Molecular and Biochemical Parasitology, 53 (1992) 169-184 (.~ 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00

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MOLBIO 01761

Cloning of the gene encoding Le&hmania donovani S-adenosylhomocysteine hydrolase, a potential target for antiparasitic chemotherapy Debbie M. Henderson a. Sheri Hanson a, Thomas Allen a, Keith Wilson a, Donna E. Coulter-Karis b, Michael L. Greenberg b, Michael S. Hershfield b and Buddy Ullman a aDepartment of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, OR. USA; and bDepartment of Medicine, Duke University Medical Center, Durham, NC, USA (Received 10 December 1991; accepted 14 February 1992)

A full-length gene encoding the S-adenosylhomocysteine hydrolase (AdoHcyase) enzyme has been isolated from a genomic library of Leishmania donovani DNA in 2GEM-II by cross-hybridization to the full-length human AdoHcyase cDNA. The nucleotide sequence of the Sail fragment contained a single open reading frame that encoded a polypeptide of 438 amino acids (47 712 Da). After maximum gap alignment, the predicted amino acid sequence of the leishmanial AdoHcyase was 70-73% identical to AdoHCyases from higher eukaryotes. In addition, a data base search revealed that the primary structure of all AdoHcyase proteins was highly homologous to that of a protein encoded by a mRNA from Drosophila melanogaster that maps near the r element function of the Abd-b homeotic gene. In Northern blots, the Sail fragment hybridized to a 3.0-kb transcript that presumably encodes the parasite enzyme. Southern blot analysis of genomic DNA revealed that the AdoHcyase gene did not exist as a tandemly repeated array within the L. donovani genome. Moreover, monoclonal antibodies generated against human AdoHcyase recognized a leishmanial protein on immunoblots. Finally, the growth of L. donovani promastigotes could be arrested by micromolar concentrations of 3-deazaaristeromycin (C3Ari) and 9-(trans-2', trans-3'-dihydroxycyclopentanyl)adenine, 2 known inhibitors of mammalian AdoHcyase. CaAri also induced a substantial expansion of the intracellular pools of both AdoHcy and S-adenosylmethionine (AdoMet), as well as a significant diminution of the AdoMet/AdoHcy ratio. Thus, AdoHcyase may have therapeutic potential for the selective treatment of diseases of parasitic origin. Key words: Leishmania donovani; S-Adenosylhomocysteine hydrolase; Methylation; S-Adenosylmethionine;Chemotherapy

Introduction Correspondence address: Buddy Ullman, Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, OR 97201-3098, USA. Tel.: 503 494 8437; Fax: 503 494 8393 Note: Nucleotide sequence data reported in this paper have been submitted to the GenBankT M data base with the accession number M76556. Abbreviations: AdoHcy, S-adenosylhomocysteine;AdoMet, Sadenosylmethionine; AdoHcyase, S-adenosylhomocysteinehydrolase; PCR, polymerase chain reaction; RT, reverse transcriptase; C3Ari, 3-deazaaristeromycin; DHCaA, 9-(trans2',trans-3'-dihydroxycyclopentanyl)adenine; PBS, phosphate buffered saline; HPLC, high performance liquid chromatography; MI, methylation index.

S-adenosylhomocysteine hydrolase (adenosylhomocysteinase; EC 3.3.1.1.; AdoHcyase) catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) to adenosine and L-homocysteine [1]. The AdoHcyase reaction is indispensable in eukaryotic cells for the normal function of all S-adenosylmethionine (AdoMet)-dependent transmethylation reactions, all of which generate as a product and are inhibited competitively by AdoHcy. Although AdoHcyase appears to be ubiquitously expressed among eukaryotes [2-7], the enzyme is not found in Escherichia coli or in many other

