17

Molecular and Biochemical Parasitology, 48 (1991 ) 17-26 © 1991 Elsevier Science Publishers B.V. / 0166-6851/91/$03.50 A D O N I S 016668519100284V MOLBIO 01581

Cloning of a cDNA encoding phosphofructokinase from Haemonchus contortus R o n a l d D. Klein 1, Eric R. Olson 1, M. A n n e F a v r e a u 1, Christal A. W i n t e r r o w d 2, Nicole T. H a t z e n b u h l e r 3, M a r y H. Shea 3, Susan C. N u l f 2 and T i m o t h y G. G e a r y 2 1Molecular Biology Research, 2Animal Health Therapeutics Research, and 3Biopolymer Chemisto', Upjohn Laboratories, Kalamazoo, M1, U.S.A. (Received 27 December 1990; accepted 29 March 1991)

Phosphofructokinase (PFK), the key regulatory enzyme in glycolysis, has been cloned from the pathogenic parasitic nematode Haemonchus contortus by functional complementation in Escherichia coli. An E. coli strain deleted for both PFK loci (strain DF1020) was transformed with plasmid D N A from a 2ZAP II H. contortus c D N A library. Two out of 3 x l07 transformants were able to grow on minimal medium with mannitol as the sole carbon source. A plasmid, pPFK, containing a 2.7-kb insert, was isolated from one of these transformants and conferred on DFI020 the ability to grow on mannitol (the PFK phenotype). The complemented cells contain PFK enzyme activity, absent in the E. coli mutant, at levels considerably higher than in wild type E. coli. Sequence analysis of the 2.7-kb insert shows an open reading frame that predicts a 789-amino acid protein that has approximately 70% similarity to mammalian PFKs. The amino acid sequence around asp182, thought to be the catalytic site, is completely conserved from nematodes to mammals. Key words: Haemonchus contortus; Phosphofructokinase; Cloning; Complementation; Expression

Introduction Haemonchus contortus, an important pathogen of sheep and cattle, is representative of a large number of economically significant gastrointestinal nematodes which parasitize ruminants. Discovery of drugs effective against these organisms is hampered by the inability to recover them in sufficient quantity to isolate proteins for study and screening.

Correspondence address: Ronald D. Klein, Molecular Biology Research, Upjohn Laboratories, Kalamazoo, MI 49001, U.S.A. Note." Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M59805. Abbreviations." PFK, phosphofructokinase; pfu, plaque-forming units; PEPS, phosphoenolpyruvate synthase; PEPCK, phosphoenolpyruvate carboxykinase; IPTG, isopropylthiogalactoside; PEG, polyethylene glycol; PTS, phosphotransferase system; amp R, ampicillin resistance; ORF, open reading frame.

Ruminant parasites grow poorly in convenient laboratory animals and are as yet intractable to culture. Obtaining them routinely from ruminants, even in small quantities, is tedious and expensive. These difficulties can be partially circumvented by the identification, characterization and cloning of genes encoding potential drug targets and their expression in an appropriate system for the production of material for biochemical study. Pathways for energy generation in parasitic helminths differ from those in mammals and have long been proposed as targets for anthelmintic drugs. Nematodes which parasitize the gastrointestinal tract are thought to be completely dependent upon glucose for the generation of energy [1-3]. These parasites share an anaerobic mitochondrial pathway for energy production which results in the excretion of succinate and other fatty acids [1 5]. Some, including H. contortus, are also capable of aerobic metabolism [6,7], while others,

18

including the best-studied parasitic nematode, Ascaris suum, are not [4]. In any case, gastrointestinal parasites are apparently incapable of using amino acids or fatty acids as energy sources. Several enzymes in parasite energy metabolism appear to be potential targets for selective inhibition, including phosphofructokinase (PFK; ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11), the key regulatory enzyme in glycolysis [8-10]. The enzyme from A. suum and other nematodes is, like mammalian PFK, inhibited by ATP and stimulated by several cofactors, including A M P and hexosebisphosphate [8-12]. Phosphorylation of PFK occurs in mammals and nematodes, but the nematode enzyme is different in that phosphorylation markedly enhances activity. In mammals, phosphorylation of PFK has variable effects, ranging from inhibition to mild stimulation (see refs. 13 15 and references therein). Based on this and other criteria, PFK from a variety of parasitic helminths is biochemically distinct from those of mammals. Pharmacological data support the argument that helminth PFK represents a selective drug target. Trivalent antimonials, especially stibophen, are effective but toxic anthelmintics [16]. Considerable evidence exists to suggest that these drugs inhibit PFK from nematodes, cestodes and trematodes 80 100x more potently than mammalian PFK, and that lethal effects in parasites are associated with the inhibition of PFK [17-20]. We based our decision to clone PFK on 3 factors: its demonstrably critical role in parasite glycolysis; clear evidence for biochemical differences between the enzymes from host and parasite; and pharmacological data suggesting that selective inhibition of the parasite enzyme is possible and is lethal. This report describes the isolation, via functional complementation cloning in Escherichia coli, of a c D N A encoding H. c o n l o r t u s PFK.

