PRECURSORS OF PYRIMIDINE NUCLEOTIDE BIOSYNTHESIS FOR GRAVID ~~G~~~T~O~GY~U~ CA~~~~ENS~~ (NEMATODE METASTRONGYLOIDEA) NELLIE N. C. So,* PATRICK C. L. WONG*~

and RONALD C. Ko$

Departments of *Biochemistry and $Zoology, Faculty of Medicine, University of Hong Kong, Hong Kong (Received 201Augmt I991; accepted 27 January 1992) N. C., WONG P. C. L. and Ko R. C. 1992. Precursors of pyrimidine nucleotide biosynthesis for gravid Angiostrongyha cantonensis (Nematoda: Metastrongyloidea). International J5wnal fm Pamsit&gy 22: 427-433. Gmvid Angiosirungylur cantonens& can utilixe radiolabelled bicarbonate, orotate, uracil, uridine and cytidine but not cytosine, thymine and thymidine for the synthesis of RNA and DNA. In cell-free extracts of the worm, a phospho~bosyltransfer~ was shown to convert orotate to OMP and uracil to UMP. A similar reaction was not observed with cytosine and thymine. Uridiae was readily phosphorylated by a kinase but a similar reaction for thymidine and deoxyuridine was not found. Cytidine could be phosphorylated by a kinase or be deaminated by a deaminase to uridine. No deaminase for cytosine was detected. There was also no phosphotransferase activity for pyrimidine nucleosides in the cytosolic or membrane fractions. Pyrimidine nucleosides were, in general, converted to the bases by a phosphorylase reaction but only uracil and thymine could form nucleosides in the reverse reaction. The activity of th~idyla~ synthetase was also measured. These results indicate that the nematode synthesizes pyrimidine nucleotides by de 110~0synthesis and by uti~~tion of uridine and uracil and that cytosine and thymine nucleotides are formed mainly through UMP. The thymidylate syntbetase reaction appears to be vital for the growth of the parasite. Aim&a&--SoN.

INDEX KEY WORDS: Angiastrongyius cantonensis; pyrimidine; lie nova biosynthesis; salvage; DNA; RNA; thy~dylate synthetase; phospho~~syltransfe~e; kinase; deaminase; phosphorylase.

INTRODUCTION ANGIOSTRONGYLIMIS is an

important zoonotic disease in South-east Asia and the south Pacific region. This nematode develops from the third-stage larvae to young adults in the brain of the rat host, which then migrate to the pulmonary vessels and heart where they become gravid and oviposition occurs. More recently, the parasite has been reported in rodents in New Orleans (Campbell & Little, 1988). In humans, the parasite has been implicated as the causative agent of eodnophihc mening~n~phalitis (Alicata & Jindrak, 1970). Active biosynthesis of DNA and RNA requires a balanced supply of purine and pyrimidine nucleotides. Some of these nucleotides also take part in sustaining and regulating various metabolic activities. In general, parasitic organism5 can be divided into three categories according to their ability to synthesize py~midine nucleotides from different precursors (Wang, 1989). The plasmodia, such as

7 To whom all correspondence should be addressed.

Plasmodium knowlesi and P. falciparum,

were unable to utilize preformed pyrimidines by salvage pathways and must depend on de nova synthesis (Gutteridge & Trigg, 1970; Gero, Brown & O’Sullivan, 1984). Members of the Kinetoplastida can make pyrimidine nucleotides both by salvage and de now synthesis (Hammond & Gutteridge, 1984), whereas in trophozoite extracts of Tritrichomonas foetus and Giardia lamb&a the activities of some of the enzymes in the de nova pathway could not be detected (Jarroll, Lindmark & Paolella, 1983; Lindmark & Jarroll, 1982). Among parasitic helminths, Qhistosoma mans& was found to contain all the enzymes of de nova UMP biosynthesis (Hill, Kilsby, Rogerson, McIntosh L Ginger, 1981) and incorporate preformed pyrimidines, especially cytidine, into nucleic acids (el Kouni & Naguib, 1990). Very little is known about py~midine nucleotide biosynthesis in nematodes. We have previously studied the pathways of purine nucleotide metabolism in Angiostrongylus cantonensis and reveabd some unusual features (Wang & Ko, 1979, 1980). Since the biosynthetic process for 427

428

N. N. C. So, P. C. L. WONG and R. C. Ko

pyrimidine nucleotides in parasites may differ substantially from that in their mammalian host, identification of key metabolic differences is useful for the targeting of selective drugs. We have therefore investigated the ability of A. cantonennsis to utilize various precursors for pyrimidine nucleotide synthesis. MATERIALS