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prokaryotes [2]. The primary structures for the human [8], rat [9], and Dictyostelium diseoideum [10] AdoHcyases have been inferred from the nucleotide sequences of their encoding cDNAs and are highly conserved. However, the quartenary structures of AdoHcyase enzymes from phylogenetically diverse species vary considerably [2-7]. Each subunit of AdoHcyase contains 1 molecule of tightly bound N A D - cofactor which is required for catalysis [11,12]. AdoHcyases from mammals are also high affinity adenosine [13,14] and cyclic AMP [15] binding proteins and are inactivated by deoxyadenosine [ 16,17], whereas the enzyme from Dictyostelium is inactivated by cyclic AMP [18]. The critical role of AdoHcyase in regulating the overall methylation status of the cell has made the enzyme a natural target for drug design. Indeed, a variety of nucleoside analogue inhibitors of AdoHcyase activity have been developed which exhibit potent antiviral, antitumor, and antimalarial activity [19-24]. Thus, there is a rational basis for examining inhibitors of AdoHcyase activity for the chemotherapeutic manipulation of leishmaniasis and other parasitic diseases. In this manuscript, we describe the cloning and sequencing of the AdoHcyase gene from a genomic library of L. donovani DNA. In addition, we have demonstrated that the leishmanial AdoHcyase can be pharmacologically blocked, since inhibitors of mammalian AdoHcyase were potent inhibitors of L. donovani growth and induced a predictable augmentation of the AdoMet and AdoHcy pools and a notable reduction in the AdoMet/ AdoHcy ratio in the intact parasites.

Materials and Methods

Chemicals and reagents. 3-deazaaristeromycin (C3Ari) and 9-(trans-2', trans-3'-dihydroxycyclopentanyl)adenine (DHCaA) were generously provided by J.A. Montgomery of the Southern Research Institute (Birmingham, AL) and R.T. Borchardt of the University of Kansas (Lawrence, KA), respectively.

[TYP]dCTP (3000 Ci mmol l) and [~-35S]dATP (1320 Ci mmol i) were bought from New England Nuclear Corp. (Boston, MA). [laC]Adenosine (56-59 mCi mmol 1) was purchased from Moravek Biochemicals (Brea, CA). The human cDNA encoding the entire AdoHcyase protein was isolated as described [8]. All restriction and DNA modifying enzymes were obtained from either BRL Life Technologies Inc. (Gaithersburg, MD) or Boehringer Mannheim Biochemicals (Indianapolis, IN). All other materials, reagents, and chemicals were of the highest quality commercially available. Cell culture. L. donovani promastigotes were cultivated in the completely defined Dulbecco's modified Eagle - Leishmania (DME-L) culture medium initially described by Iovannisci and Uilman [25]. The parasites were grown continuously in a humidified 10% CO2 atmosphere at 26"'C. The strain of parasites used in all experiments was the DI700 clone of the IS Sudanese strain of L. donovani. Nucleic" acid isolation. Genomic DNA was prepared from the DI700 L. donovani strain as described by Davis et al. [26]. To prepare RNA, DI700 cells were resuspended in 50 mM Hepes, pH 7.5/150 mM NaCI/i mM EDTA and lysed by the addition of Sarkosyl to a concentration of 0.1%, and the RNA was isolated by the phenol/chloroform extraction procedure described by Landfear and Wirth [27]. Preparation of leishmanial genomic library. A genomic library of DI700 DNA was constructed by partial digestion of genomic DNA with Sau3A restriction endonuclease and size selecting the DNA fragments on sucrose gradients. 16--24-kb DNA fragments were ligated into the BamHI site of the bacteriophage vector 2GEM-11, a derivative of EMBL3, using the protocol described in the brochure provided by Promega (Madison, WI). Isolation of the leishmanial AdoHcyase gene. 50000 plaques from the L. donovani library