Materials and Methods

Materials Materials, including restriction enzymes and DNA modifying enzymes, have been described previously [21,22]. Cloning cDNA library construction. Immature (21day infections) H. contortus adults obtained from the abomasum of an experimentally infected sheep were used for m R N A isolation. Both genders of worms were used. Immature organisms were selected to avoid the preponderance of messages for egg and sperm production anticipated from sexually competent adults, m R N A was isolated by guanidinium isothiocyanate (Sigma Chemical Co., St. Louis, MO) extraction [23] of 2.44 g worms frozen and crushed in liquid N2. Following CsC1 centrifugation and poly(A) ~ m R N A selection on oligo (dT) cellulose (Pharmacia, Piscataway, N J) as described [24], a c D N A library was constructed by Stratagene, Inc. (LaJolla, CA) in 2ZAP II [25]. The titer of the amplified library was 5 x 10 9 pfu ml-~, with insert sizes ranging from 500 bp to 10 kb and an insert frequency of approximately 95% as determined by the manufacturer. Strains, media and growth conditions. E. coli strain DFI020 ((F , pro82, supE44, 2 , ApJkB201, endA1, recA56, A(rha-pjkA)200, thi-1, hsdR17), CGSC #6194, deposited by D. Fraenkel, Harvard University) was obtained from the E. coli Genetic Stock Center (Yale University, New Haven CT). E. coli strain E1607 is a derivative of H3-5 (K37 PEPS , PEPCK ; ref. 26) containing an F' that carries LACI q and chloramphenicol resistance (unpublished data). E1628 is DF1020 containing the F' from E1607. E. coli strain W3110 was a gift from F. Neidhart (University of Michigan; ref. 27). M9 and M63 based minimal media [28] and YT medium [24] were used as indicated. Succinate, mannitol, mannose, glycerol and glucose were used at 0.4% final concentration and proline was at 0.023%.

19

Ampicillin, chloramphenicol and IPTG were used at 50 /xg ml-~, 20 /tg ml-1 and 1 mM, respectively. For growth curves, cells were grown overnight in the specified medium at 37°C, subcultured into fresh medium the next day (1:100 dilution) and the density of the culture monitored over time at A550. Doubling times were calculated as described [29]. 2 Z A P II-cDNA library excisions and transductions. Ten separate in vivo excision reactions with the 2ZAP II H. contortus c D N A library were done, as per the manufacturer's instructions, to achieve a complete representation of the library. Each excision reaction was carried out as described [25]. E. coli XL-1 Blue (0.2 ml of Ass0 = 1, grown in 2 x YT medium) were infected with 0.2 ml of the c D N A library (2 x 10 9 p f u m l - l) and 1/,1 of R408 helper phage (7 x 10 I° pfu m l - l ) . The infected cells were incubated at 3T'C for 15 min, added to 5 ml 2 x YT and shaken at 37°C for 2 h. The resulting lysate was heated at 70°C for 20 min and stored at 4°C. 0.2 ml of each 'excision pool' was then used to infect 0.5 ml of E. coli strain E1628 (Asso = 1) in 2 x YT medium at 37°C for 15 min. The cells were then centrifuged, washed with 1 x M9 salts, plated onto a 150-mm selection plate (M63-mannitolproline-ampicillin) and incubated at 37~C. Aliquots from 2 of the samples were titered on AB2 plates (antibiotic medium 2, Difco Laboratories, Detroit, MI) supplemented with ampicillin to determine the efficiency of excision and infection reactions. Each pool resulted in 1-5 x 106 amp r transductants. Colonies which grew on the mannitol selection plates were purified on the same medium.

Kemerer et al. [31]. The oxidation of N A D H catalyzed by ~-glycerophosphate dehydrogenase was monitored at 340 nm in a Gilford Response spectrophotometer. All enzymes and reagents were obtained from the Sigma Chemical Company (St. Louis, MO). Cells were harvested from log phase cultures grown on mannitol in minimal medium by centrifugation. The cells were resuspended in an equal volume of 0.1 M sodium phosphate, pH 7.2/1 mM E D T A and disrupted by sonication with a Vibracell microtip (Sonics and Materials, Inc., Danbury, CT) at the maximum setting. Supernatants were obtained by centrifugation at 10000 rev./min for 10 min using an SS34 rotor in a Sorvall RC5C centrifuge. Protein concentration was estimated with a Total Protein Kit (Biuret method) using the protocol provided by the manufacturer (Sigma Chemical Company, St. Louis, MO). Preparation of DNA. Large and small scale D N A isolations were carried out using the alkaline lysis method [21,24]. Plasmid D N A for sequence analysis was prepared from 10 ml cultures using the alkaline lysis [21,24] or PEG precipitation method [32]. D N A restriction fragments were isolated by electroelution as described previously [33] or using a Gene Clean kit (BIO 10, LaJolla, CA) following the protocol provided by the manufacturer. Sheep genomic D N A was purchased from Clonetech, Inc. (Palo Alto, CA).