AND METHODS

Chemicals. Na[‘4C]HC0, (2.07 TBq mall’), [2-‘4C]uracil (2.00 TBq mall’), [2-‘4C]thymidine (1.90 TBq mall’), [2-14C]uridine (1.87 TBq mall’) and deoxy[5- ‘H]UMP (740 TBq mall’) were obtained from Amersham International plc, U.K. [6-?]Orotate (1.74 TBq mall’) was from New England Nuclear Corp., Boston, MA. [5-‘H]Cytosine (736 TBq mall’), [5- ‘Hlcytidine (851 TBq mall’), 2-deoxy[2Y$tridine (2.15 TBq mall’), and [f-‘%]thymine (2.09 TBq mall’) were supplied by Sigma Chemical Co., St. Louis, MO and so were 5-phosphorylribose-I-pyrophosphate (PRPP) and non-radioactive pyrimidines. Polyethyleneimine cellulose sheets (300 PEl) were supplied by Brinkman, Westbury, NJ and Silica gel u.v.~~ sheets by Schleicher & Schnell, Germany. All other reagents were of analytical grade from various sources. Source and maintenance of parasites. A. cantonensis was maintained as describe.d by Wong & Ko (1979). Briefly, the first-stage larvae from the lungs of infected Wistar albino rats were injected into Achatinajiilica. After 5-6 weeks, the thirdstage larvae were obtained by digesting the snail with 0.6% pepsin in 0.1 M-HCl at 37°C for 3 hand fed to rats by stomach intubation. The worms used in these experiments were obtained from the pulmonary vessels and hearts of sacrificed rats which had been infected for at least 4 weeks. Incubation. The collected worms were rinsed in 0.9% NaCl at room temperature, blotted lightly and weighed. About 40 mg of worms, irrespective of sex, were incubated with shaking for 10 mm at 37°C in 2 ml of Krebs-Ringer phosphate solution pH 7.4 (110 mM-NaCL4.9 mM-KCl, 1.2 mr+MgSO,, 25 mM-sodium phosphate) containing 5.5 mMglucose. Various radiolabelled precursors were added and the incubation continued for 2 h. The worms were then removed, rinsed in 50 ml ofKrebs-Ringer phosphate, blotted with filter paper and homogenized in 1 ml of 5% (w/v) ice-cold TCA. An aliquot of 50 ~1 of the homogenate was counted for radioactivity (total uptake) in Aquasol 2. A control experiment was performed in parallel in which worms were removed and homogenized immediately after addition of precursor (zero-time control). All worms remained lively with wriggling motion throughout the incubation period. Incorporation of precursors into nucleic aciak After incubating with various radiolabelled precursors as described above, the worms were washed and homogenized in 1 ml 0.25 M-NaOH containing 0.5 mg of calf thymus DNA and 0.05 mg of the corresponding unlabelled precursor. The contents were incubated at 37°C for 16 h. DNA and proteins were precipitated by addition of 2 ml ice-cold 5% TCA. The precipitate was sedimented by centrifugation, washed once with 5% TCA and collected onto a cellulose nitrate disc (0.45 pm in diameter). The radioactivity in the filter disc was used

to represent the amount incorporated into DNA of the worms. For measurement of incorporation into RNA, worms incubated with various radiolabelled precursors described above were collected, washed and homogenized in 1 ml of 1% (w/v) SDS containing 0.5 mg of baker’s yeast transfer RNA and 0.25 mg of the corresponding unlabelled precursor. The homogenate was mixed immediately with 1 ml of 5% ice-cold TCA and kept at 4’C for 16 h. The resulting RNA precipitate was washed with 5% TCA and treated with 1 ml of 0.3 M-KOH at 37°C for 16 h. After that, 0.1 ml of 3 Mperchloric acid was added and the radioactivity in an aliquot of 0.1 ml of the supernatant was determined. Separation of nucleobases of RNA. The nucleobases containing radiolabel in the RNA precipitate obtained as described above were identified. The RNA fraction was hydrolysed at 100°C in 1 ml of 12 M-perchloric acid for 1 h. After neutralizing with KOH, the mixture was kept in ice for 2 h and centrifuged. The supernatant was freeze-dried and the contents redissolved in 100 ~1 of 0.05 M-HCl. A sample of 10 ~1 was chromatographed on thin layer silica gel plates (t.1.c.) using a mixture of chloroform and methanol (85:15). The R, values for cytosine, uracil and thymine were 0.02.0.2 and 0.32, respectively. The bases were visualized under u.v., cut out from the chromatogram and the radioactivity determined directly. Alternative methodsfor isolating DNA and RNA. For some preparations, a method used to prepare ‘genomic’ DNA was employed. Worms (40 mg) incubated with radiolabelled precursors (as before) were suspended in 400 ~1 of a solution containing 20 mmTris-HCl pH 7.5, 1 mM-EDTA, 2% (w/v) SDS, 0.5 mg mll’ proteinase K and then digested at 50-55°C for 16 h. The digest was extracted twice with 400 ~1 of phenol and then once with 400 ~1 of a mixture of phenol, chloroform and isoamylalcohol (50:49:1). Absolute ethanol (1 ml) was then added followed by sodium acetate pH 5.6 to a final concentration of 0.2 M. The precipitated DNA, referred to as genomic DNA, was washed with 70% ethanol, dried and suspended in 100 ~1 of H,O for the determination of radioactivity. A method different from the one described above was used to prepare RNA in some instances. Radiolabelled worms (40 mg) were mixed with 200 ~1 of guanidinium thiocyanate containing 0.75 M-sodium citrate pH 7.0, 10% (w/v) N-lauroylsarcosine (sodium) and 14.2 M2-mercaptoethanol. After digesting at 2o’C for 10 min, 20 ~1 of sodium acetate pH 4 was added. The mixture was extracted once with 100 ~1 of phenol followed by 20 ~1 of chloroform/isoamylalcohol (49 1). The final aqueous layer was transferred to 240 ~1 of propan-2-01. The precipitated RNA was collected by centrifugation and washed in 70% ethanol. This RNA was then treated with 40 units of ribonuclease-free deoxyribonuclease (DNase; in 40 mM-TrisHCl pH 8.5 and 6 mM-MgCl,) for 1 h at 37’C, precipitated with 1 ml of absolute ethanol and washed with 80% ethanol. The radioactivity in this RNA fraction (DNase-treated RNA) was determined. Enzyme extract. Freshly collected worms were washed in 0.9% NaCl, and homogenized in 4 vol of a solution containing 0.1 M-Tris-HCl pH 7.5, 0.1 mM-EDTA and 5 m&r-2-mercaptoethanol. After centrifugation at 3O,C@Og for 30 min, the supernatant was further centrifuged at 105,000 g