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were plated and blotted onto Nytran filters, and the filters were prehybridized in 5x Denhardt's solution/0.1% SDS/5 x SSC (0.3 M NaCI/0.03 M sodium citrate)/100/~g m l - i salmon sperm DNA/50% formamide for 4-8 h at 42°C. The filters were then hybridized for 16-20 h at 42°C in the same buffer to 106--107 cpm of the [32p]-labeled human cDNA that encompassed the entire protein coding portion of the human AdoHcyase mRNA. The human AdoHcyase cDNA was radiolabeled by nick translation with [ct-32p]dCTP as described [28]. The filters were washed twice with 2 x SSC/ 0.1% SDS at room temperature, followed by one wash in 1 x SSC/0.1% SDS at 42°C for I h and one wash at 0.1 x SSC/0.1% SDS at 50°C for 30 min. The hybridization signals from positive bacteriophage were detected by autoradiography. DNA was isolated by CsCI density gradient centrifugation as described by Sambrook et al. [29]. Subcloning and sequencing of the L. donovani AdoHcyasegene. The isolated DNA from the bacteriophage 210-12 was digested with a multitude of restriction enzymes, fractionated by electrophoresis on 0.8% agarose gels, and transferred to Nytran membranes by the method of Southern [30] as described by Ausubel et al. [31]. A 1.6-kb Sali fragment from the plaque purified bacteriophage that hybridized to the human AdoHcyase cDNA was ligated into the Bluescript KS + plasmid acquired from Stratagene (San Diego, CA) and transformed into the E. coli strain XLI-Blue. Large amounts of Bluescript vectors containing the 1.6-kb Sail fragment in opposite orientations were prepared by the large-scale alkaline lysis method described by Davis et al. [26]. The Sail fragment was then sequenced using the dideoxynucleotide chain termination method [32] with [3sS]dATP as the radiolabel using the Sequenase 2.0 sequencing kit from United States Biochemical (Cleveland, OH). Both single stranded and double stranded DNA sequencing were performed. Single stranded DNA was prepared from plasmids containing the Sail fragment as described by Sambrook et al. [29]. The DNA sequence was

obtained for the entire 1628 nucleotides from both strands of the Sail fragment using synthetic oligonucleotide primers provided by Research Genetics (Huntsville, AL). Analysis of nucleotide and deduced amino acid sequences was performed on an IBM compatible AT computer using the Sequence Analysis Program from International Biotechnologies, Inc. (New Haven, CT). Searches of the National Biomedical Research Foundation protein data base were conducted on the VAX located at the Oregon Health Sciences University. Southern and Northern hybridizations. Restriction enzyme digested DNA was electrophoresed on 0.8% agarose gels and transferred to Nytran membranes by the method of Southern [30]. For identification of specific AdoHcyase transcripts, total RNA was electrophoresed on 1.2% agarose gels containing 2.2 M formaldehyde [34] and transferred to Nytran membranes as described [33]. For hybridization of DNA to homologous probes or to the human AdoHcyase cDNA, Nytran filters were prehybridized, hybridized, and washed under high stringency conditions described above for the library screening. All Southern and Northern blots were visualized by autoradiography. Mapping the 5' terminus of the mature AdoHcyase transcript. The 5' end of the AdoHcyase mRNA was defined by amplification of the 5' terminus using the polymerase chain reaction (PCR) protocol described by Kawasaki [34]. The reverse transciptase (RT) reaction was performed in l x PCR buffer (Bethesda Research Laboratories, Bethesda, MD) containing 100 pmol of random hexamer/20 nmol of each dNTP/20 units RNasin/2 /ag total RNA from DI700 cells/200 units Superscript RNase H - RT (Bethesda Research Laboratories) in a volume of 20/~1 for 1 h at 37°C. To amplify the 5' end of the AdoHcyase mRNA, the RT reaction cocktail was adjusted to 10 mM Tris-HC1, pH 8.3/50 mM KCI/1.5 mM MgC12, and 20 pmol of each oligonucleotide primer and 5 units of Taq DNA polymerase (Perkin Elmer Cetus) were added. The sense primer, 5'-CTCGGGATCCCAACGCTATA-