Bacterial matings. Cells were grown in LB medium [28] to A550 = 0.5; donor and recipient cells were mixed in an equal ratio and incubated for 2 h with gentle shaking at 37°C. This mating mixture was centrifuged, washed in 1 x M9 salts, plated onto selective plates and incubated at 37°C.

Southern hybridization analysis. Approximately 5 /tg of H. contortus D N A and 15 #g sheep genomic D N A was fractionated in 0.7% agarose gels as described [22] and transferred to Gene Screen Plus (Dupont, Boston, MA) as per the manufacturer's instructions. Isolated restriction fragments to be used as hybridization probes were labeled with [~-32p]dATP (Amersham Corp., Arlington Heights, IL: 3000 Ci mmol l) by random priming as described [22],

Enzyme assay. P F K activity was measured by a linked enzyme assay [30] as modified by

Plasmid construction. The plasmid p P F K contained a 2.7-kb c D N A insert cloned into

20

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Fig. 1. Restriction map and strategy for sequencing the pPFK c D N A . The restriction fragments used in subcloning are shown at the top of the figure. EcoRl fragments A and B are 2.1 and 0.6 kb, respectively, and were used to construct plasmids p P F K A and pPFKB. HindIIl fragments C and D are 1.4 and 1.3 kb, respectively, and were used to construct plasmids p P F K C and p P F K D . The BamHl fragment E is 0.3 kb and was used to construct plasmid pPFKE. The open box indicates the open reading frame encoding PFK with relevant restriction sites in the pBluescript II SK polylinker indicated to the left and right. The independent gel readings used to construct the final sequence are indicated by horizontal arrows. The scale in bp is indicated. Restriction enzyme cleavage sites are indicated by vertical lines and are: EcoRl, BamHl, Sall, Clal, Hindlil (Hdlll), Xbal and Xhol.

the EcoRI site of the plasmid vector pBluescript II SK. Fragments for subcloning were identified by restriction enzyme mapping (see Fig. 1). The following subclones were used: pPFKA, with a 2.1-kb EcoRI fragment which included the amino terminus of the cDNA; pPFKB, with a 600-bp EcoRI fragment which included the carboxy terminus; pPFKC, with a 1.4-kb HindIII fragment from the 5' half of the clone; p P F K D , with a 1.3-kb HindIII fragment from the 3' half; and pPFKE, with a 300-bp BamHI insert that contained part of the 5' region. The plasmid pBluescript KS (Stratagene, Inc., La Jolla, CA) served as the vector for these constructions.

DNA sequencing. All DNA sequence analysis was by the dideoxy nucleotide chain termination method of Sanger et al. [34] with double

stranded plasmid D N A as the template using a Sequenase Kit (United States Biochemical Corp, Cleveland, OH) as per the manufacturer's instructions and as described before [35]. Both strands of the cDNA insert were sequenced.

Computer analysis. Analysis of nucleotide sequences were performed using a VAX computer and D N A program package available from the University of Wisconsin Genetics Computer Group [36]. DNA sequences were from GenBank (release 59.0); peptide sequences were generated using the DNA program package, and protein sequence identities were assessed using the BESTFIT and GAP programs and FASTP algorithm [37].

21

Results and Discussion E. coli has 2 genes coding for PFK, designated P F K A and PFKB. A mutant deleted for both of these (DF1020) is unable to utilize any sugar entering glycolysis above fructose-l,6-diphosphate. The rationale for isolating an H. contortus P F K gene was based on the idea that this P F K gene would complement E. coli DF1020 for growth on mannitol. Due to the low efficiency of transformation of our library into DF1020, we decided to screen for functional complementation by transduction. Since the transducing particles generated from the )~ZAP II library require the presence of an F' in the recipient cell, ail F' was first introduced from E1607 into D F I 0 2 0 as follows. The donor strain, E. coli E1607, cannot grow on 4-carbon dicarboxylic acids and contains an F' carrying chloramphenicol resistance and LACI q, whereas the recipient, DFI020, is chloramphenicolsensitive and can utilize 4-carbon dicarboxylic acids as a sole carbon and energy source. The strains were mated and transconjugants selected on M9 plates with succinate, proline and chloramphenicol. One of these transductants, E1628, was purified and found to be unable to grow on M9 plates supplemented with mannitol and proline and was used in the complementation experiments. The phage cloning vector 2ZAP II has the unique property of allowing the in vivo excision of a plasmid containing the recombinant D N A insert of interest, a ColE~ origin for plasmid replication, and an fl bacteriophage origin of replication for positive strand synthesis. The plasmids can then be introduced into the desired E. coli recipient by transformation, where they will replicate via the plasmid origin of D N A replication and confer ampicillin resistance to the cell. If one of these phagemids contains a c D N A insert that confers a functional complementing phenotype on a suitable E. coli host when grown on the appropriate selective medium, that plasmid can be isolated from the library. Ten aliquots of the 2ZAP II H. contortus c D N A library were used to generate transduIsolation o f pPFK.

