EL S E VI E R Molecular and Biochemical Parasitology 63 (1994) 171 174

MOLECULAR AND BIOCHEMICAL PARASITOLOGY

Short Communication

Expression and cellular localisation of hexokinase during the bloodstage development of Plasmodium falciparum

P~tur Olafsson, Ulrich Certa*

Department PRTB, F, Hoffmann-La Roche, Ltd., Grenzacherstrasse 124, CH-4002 Basel, Switzerland

Received 12 November 1993; accepted 15 November 1993

Key words: Hexokinase; Transcription; Translation; Subcellular localisation

Recently, the molecular cloning and analysis of a number of glycolytic enzymes of the malaria parasite Plasmodium falciparum has suggested this pathway as a novel target for the rational design of antimalarials. In the past it was supposed that the enzymes of the parasite and the host are too similar for them to be exploited for the develop- ment of selective inhibitors. Aldolase, phosphogly- cerate kinase, lactate dehydrogenase, hexokinase and glucosephosphate isomerase have a surpris- ingly low sequence identity (26-60%) with the equivalent host enzymes, although they catalyse the same biochemical reactions [1-5].

In the case of aldolase, antibodies and espe- cially peptide inhibitors have been discovered which have virtually no influence on the activity of the human enzyme [6]. As expected, the bind-

*Corresponding author. Tel.: +41-61-688-5340; Fax: +41-61- 688-4575

Abbreviations: PfHK, Plasmodium falciparum hexokinase; SDS, sodiumdodecyl sulfate.

0166-6851/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 6 - 6 8 5 1 ( 9 3 ) E 0 1 8 3 - 9

ing site for the inhibitor peptide which is based on P. falciparum ~-tubulin, is formed by two parasite- specific lysine residues which were identified by in vitro mutagenesis of the carboxy terminus of re- combinant aldolase [7].

We have recently cloned and analysed hexoki- nase, which is the first enzyme in the glycolytic pathway and therefore of great importance [4]. Hexokinase represents another parasite enzyme for which the search for inhibitors appears pro- mising. The sequence identity to the human equivalent is as low as 26%, and the highest homology appears in functionally important do- mains of the enzyme. In addition, neither polyclo- nal nor monoclonal antibodies inhibit or cross- react with vertebrate hexokinase, which demon- strates indirectly the possibility of finding selec- tive inhibitors [4]. For the future development of hexokinase-based inhibitors we thought it impor- tant to establish the expression mode and the cel- lular localisation of the enzyme in asexual blood- stage parasites.

172 P. Olaf~son, U. Certa/Molecular and Biochemical Parasitology 63 (1994) 171 174

Activity and expression of hexokinase and aldolase during bloodstage development. We used highly synchronised parasites to prepare either protein or RNA samples for Western and Northern blot analysis. First, the relative hexokinase and aldo- lase activity was determined (Fig. l-I). In ring stages (0 h) and after schizont burst (48 h) both enzymatic activities are low and both reach a max- imum in the trophozoite and schizont stage (32-36

h). The activity ratio of hexokinase to aldolase remains virtually constant during this phase of development which may be of functional rele- vance for glycolysis (Fig. l-I). For both enzymes, the activity changes are proportional to the rela- tive amounts of protein detectable by antibodies (Fig. 1-II; blots A and B; [8]) which demonstrates regulation of protein synthesis during asexual de- velopment. This control depends apparently on

A O D A B

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P \ .o / \

/ / \

25.7

/ \ / \

/ \ 19.4

~..3

0 ~

¸¸¸¸5

(kb) 0 24 32 36

9,5 _

r,5 _

4 . 4 - -

2 . 4 - -

rRNA

i i i i b rRNA

Activity ratio: a,w,ase/hex_,_naseb4nl / nki 1.4 _

2.o~0 t ~ " - ~ - - " ~ " - ' ~ ~ ........

0 24 32 36 48 t {h) 0 .2__

Fig. 1. Hexokinase and aldolase activity, protein and RNA levels during asexual intraerythrocytic parasite development. (1) Relative hexokinase (triangles) and aldolase (dots) activities during the intraerythrocytic, asexual parasite development (ring stages - 0 and 48 h; young trophozoites = 24 h; old trophozoites = 32 h; schizonts = 36 h). To demonstrate the correlation between the aldolase and hexokinase activity the ratio is plotted below. Aldolase and hexonkinase activity was determined as described [1,4] using constant amounts of protein per assay. (II) Western blot analysis of the same samples (A, hexokinase serum; B, aldolase serum). The position of molecular weight markers is indicated on the left. (III) Northern blot analysis of the same samples using a hexokinase (A) or aldolase (B) structural gene probe. The position of RNA markers (from BRL) and rRNA is indicated at the left. The amount of total protein was determined by using a commercial antibody to ~-tubulin (Sigma) and the amount of RNA was estimated by using the intensity of the ribosomal RNAs in an ethidium bromide gel as a reference (data not shown). Rabbit antibodies to aldolase [1] or hexokinase [4] were used in Western blots and a structural gene probe for northern hybridisation under stringent conditions [4]. Parasite synchronisation was carried out by the sorbitol method essentially as described [10].