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TAAGTATCAGTTTCTGTACTTTATTGY, contained a short leader with a BamHI restriction site followed by 31 nucleotides corresponding to positions 5-35 of the L. donovani mini-exon nucleotide sequence [35] that is trans-spliced onto the 5' end of mRNAs from the Trvpanosoma and Leishmania genera [36,37]. The antisense primer, 5'-GTTATCCTGAGTGGA-Y, was constructed from the nucleotide sequence corresponding to amino acids 80-84, STQDN, of the L. donovani AdoHcyase. The PCR mixture was overlayed with 50/al of mineral oil, and the sample was incubated in a Coy Instruments (Ann Arbor, MI) thermocycler at 94~'C, 55~C, 72~C, for 1 min, 1.5 rain, and 1.5 min, respectively. This cycle was repeated 30 times. The aqueous samples were loaded onto a 3% Nusieve agarose/l% HGT agarose (FMC BioProducts, Rockland, ME) gel, and a single DNA fragment of approx. 430 bp was excised and isolated by the method of Geneclean provided by Bio 101 Inc. (La Jolla, CA). The purified DNA product was digested with Sau3A at 37~C for 2 h, and an approx. 230-bp fragment was ligated into BamHl-digested pBluescript KS " and sequenced as described above. AdoHcvase assay. AdoHcyase was partially purified from extracts of L. donovani promastigotes using a Sephacryl-200 gel sieving column followed by ion exchange chromatography on a DEAE-cellulose resin as described [38]. AdoHcyase activity in the L. donovani cell lysates was then measured by the radiometric assay described by Hershfield [16]. lmmunoblotting. 30 #g of leishmanial extract was fractionated by sodium dodecyl sulfate (SDS) gel electrophoresis under the reducing conditions described by Laemmli [39]. In a parallel lane, 5 ng of affinity purified AdoHcyase from human placenta [38] was electrophoresed. After electrophoresis, the proteins were electroblotted onto Nytran filters (Schleicher & Schuell, Keene, NH) as described by Towbin et ai. [40]. The Nytran filters were incubated with a panel of 4 monoclonal antibodies that were generated against the

human AdoHcyase protein [41]. The antigenantibody complex was detected using the VECTASTAIN ABC kit from Vector Laboratories (Burlingame, CA), which utilizes biotinylated anti-human horse and mouse IgG as the secondary antibody and horseradish peroxidase as the substrate. Protein determinations. The amount of protein in the cell lysates was quantitated by the method of Bradford [42]. Growth rate determinations. The growth inhibitory and cytotoxic effects of C3Ari and DHCaA on wild type L. donovani promastigotes were determined in Costar 24-well plates using the protocol described by Wilson et al, [43]. AdoHo, and AdoMet measurements. To measure the effects of C3Ari on the intracellular pools of AdoHcy and AdoMet, exponentially growing L. donovani promastigotes were exposed for 4 h to concentrations of C3Ari between 12.5 and 100 /aM in the absence and presence of 100 /aM AdoMet. Two flasks lacking C3Ari and AdoMet were maintained in parallel as controls. Parasites were harvested by centrifugation, washed several times with PBS, and stored at - 8 0 ° C until further analysis. The intracellular AdoHcy and AdoMet levels in HCIO4 extracted cells were determined by high performance liquid chromatography (HPLC) employing the complex elution scheme described by Greenberg et al. [44].

Results

Isolation and sequencing of the AdoH O, gene. From the initial screening of the L. donovani genomic library, 4 bacteriophage that were recognized by the human AdoHcyase cDNA under conditions of stringent hybridization were carried through three steps of purification. From one of these, 210-12, a 1628-bp SalI fragment was subcloned into pBluescript K S ' A partial restriction map of this SalI

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GTCGACTAGA AAAAGTGAAA GTCTCTCTTC ATATCTCCCT CTCCTACTCA CCCTTTCATC TTCCCATTGT CCACGCTC~: CACCTGACC~: ATCGGAAGCG CTGCCGTGTT TTCTTCTTTC CCGTTTCAGT CGAACTGCAA TC

i

ATG GCG GAC TAC AAG GTA AAG GAC ATC AGC CTC GCG GAG TGG GGC CGC AAG GCG ATC GAG CTG GCT GAP, AAC GAG HET A l a Al~p Tyr Lyn V a l Lys Asp I l e S e t Lou A I a G l u Trp Gly Ar 8 Ly* Ala Ile Glu Leu Ale GLu Asn Glu

25

26

ATG CCT C.~T CTG ATG GAG CTA CGC C~. GAG TAC GGC CCC TCC CAG CCT TTG AAG GGC GCC AAG AI~ GCC GGC TGC Met Pro GIy Leu M*t Glu Lou At& ArK Glu Tyr Gly Pro S e r GI~ P r o L e u Lys G I y A l a Lys 1 1 , Ala Gly Cys

50

51

CTG CAC ATG ACT GTG CAG ACT GCC GTG CTG ATC GAG ACC CTC A / ~ GCC CTC GGT C-CG ~ GTG CGC TGG "I'CG TCG Leu 8is Met Thr Val Gln Thr Ale Val Leu II* Glu Thr Lou Lya A1a L*u G1y I f * Aap V a l Ar& Trp S * r S * r