cing particles containing the excised c D N A phagemids. These phagemids were introduced into E1628. A portion of 2 of the aliquots was used to determine the number of amp R transductants and the rest were spread onto M63 plates supplemented with mannitol, proline, chloramphenicol, ampicillin and IPTG. Two aliquots, 3 and 7, gave 4 x 106 and 2 x 106 transductants, respectively; thus, approximately 3 × 107 clones were screened for PFKcomplementing activity (10 aliquots). After incubation at 37°C for 3-4 days, 2 colonies appeared on the selection plates. These colonies were purified on identical media and plasmids isolated from each were used to retransform E1628. A transformant from one of these plasmids, strain E1688, grew on mannitol minimal media and was shown to contain a plasmid isolated from the 2ZAP II c D N A library. The plasmid from E1688, p P F K , contained a 2.7-kb insert and, when introduced by transformation into DF1020, was found to confer the same growth phenotype. These data demonstrate that the P F K phenotype was mediated by the plasmid pPFK. Table I shows the growth characteristics of E. coli strain DF1020[pPFK] on mannitol and glycerol (which enters the glycolytic pathway TABLE I Growth of strains on glycerol or mannitol Host ~

Plasmid

Carbon b source

Doubling c Final Ass0 time (h)

DF1020 DF1020 DF1020 DF1020 W3110

None None pPFK pPFK None

Glycerol Mannitol Glycerol Mannitol Mannitol

1.5 3.5 1.5 1.5 1.1

4.0 0.08 2.8 3.8 4.6

a Overnight cultures of W3110, DF1020 and DFI020[pPFK] were grown on M9 supplemented with glycerol and proline and M9 supplemented with mannitol, proline and IPTG, respectively. They were diluted I:100 into the indicated media and the A55o monitored. E. coli strain W3110 is F , IN (rrnD-rrnE) and has both loci (PFKA and PFKB) encoding PFK. Media was M9 supplemented with proline at 0.023%. IPTG was added to the DF1020[pPFK] culture grown in mannitol. CDoubling times were calculated from growth curves during logarithmic growth.

22

below PFK). The transformed strain, DF1020[pPFK], had growth characteristics comparable to that of a wild-type E. coli strain, W3110, when mannitol was the sole carbon source. Interestingly, DF1020[pPFK] grew very slowly on plates containing glucose as a carbon source, even though it should be able to bypass the PFK mutation. The inability to utilize glucose is thought to involve a defect in the phosphotransferase system (PTS) that is regulated by PFK [38] by a poorly understood mechanism. Neither DF1020[pPFK] nor E1688 grew well on glucose, indicating that the defect in PTS is not overcome by the restoration of glycolysis per se (data not shown). Apparently, H. contortus PFK does

not possess the PTS regulating activity of the E. coli enzyme. We did not observe an increase in cellular PFK activity or the rate of cell growth when IPTG was present. Phosphorylation of PFK is a general phenomenon, and PFK from A. suum is activated by phosphorylation [39]. We do not know if phosphorylation activates the H. contortus enzyme or if it is phosphorylated in E. coli. Phosphorylation can be detected on serine and threonine residues of E. coli proteins [40], but there is no evidence that E. coli possesses a cyclic AMP-dependent protein kinase similar to that which phosphorylates PFK in Ascaris [11,39]. It will be interesting to

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Fig. 2. The nucleotide sequence of the entire PFK cDNA. The most probable initiation codon is indicated at position 254 bp (the first bp 3' to the EcoRI site in the pBluescript I1 SK polylinker is designated ~l ') The amino acid sequence of the translated OR F beginning at the A T G at position 264 bp is shown below the nucleotide sequence.

23 determine the phosphorylation status of the enzyme obtained from the complemented E. coli strains and how this influences its activity. In situ, PFK from nematodes, mammals, yeast and bacteria is active in an oligomeric (generally tetrameric) configuration [8-10,41]. Whether or not the nematode PFK is assembled into tetramers in E. coli and how its activity is regulated by cofactors such as ATP, A M P and hexose bisphosphates remains to be determined. Enzymatic activity corresponding to PFK was measured in P F K - E. coli transformed with pPFK. Under the conditions employed, no ATP/fructose-6-phosphate-dependent oxidation of N A D H was detectable in DF1020. However, PFK activity of 0.12 U/mg was found in the wild type strain. Kemerer et al. [31] report a value of 0.36 U/mg in a crude homogenate of E. coli. Their disruption procedure was more thorough than that used here, perhaps accounting for the discrepancy. The corresponding value for DFI020[pPFK] was 2.04 _+ 0.2 U/rag (N = 3). The wild-type strain grows somewhat better on mannitol than the complemented strain (Table I). This strain is not isogeneic with DF1020, so the basis for this phenomenon, in spite of the much higher levels of PFK found in DF1020[pPFK], is unclear. It may reflect an inadequate regulation of the nematode enzyme in situ or compartmentalization of PFK in inclusion bodies, which we have observed by Nomarsky microscopy in DF1020[pPFK] (not shown). More work will be required to resolve this discrepancy.