429

Pyrimidine nucleotide precursors in A. cantonensis for 1 h. The activities of enzymes in the supernatant (cytosol) and pellet (microsome) obtained in the second centrifugation step were determined. The pellet was suspended by sonication in 200 ~1 of extraction buffer. Proteins were assayed by the method of Bradford (1976) using bovine serum albumin as standard. Enzyme aways. All enzyme activities were determined at 37’C using the linear portion of a plot of product formation against time. Unless stated otherwise, reactions were. started by addition of enzyme and terminated by rapidly applying 5 ~1 of the reaction mixture onto the t.1.c. plate. After the chromatograms were developed, radioactivities associated with the spots containing both product and substrate were determined by counting the cut-out areas directly. For nucleoside kinases, the reaction mixture contained 0.1 MTris-HCl pH 7.5,s mM-MgCl,, 5 mM-ATP, either 200 PM of [2-“Cluridine (5000 d.p.m. nmol- ‘), or 40 fly of [2-14C] 2-deoxyuridine (28,600 d.p.m. nmol-‘), or 400 FM of [S- ‘Hlcytidine (1250 d.p.m. nmol-‘) or 200 PM of [2-‘Qthymidine (4730 d.p.m. nmolV’) and enzyme extract in a total volume of 100 ~1. The nucleotides (remaining at origin) and nucleosides were separated in a sheet of PEI cellulose developed in methanol/H,0 (1:l). Redevelopment of the chromatogram in 0.55 M-LiCl containing 0.2% (v/v) formic acid separated the mono-, di- and triphosphates of the nucleosides. For nucleoside phosphorylase measured in the direction of nucleobase formation (catabolic), the reaction mixture contained 0.1 M-Tris-HCl pH 7.5, 5 mM-MgCl,, 5 mM-Na,HPO, either 200 pM-[2-Y]uridine or 200 PM-[~‘Qthymidine, or 400 PM-[5-‘Hlcytidine all having specific radioactivities similar to the nucleoside kinase reaction, or 200 flM-[2-14C]2’-deoxyuridine (20,000 d.p.m. nmol-‘) together with enzyme extract in a final volume of 100 ~1. The nucleosides and bases were separated by chromatography in silica gel thin layers. For uridine and thymidine, the chromatograms were developed in chloroform/methanol/ glacial acetic acid (44:3:3 by volume), the respective R, values for uracil, uridine, thymine and thymidine being 0.28, 0.03, 0.45 and 0.14. For cytidine, the developing solvent was chloroform/methanol/acetic acid in proportions of 28: 12:2. For this system, the R, values were cytosine, 0.23; cytidine, 0.15; uracil, 0.58 and uridine, 0.40. For the anabolic direction, the reaction mixture contained 0.1 M- Tri-HCl pH 7.5, 10 mM-o-ribose-l-phosphate, 5 mM-MgCl,, either 200 j&+[2-14C]thymine (21,200 d.p.m. nmol-‘), or 200 PM-[2“C]uracil (4352 d.p.m. nmol-I), or 400 PM-[5-‘Hlcytosine (4672 d.p.m. nmol-‘) and enzyme extract in a final volume of 100 ~1. The chromatographic systems were the same described for the catabolic reaction. For the phosphoribosyltransferases, the reaction mixture contained 0.1 M-Tris-HCI pH 7.5, 5 mM-PRPP, 5 mM-MgCl,, either 20 pM-[2-‘4C]uracil (40,700 d.p.m. nmol- ‘), or 20 PM-[2‘Qhymine (240,000 d.p.m. nmol-‘), or 20 p~-[5‘Hlcytosine (3 1,800 d.p.m. nmol-I), or 40 pM-[6-‘4C]orotic acid (34,500 d.p.m. nmol-‘) and enzyme extract in a total volume of 100 ~1. Substrate and products were separated as described for the nucleoside kinases except for the orotate assay in which the chromatogram was developed in 0.2 MLiCl pH 5.5. R, values were OMP, 0.00; UMP, 0.11 and orotate, 0.35. The nucleoside phosphotransferase activity