75

76

TGC AAC ATC TTT I~C ACT CAG GAT AAC GCC GCT GCG GCC ATT GCC AAG GCT GGC GTA CCG GTG TTC GCG TGG AAG Cys Ash Ile Fho Set Thr Gin Asp Ash AIs Ala Ala Ala lie A l a Lys A l a G l y V a l P r o V a l F h . Ala Trp Lys

100

I01

C~3T GAG ACA GAC GAG GAG TAT GAG TGG ~ ATC GCC CAG ACA GTG AAG GGC TTC AEC GGT GAC GGT CTG CCG AAC GIy Glu Thr Asp Glu Glu Tyr Glu Trp Cys If* Ala GI~ Thr V a l L y s G l y P~* S i r G1y Asp G1y Leu P r o Asn

125

126

ATG ATC CTC GAC GAC GGT GGC GAC CTC ACG AAT CTG GTG ATC GAC CAC CAC CCC GAG CTC GTG CCC AAG ATC TTC Het Ile Lou A~p Asp Gly GIy Asp Leu Thr Ash Leu V s l Iio Asp H i a H i s P r o G1u Lou V a l P r o Lya II.* Phe

150

151

GGT ATC TCC GAG GAG ACC ACA ACG GGT GTG AAG AAC CTG TAC AAG ~ CTG AGC AAG GGC AAC CTC CCC ATG TGC G l y If* Sot GIu G~u Thr Thr Thr Gly Val Lys ASh Leu Tyr Lya A t 8 L e u S * r Lya G1y Asn L*u P r o Met Cy*

175

175

GCT ATC AAC GTT AAC GAC AGC GTG ACG AAG AGC AAG TTC GAC AAC CTT TAE ~ TGC CC.A GAG TCC TTG GTG GAC A l a I i o Ash V a l Asn Aap S e r V a l T h r Lys S e t Lys Ph* Asp Ash L e u T y r G1y Cys Arg G I u S * r Leu V&1 AJp

200

201

GGC ATG AAG CGC GCC A C G CAT GTA ATG ATT (;CC GGC A A G ACG TGC TGC GTG TUC GGA TAE GGT GAT GTT GGT AAG Gly Met Lyu Ar$ AI& ThE Asp Va~ Met Iio A1a Gly Ly8 Thr Cya Cys Va~ Cys G l y T y r G l y Asp V s l G l y Lys

225

226

GGC TGC GCC GOC GCC CTG ( ~ GCC TTC GGC GCT CGC GTC GTG GTA ACG GAG GTG GAC CCG AT(: AAC GCC CTG CAG GIy CTs Ala Ala Ala Lou Az8 Ale Fh* Gly A1a Az 8 V*I V*I Val Thr Glu Val A s p Pro Ile Ash AI* L*u Gln

250

251

GCC TCC A T G GAG GGC TAC CAG GTC GCT CTG GTC GAG GAC GTC A T G GCC GAT GOG CAC ATC TTC G T G ACG ACC ACC Ala SeE Her Glu Gly T y r Glm Val A1a Lou Val G~u Asp Val Met A l a Asp / t t a H i s I l e Phe V a l ~ Thr Thr

275

276

GGC AJ~ GAT GAE ATC ATC ACC TCT GAA CAC TTC CCG CAC A T G CIGC GAC GAC CK~ ATT GTG TGC AAC ATC GGC CAC G l y A s n Asp A s p ][~e I l e Th~ S e t G l u H i s Fhe Pro H i s H e r Ar 8 Asp Asp A l a 11o V a l Cys Ash I 1 e G~.y H I s

300

301

TTC GAC A C G GAG ATC CAG GTG GGA TGG CTG GAG GCA AAC GCC AAA GAG CAC GTG GAA ATC AAG CCT CAG GTG GAC Phe Asp T h r G l u l L e GLn V a l G l y T r p L e u G l u A l a A~n A l a L y s G l u H i s V a l G l u I i * Lys P r o G l n V a l Aap

325

326

CGT TAT ACC ATG GAG AAC GOC CGC CAC ATC AT