Sequence of the cDNA. The plasmid p P F K was shown to contain a 2.7-kb insert (Fig. 1). D N A sequence analysis showed that the insert contained an O R F of 789 amino acids with approximately 70% similarity to both human muscle [42] and mouse liver [43] PFK. Further sequence analysis was conducted on pPFK, pPFKA, pPFKB, pPFKC, p P F K D and pPFKE. The final assembly of the composite sequence was confirmed by sequencing through the boundaries of each of the sites defining these plasmids with pPFK as a

template. The gel readings used in constructing the final sequence are summarized in Fig. 1, with the entire nucleotide sequence and the amino acid sequence predicted from the O R F presented in Fig. 2.

DNA hybridization analysis. Fig. 3 shows the results of Southern hybridization analyses of H. contortus genomic DNA digested with various restriction enzymes and probed with either the entire 2.7-kb insert isolated as an XbaI/XhoI fragment (Fig. 3A) or the 1.5-kb BamHI/EcoRI fragment from the central portion of the c D N A clone (Fig. 3B). Complex banding patterns were found with both fragments. For example, SalI cleaves the c D N A once near the 5' terminus and a genomic blot probed with the entire c D N A insert would be expected to identify only two bands. Instead, 3 bands, at approximately 5.2, 3.5 and 2.0 kb, are seen (Fig. 3A, lane 1). Cleavage with both EcoRI and BamHI would be expected to produce at least three bands probing with the entire cDNA. One band should be seen at 1.5 kb; instead, 5 bands are seen, migrating at approximately 3.7, 3.5, 2.5, 2.1 and 1.5 kb (Fig. 3A, lane 2). When the EcoRI/BamHI digest is probed with the 1.5 kb insert (Fig. 3B, lane 4), only one band, at 1.5 kb, is expected. Instead, bands at approximately 4.15, 3.0 and 1.6 kb are seen. An EcoRI/HindIII digest would be expected to produce two bands using the 1.5-kb probe, one of which should migrate at 850 bp. Instead, three bands are seen, at approximately 3.75, 1.7 and 1.55 kb (Fig. 3B, lane 3). These and the additional data presented in Fig. 3 suggest the presence of introns (containing additional restriction sites) within the genomic locus encoding PFK. In addition, several of the bands seen in Fig. 3 could be attributed to D N A sequences with significant identity to the PFK c D N A and represent possible isoforms of this gene. Attempts to show hybridization of the c D N A probe to sheep genomic D N A cleaved with EcoRI or HindIII (panel A, lanes 6 and 7) were not successful, even at low stringency.

24

A

1

2

3

4

5

6

7

B

1

2

3

4

1 USLKRNIRRLDSIVPTAGREGSDIQFNLYKGRGVAVFTS~GDSQGiw(NGAV Be

I I I IIIIIIIIIli IIIIItl MATVDLEKLRMSGAGKAIGVLTSGGDA~GMNAAV 34

1 ................

23 -

:

Sl HSVVRUGIYLGCKVCFINEGYQGUVDGGDNIVEAS~SGSDIIQKGGTII l e e

~

I I IIIIIll II II I l l l l l t l l l l l l I I I II II I I I I I 35 RAVTRMGIYVGAKVFLIYEGYEGLVEGGENIKPAN~-SVSNII~LGGTZI 84 1Q1 GSARCTDLRQREGRMKAAYNLIEKG~TNLVVIGGDGSLIGANQFRKD~G ]5~

239.4 -

,

I

IIIII

IIIIIII

IIIII

IIIIIIII

II

II

II

I

151LVKELVDTKKITPEAAKSYPNI~IVGLVGSIDNDFCGTDMTIGTDSAL~R 2 ~ II Ill II I III] I Illlllllllllllll IIIIItll 135 LLEELVKEGKISESTAQNYAHLTIAGLVGSIDNOFCGTDMTICTDSALHR 184

6.6-

4.4-

lllll

85 GSARCKAFTTREGRLAAAYNLLQHGITNLCVIGGDGSLTGANIFRNEWGS 134

9.4-

201 IIESIDAVVATAQSHQRAFVVEVUGRHCGYLALVAALACEADFCFIPE~ 250

llJ

6.6-

IIII

lilltll

llllilllllllllll

IIIIII

I illl

lBB IMEVIDAITTTAQSHQRTFVLEVMGRHCGYLALVSALASCADWLFIPEAP

I

234

261 PPVNWREILCKKLQEMRAEGQRLNIIVVAEDD.DRDGTPISSDLVKDVVA 299

[

2.22.0-

4.4-

I [ill

II

I I

I tll

IIIlll

II

I tll

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235 PEDG~ENFMCERLGETRSRGSRLNIIIIAEGAIDRHGKPISSSYVKDLVV 284 300 KTLKYDTRVTVLGHVQRGGSPSAFDRLLGCRMGAEAVLACUEMNEESEPC 349