P. Olafsson, U. Certa/Molecular and Biochemical Parasitology 63 (1994) 171-174

the gene and on the point during development at which the gene product is required. The quantities of tubulin are virtually identical throughout the asexual cycle suggesting constitutive protein synthesis (data not shown). It remains to be clar- ified whether the genes of the glycolytic enyzmes hexokinase and aldolase contain specific control elements responsible for their differential expres- sion during the asexual development of the para- site.

We next used a hexokinase or aldolase structur- al gene probe to measure the mRNA levels during the asexual development in the same parasite sam- ples used for the protein analysis above. We were not surprised that the highest amounts of aldolase and hexokinase transcripts are found in tropho- zoite stages (Fig. 1-III) and only minor amounts of mRNA are detectable in ring stages and late schizonts. It thus appears that the expression of the enzymes is regulated at the transcriptional le- vel.

Cellular localisation of hexokinase. Glycolytic en- zymes including hexokinase are commonly found in the cytoplasm. A unique feature of PfHK is a potential hydrophobic membrane anchor sequence at the carboxy terminus [4]. In rat brain hexoki- nase, for example, such an anchor sequence is lo- cated at the amino terminus, which is necessary for the integration into the mitochondrial mem- brane [9]. We thus found it interesting to find out if this hydrophobic sequence is functional, which would represent an additional difference between parasite and host hexokinase. We first prepared crude subcellular parasite fractions and assayed those for the presence of hexokinase and aldolase as control (Fig. 2-I). Both enzymes are clearly pre- sent in the soluble fraction, but virtually absent in the buffer insoluble membrane containing frac- tion. Nevertheless, we used the more sensitive im- mune electron microscopy technique to detect the enzyme in situ. Out of 947 gold particles counted, a statistically significant amount (65.7%) was found in association with parasite membrane structures (Fig. 2-II). In order to establish the ex- perimental conditions, we used initially Escheri- chia coli cells expressing PfHK as specimen because the protein is abundantly made [4]. Inter-

173

estingly recombinant PfHK, made as an insoluble protein in E. coli, is found in association with the plasma membrane rather than in inclusion bodies (data not shown). These experiments favour the possibility that P fHK is indeed associated with the parasite membrane which could improve the efficiency of glucose phosphorylation at the site

I 1 2 3 4 5

~ k - - e

Aldo -..- ~

lI

Fig. 2. Subcellular localisation of hexokinase. (I) Aldolase (Aldo) and hexokinase (Hk) were co-detected in cell fractions by western blotting. Lane 1, total lysate; lane 2, soluble, cyto- plasmic fraction; lane 3, buffer insoluble fraction; lane 4, Tri- ton-X-100 soluble fraction; lane 5, detergent insoluble pellet). Fractions were prepared by differential centrifugation of para- site (K1) saponin lysates. (II) Cellular localisation of hexoki- nase. Sections of blood stage forms of the parasi te were labelled with monospecific hexokinase serum followed by pro- tein A gold labelling and silver enhancement [11]. Slide I, ring stage; slide 2, trophozoite; slide 3, schizont; slide 4, schizont at higher magnification. Bars indicate 1 #m (slides 1 3) or 0.1 /~m (slide 4).

174 P. Olafsson, U. Certa/Molecular and Biochemical Parasitology 63 (1994) 171-174

of uptake. In conclusion, the presence of hexokinase

throughout the asexual cycle and its cellular loca- lisation suggest that novel hexokinase-based anti- malarial drugs could be developed.

Acknowledgements

We thank Prof. R.M. Franklin, Dr. H. Matile and Dr. W. Rudin for suggestions and advice. We are grateful to A, Soederberg for expert advice and help during the preparation of synchronised parasites and to Y, Burki for technical assistance.

References

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[ 2] Hicks, K.E., Read, M., Holloway, S.P., Wendehals, G., Sims, P.F.G. and Hyde, J.E. (1991) Glycolytic pathway of the human malaria parasite Plasmodium falciparum: primary sequence analysis of the gene encoding 3- phosphoglycerate kinase and chromosomal mapping studies. Gene 100, 123-129.

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[8] Torii, M., Aikawa, M., Srivastava, I.K., Schmidt, M., Nakamura, K. and Perrin, L.H. (1992) Expression and subcellular location of Plasmodium Jalciparum aldolase. J. Protozool. Res. 2, 1(~15.

[9] Polakis, P.G. and Wilson, J.E. (1985) An intact hydro- phobic N-terminal sequence is critical for binding of rat brain hexokinase to mitochondria. Arch. Biochem. Biophys. 236, 328-337.

[10] Lambros, C. and Vanderberg, J.P. (1979) Synchronization of Plasmodiumfaleiparum erythrocytic stages in culture. J. Parasitol. 65, 418~420.

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