was assessed by a method modified from Nelson, LaFon, Tuttle, Miller, Miller, Krenitsky, Elion, Berens & Marr (1979). The reaction mixture contained 0.1 M-sodium acetate pH 5.4, 50 mi+MgCl,, 10 mw-p-nitrophenyl phosphate, either 200 pM-[2-‘4C]thymidine, or 200 pM-[2-14C]uridine, or 400 phi-[S’H]cytidine with specific radioactivities described for the nucleoside kinase reaction, together with enzyme extract in a total volume of 100 ~1. Products and substrate were separated by t.1.c. as described for the kinase. The deaminases were determined by incubating the enzyme extract in 0.1 M-Tris-HCl pH 7.5, 5 mM-MgCl, with either 400 PM-[5-‘Hlcytosine or 400 pM-[5-‘Hlcyytidine (specific radioactivity described for the phosphorylase reaction) in a total volume of 100 ~1. The products were separated in silica gel developed in chloroform/methanol/acetic acid (28: 12:2) as described before. Thymidylate synthetase was determined by the method of Hunston, Jones, McGuigan, Walker, Balazarini & De Clercq (1984). The reaction mixture contained 0.1 M-Tris-HCl pH 7.4, 15 mh+Zmercaptoethanol, 5 mM-formaldehyde, 1 mM-tetrahydrofolate, 10 PM-~'deoxy[S-‘H]UMP (11,200 d.p.m. nmol-I) and enzyme extract in a total volume of 100 ~1. The reaction was terminated by addition of 150 ~1 of a suspension of activated charcoal (100 mg in 1 ml of 20% TCA). After centrifugation, 50 ~1 of the supernatant was counted for radioactivity. All enzyme activities were expressed as pmol of product formed per min per mg of protein.

RESULTS

Uptake and incorporation into nucleic acid The uptake of radiolabelled precursors into A. cantonensis was determined by measuring the total radioactivity in intact worms. In Table 1, the amounts accumulated after 2 h of incubation are described. Radioactivities with associated the worm homogenates in zero-time control samples were < 2% of that at 2 h. Approximately 10-20, 9-l 1, 7-8, G-8 and 4-S’/& respectively of the added bicarbonate, orotate, uracil, uridine and cytidine were taken up whereas ~2% of the added cytosine, thymine or thymidine were incorporated. Radioactivities incorporated into DNA and RNA fractions are also shown in Table 1. These results represented the lower limits of the rates of the biosynthetic processes as no attempt was made to determine the specific radioactivities of the end products. About 27% of the radioactivity associated with the bicarbonate that was taken up was found in the ‘nucleic acid’ fractions while the figure for orotate was about 13%. Approximately 12% of the radiolabelled uridine taken up was incorporated into nucleic acids compared with only about 6% for uracil. A small amount of the radioactivity associated with cytidine was found incorporated into RNA. Very little radioactivity was found in DNA or RNA when cytosine, thymine or thymidine was presented to the worms. The results for the incorporation of uridine

430

N. N. C. So, P. C. L. WONCand R. C. Ko TABLE I-INCORPORATION

OF RAD~OLABELLED PRECURSORS INTO NUCLEIC ACIDS OF GRAVID CcPltOnWlSiS

Precursor

A.

AFTER2HlNCUBATlON Incorporation(nmol

Concentration

per g fresh weight)

(flM

Bicarbonate Orotate Uracil Uridine Uridine Cytosine Cytidine Thymine Thymidine Thymidine

;; 4 4 4 4 4 4 4

Total uptake

DNA fraction

RNA fraction

57.83 f 26.76 i 10.10 f 16.90 f 13.93 + 2.07 f 5.95 f 2.24 f 2.74 f 1.61 f

1.50 * 0.13 0.90 f 0.10 0.03 f 0.01 0.30 f 0.03 0.68 f O.IO*