I III[IIIIIIIIIII

llll

IIIII~II

llllill

I

II

286 QRLGFDTRVTVLGHVQRCCTPSAFDRILSSKMCMEAVMALLEATPDTPAC 334 350 VISIUVTRWY.VPLUQCVERTKAVQKAMSEKDWELAVKLRGRSFQRNLET 398

llll

2,22.0-

l~lllill

II

I III

I

II

II

III

III

II

335 VVSLSGN~SVRLPLMECVQVTKDVQKAMI)EERFDEAIqLRGRSFENNWKI 384 3gg YKLLTKLRTVEKDNLSGGQNFNVAV~NVGAPACCUNAAVRSFVRUAIVPS 448

[III

i

II

II

~lllllllI[IIll

Iil

II

I

385 YKLLAHQKVSKEKS . . . . . NFSLAILNVGAPAACMNAAVPSAVRTCISEC 429

.56-

449 LYSLRYEDSFEGLANGAFKKFQWGDVTNWUHGGSFLGTQK~LPNEKNVP 498

lllilll

I

I

I

II

IIIIII[

I II

)I

43~ HTVYIVHDGFEGLAKG~VQEVGWHDVAGWt.GRGGSMLGTKRTLPK.PHLE 478 499 LIAEQLRKHNIQALLLVGGFEAYHSTLILSKNREKYPEFCIPLCVIPCTI 548

I Iil

llllllllllllJl

I I

Ill

IIII

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II

479 AIVENLRTYNIHALLVIGGFEAYEGVLQLVEARGRYEELCIVMCVIPATI 52B

Fig. 3. Southern hybridization analysis of total D N A from H. contortus probed with a -~2P-labeled XbaI/Xhol fragment containing the entire e D N A insert (panel A) or a 1.5 kb EcoR1/BamHI subfragment of the PFK insert (panel B) (A) D N A was digested with Sall (I), EcoR1/BamHl (2), EcoRI/ tfindIII (3), EcoRl/Sall (4) and HindIIl/BamHl (8). Lanes 6 and 7 contain sheep genomic D N A digested with EcoRl (6) or Hindlll (7). (B) Total H. contortus D N A digested with Hindlll (1), EcoRl (2), EcoRl/Hindlll (3) and EcoR1/BamHl (4). Molecular size markers in kb were derived from a HindIll digest of bacteriophage )~ DNA.

549 SNNVPGTSISLGSDTAINEICTUIDKIK~SATGTKRRVFIIETUGGYCCY59B

Ilillll

lillllllll

llllll

lilllllllilllllll

529 SNNVPGTDFSLCSDTAVNAA~ESCDRIK~SASCTKRRVFIVETMGCYCCY 578 599 LAILSALASGADNAYIFEEKFTVEISLRRGSHRC.KUAQGVQRYLIVRNE 647

llll

III

III

llllll

I I

II

flll

lllIll

57g LATVTCIAVGADAAYVFEDPFNIHDLKANVEHMTEKMKTDIQRGLVLRNE 628 64B YANKNFTTEFVKQLFAEECKCEFSTRINILCHAQQGCSPTPFDRNMGTKL 697

i llllI[l

II

I~II

I

I fill

IIII

III~III

IIII

829 KCHEHYTTEFLYNLYSSECRCVFDCRTNVLCHL~QCCAPTPFDRNYCTKL 67R 698 AARALEYIITQIKESUVNGVVSTKSPERATLLGLTGRRVVFTPVEELAAE 747

I III

II

III

~ I

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I ~I I

i I I II

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I

679 GVKAMLWVSEKLRDVYRKGRVFANAPDSACVIGLRKKVVAFSPVTELKKE 728 74B TDFDKRLPCDQ~fLKLRPLCRILAKHTSIYHTEAMEDTEDYD. . . . . . . .

IIII

~II

IIIII

II

IIIIII

il

789

I

729 TDFEHRMPREQ~LNLRLMLKMLAHYRISUADYVSGELEHVTRRTLSIDK 77B

Four in-frame A T G codons precede the O R F which predicts a protein with significant homology to other PFKs. We have chosen to represent the A T G at position 254 bp as the initiation site based solely on size comparisons with other PFKs. Determination of the amino-terminal peptide sequence of the recombinant P F K and the purified H. contortus enzyme will be required to define the actual initiation codon. The comparison of H. contortus and mouse liver P F K [43] at the amino acid level (Fig. 4), matching for similar and conserved residues, reveals striking similarities and differences. Of particular interest is the absolute conservation of the amino acid sequence between residues 176-203. This sequence comprises the active Sequence analysis'.

