 50%respectively. Adding the yeast and various bacterial ATases tothe comparison (Figure 1, line Y) reveals that the core regionis still the area of most homology although on a much reducedscale with the pentapeptide PCHRV being the longest motif totallyretained by all species except yeast which has a closely relatedsequence of PCHRI. A 16 residue core is located just N-terminalto this region and can be represented asGXPXA(A/V)RAV(G/A)XAXXXN. Homologies within the N-and C-terminal domains are dramatically reduced. It is knownthat the E.coli ada and ogt proteins and the human proteindifferentially repair C^-alkylguanine and (7*-alkylthymineresidues (36,37,38) and also show different sensitivities toinhibition by the free base C^-benzylguanine (Margison GP,


Moschel RC, Rafferty JA and Elder RH, unpublished data; 39).Whether or not such characteristics are a function of thesedivergent domains or are a consequence of the fewer differencesin the core region (especially between the mammals) has yet tobe determined.

In order to address these possibilities we have started toinvestigate the structural relationship between the mammalianATases using anti-human and anti-rat ATase pAbs inimmunoblotting and functional inhibition assays. The anti-humanpAb only inhibits the human ATase while the anti-rat pAb hasno effect on this but is an effective inhibitor of both rat and murineATase (21). However, the hamster protein does not interact witheither pAb in a way which influences ATase activity. A furtherinteresting observation is that, under the conditions used, the anti-rat pAb almost completely inhibits the rat ATase but the anti-human pAb only achieves <50% inhibition. This could beexplained by the fact that the epitope(s) involved in inhibitionof function clearly differ between the two ATase proteins. Thevariations observed between the mammalian proteins have alreadybeen discussed (see above) and these might provide someexplanation for the specificity of functional inhibition.Nevertheless, because the antibodies are polyclonal it is possiblethat recognition of more than one epitope is involved in theinhibition process or equally that inhibition is due to the few butperhaps significant amino acid changes in the more highlyconserved core sector.

The use of the anti-human and anti-rat ATase pAbs for westernanalysis revealed that the Chinese hamster protein is likely tohave at least one common epitope which is shared across speciesbut it is presumably not in a region of the molecule closelyassociated with ATase activity. Even when equal amounts ofrecombinant protein are analysed by this method the rat pAbappears to recognise the human ATase more efficiently than thehamster ATase. This difference cannot simply be ascribed toepitope exposure as the proteins are being recognised in adenatured form. Therefore it is possible that the fewer differencesin the highly conserved regions of these proteins might have asignificant effect in determining epitope antigenicity.

Isolation of this cDNA should greatly facilitate gene regulationand biochemical studies relating to the role of ATase in chemicalcarcinogenesis in hamsters. As comparative sequence dataamasses, important structure/function relationships can be moremeaningfully studied and this may ultimately allow the designof inhibitors of ATase which could significantly improving theefficacy of cancer chemotherapy involving nitrosoureas andrelated alkylating agents.

ACKNOWLEDGEMENTS

This work was supported by the Cancer Research Campaign.We thank Mrs. Ann Hallam for excellent technical assistance.

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Isolation and partial characterisation of a Chinese hamster C