Fig. 4. Comparison of the amino acid sequences of PFK ~ o m H. contortus (top) and mouse liver. Sequences were aligned to give maximal similarity. The vertical lines indicate conserved or identical amino acids.

site [44,45]; the catalytic residue is probably asp182, which corresponds to asp127 in the rabbit muscle gene [8,9]. A nearly identical comparison is obtained with human muscle P F K [42] (data not shown). The location of the consensus phosphorylation site sequences [46] differs between mammalian P F K s and the H. contortus enzyme. The phosphorylation site in the mammalian P F K s is found at the carboxy terminus, but no suitable phosphorylation site is located in this region of the H. contortus gene. The amino acid sequence of the phosphorylation site of

25

the A. s u u m enzyme has been determined [12] and is no more closely related to the putative phosphorylation sites in the H. c o n t o r t u s gene (for example, residues 8-12 or 629-629) than to the mammalian sequences. The location of the A. s u u m site is not known. Mammalian PFKs are encoded by at least three genes which give rise to isozymes with different tissue distribution and kinetic characteristics (see ref. 43 for references). The amino acid sequence identities between mammalian P F K s is quite high, especially for enzymes isolated from the same tissue. For example, the mouse liver and rabbit muscle P F K sequences show a 68% identity at the amino acid level, while the mouse muscle and rabbit muscle genes are 96% identical [43]. Our similarity analysis showed, under the conditions specified, human muscle P F K was 69% identical to mouse liver PFK, while the identity to H. c o n t o r t u s P F K was 56% and 54%, respectively. When conservative amino acid substitutions are allowed, the similarity was calculated as: human muscle to mouse liver, 83%; human muscle to H. contortus, 72%; mouse liver to H. contortus, 69%. Overall, identities and similarities are quite high with regions of absolute identity present throughout the gene. The P F K gene in H. contortus does not preferentially resemble the amino acid sequence of either of the 2 published mammalian isozymes.

Acknowledgements We would like to thank Drs. Gopal Kulkarni and Ben Harris for their advice and very useful conversations.

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4 Saz, H.J. (1981) Energy metabolism of parasitic helminths, adaptations to parasitism. Annu. Rev. Physiol. 43, 323 341. 5 Barrett, J. (1984) The anaerobic end-products of helminths. Parasitology 88, 179 198. 6 Ward, P.F.V. (1974) The metabolism of glucose by Haemonchus contortus, in vitro. Parasitology 69, 175 190. 7 Ward, P.F.V. and Huskisson, N.S. (1978) The energy metabolism of adult Haemonchus contortus, in vitro. Parasitology 77, 255 271. 8 Uyeda, K. (1979) Phosphofructokinase. Adv. Enzymol. Mol. Biol. 48, 193 244. 9 Evans, P.R., Farrants, G.W. and Hudson, PJ. (1981) Phosphofructokinase: structure and control. Phil. Trans. R. Soc. Lond. B293, 53 62. 10 Starling, J.A., Allen, B.L., Kaeini, M.R., Payne, D.M., Blytt, H.J., Hofer, H.W. and Harris, B.G. (1982) Phosphofructokinase from Ascaris suum. Purification and properties. J. Biol. Chem. 257, 3795 3780. 11 Hofer, H.S. Allen, B.L., Kaeini, M.R. and Harris, B.G. (1982) Phosphofructokinase from Ascaris suum. The effect of phosphorylation on activity near physiological conditions. J. Biol. Chem. 257, 3807 3810. 12 Kulkarni, G., Rao, G.S.J., Srinivasan, N.G., Hofer, H.W., Yuan, P.M. and Harris, B.G. (1987) Ascaris suum phosphofructokinase. Phosphorylation by protein kinase and sequence of the phosphopeptide. J. Biol. Chem. 262, 32 34. 13 Sale, E.M. and Denton, R.M. (1985) Adipose-tissue phosphofructokinase. Rapid purification and regulation by phosphorylation in vitro. Biochem. J. 232, 897 904. 14 Kamemoto, E.S. and Mansour, T.E. (1986) Phosphofructokinase from the liver fluke Fasciola hepatica. Purification and kinetic changes by phosphorylation. J. Biol. Chem. 261, 4346 4351. 15 Kamemoto, E.S., lltzsch, M.H., Lan, L. and Mansour, T.E. (1987) Phosphofructokinase from Fasciola hepatica: activation by phosphorylation and other regulatory properties distinct from the mammalian enzyme. Arch. Biochem. Biophys. 258, 101 111. 16 Pratt, W.B. and Fekety, R. (1986) The Antimicrobial Drugs, pp. 421~425. Oxford University Press, New York. 17 Mansour, T.E. and Beuding, E. (1954) The actions of antimonials on glycolytic enzymes of Schistosoma mansoni. Br. J. Pharmacol. Chemother. 9, 459 462. 18 Beuding, E. and Mansour, J.M. (1957) The relationship between inhibition of phosphofructokinase activity and the mechanism of action of trivalent organic antimonials on Schistosoma mansoni. Br. J. Pharmacol. Chemother. 12, 159 165. 19 Beuding, E. and Fisher, J. (1966) Factors affecting the inhibition of phosphofructokinase activity of Schistosoma mansoni by trivalent organic antimonials. Biochem. Pharmacol. 15, 1197 1211. 20 Saz, H.J. and Dunbar, G.A. (1975) The effects of stibophen on phosphofructokinases and aldolases of adult filariids. J. Parasitol. 61, 794-801.

26 21 Klein, R.D. and Roof, L.L. (1988) Isolation of a gene from Schwanniomyces occidentalis which complements a ura-3 mutation in Saccharomyces cerevisiae. Curr. Genet. 13, 29 35. 22 Klein, R.D. and Favreau, M.A. (1988) Transformation of Schwanniomyces occidentalis with an ADE2 gene cloned from S. occidentalis. J. Bacteriol. 170, 5572 5578. 23 Chirgwin, J.W., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294 5299. 24 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual, pp. 197 198. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 25 Short, J.M., Fernandez, J.M., Sorge, J.A. and Huse, W.D. (1988) 2ZAP, a bacteriophage 2 expression vector with in vivo excision properties. Nucleic Acids Res. 16, 7583 7600. 26 Hansen, E.J. and Juni, E. (1974) Two routes for synthesis of phosphoenolpyruvate from C4-dicarboxylic acids. Biochem. Biophys. Res. Comm. 59, 2401 1210. 27 Hill, C.W. and Harnish, B.W. (1981 ) Inversions between ribosomal RNA genes of Escherichia coll. Proc. Natl. Acad. Sci. USA 78, 7069 7072. 28 Miller, J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 29 Koch, A.L. (1981) Growth measurements, In: Manual of Methods for General Bacteriology (Gerhardt, P., Murray, M.G.E., Costilow, R.N., Nester, E.W., Wood, W.A., Krieg, N R . and Phillips, G.B., eds.) American Society for Microbiology, Washington, DC. 30 Racker, E. (1947) Spectrophotometric measurement of hexokinase and phosphohexokinase activity. J. Biol. Chem. 167, 843 854. 31 Kemerer, V.F., Griffin, C.C. and Brand, L. (1982) Phosphofructokinase from Escherichia coli. Methods Enzymol. 90, 91 98. 32 Kraft, R., Tardiff, J., Kranter, K.S. and Leinward, L.A. (1988) Using mini-prep plasmid DNA for sequencing double stranded templates with Sequenase. Biotechniques 6, 544 549. 33 Schleif, R.F. and Wensink, P.C. (1981) Practical Methods in Molecular Biology, pp. 124 125. SpringerVerlag, New York.

34 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463 5467. 35 Klein, R.D., Poorman, R.A., Favreau, M.A., Shea, M.H., Hatzenbuhler, N.T. and Null, S.C. (1989) Cloning and sequence analysis of the gene encoding invertase from the yeast Schwanniomyces occidentalis. Curr. Genet. 16, 145 152. 36 Devereux, J., Haeberli, P. and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387 394. 37 Lipman, D.J. and Pearson, W.R. (1985) Rapid and sensitive protein similarity searches. Science 227, 1435 1441. 38 Roehl, R.A. and Vinopal, R.T. (1976) Lack of glucose phosphotransferase function in phosphofructokinase mutants of Escherichia coli. J. Bacteriol. 126, 852 860. 39 Daum, G., Thalkofer, H.P., Harris, B.G. and Hofer, H.W. (1986) Reversible activation and inactivation of phosphofructokinase by the action of tissue-homologous protein phosphorylating and dephosphorylating enzymes. Biochem. Biophys. Res. Commun. 139, 215 221. 40 Enami, M. and Ishihama, A. (1984) Protein phosphorylation in Escherichia coli and purification of a protein kinase. J. Biol. Chem. 259, 526 533. 41 Chaflbtte, A.F., Laurent, M., Tijone, M., Tardien, A., Roucous, C., Seydoux, F. and Yon, J.M. (1984) Studies on the structure of yeast phosphofructokinase. Biochimie 66, 49 58. 42 Vora, S., Hong, F. and Olender, E. (1986) Isolation of a c D N A for human muscle 6-phosphofructokinase. Biochem. Biophys. Res. Commun. 135, 615 621. 43 Gehnrich S.C., Gekakis, N. and Sul, H.S. (1988) Liver (B-type) phosphofructokinase mRNA. Cloning, structure, and expression. J. Biol. Chem. 263, 11755 11759. 44 Poorman, R.A., Randolph, A., Kemp, R.G. and Heinrikson, R.L. (1984) Evolution of phosphofructokinase-gene duplication and creation of new effector sites. Nature 309, 467 469. 45 Lee, C.-P., Kao, M.-C., French, B.A., Putney, S.D. and Chang, S.H. (1987) The rabbit muscle phosphofructokinase gene. Implications for protein structure, function, and tissue specificity. J. Biol. Chem. 262, 4195 4199. 46 Cohen, P. (1988) Protein phosphorylation and hormone action. Proc. R. Soc. Lond. B234, 115 144.