Metabolic proof-reading in Plasmodium berghei: essentiality of phosphoglycolate phosphatase

Plasmodium falciparum (Pf) 4-nitrophenylphosphatase was earlier shown to be involved in vitamin B1 metabolism by Knöckel et al., (Mol. Biochem. Parasitol. 2008, 157, 241-243). An independent BLASTp search showed that the protein had significant homology with phosphoglycolate phosphatase from mouse, human and yeast, and prompted us to re-investigate the biochemical properties of the recombinant Plasmodium enzyme. Owing to the insoluble nature of the Pf enzyme, an extended substrate screen and biochemical characterization was performed on its P. berghei (Pb) homolog that led to the identification of 2-phosphoglycolate and 2-phospho L-lactate as the relevant physiological substrates. 2-phosphoglycolate is known to be generated during repair of damaged DNA ends whereas, 2-phospho L-lactate is a product of pyruvate kinase side reaction. These metabolites are potent inhibitors of the key glycolytic enzymes, triosephosphate isomerase and phosphofructokinase, and hence clearance of these toxic metabolites is vital for cell survival and functioning. Gene knockout studies conducted in P. berghei revealed the essential nature of this conserved ‘metabolic proof-reading enzyme’.

thors had proposed a novel role for this HADSF 224 member and suggested involvement in vitamin B1 225 homeostasis (15).We found the P. falciparum 4-

Generation of P. berghei transfection vectors -
The library clone for P. berghei PGP (PbG02_B-53b06) was obtained from PlasmoGem.
The procedure for knockout and tagging construct generation was as described earlier (17,30) and is provided in detail in 'Supplementary information'.

Cultivation and transfection of P.
berghei -Male/female BALB/c mice aged 6-8 weeks were used for cultivation and transfection of P. berghei.Glycerol stock of wild type P. berghei ANKA parasites was injected into a healthy male BALB/c mouse.The parasitemia was monitored by microscopic observation of Giemsa stained smears of blood drawn from tail snip.Transfection of the parasites was done by following the protocol described by Janse et al., (31), using Amaxa 4D nucleofector (P5 solution and FP167 programme) followed by injection into 2 mice.For PbPGP knockout, drug resistant parasites were selected for by feeding infected mice with pyrimethamine in drinking water (7 mg in 100 mL), whereas parasites with PbPGP RFA-tag were selected for by feeding infected mice with trimethoprim in drinking water

8 . 7 . 4 .
226 nitrophenylphosphatase sequence to have homol-227 ogy with human, mouse and yeast phosphoglyco-228 late phosphatases.An extended substrate speci-229 ficity screen of the recombinant P. berghei en-230 zyme revealed that, indeed this protein is phos-231 phoglycolate phosphatase, which is mainly in-232 volved in detoxification, having very high activ-233 ity on 2-phosphoglycolate and 2-phospho L-lactate 234 with no activity on thiamine monophosphate.2-235 phosphoglycolate is reported to be formed during 236 repair of free radical mediated damage of DNA 237 ends (18) and accumulation of this metabolite in 238 the cell, leads to inhibition of the key glycolytic 239 enzyme triosephosphate isomerase (TIM) (Fig. 4).240 Studies on phosphoglycolic acid phosphatases from 241 yeast and mouse have demonstrated that this en-242 zyme also performs metabolic proof-reading by ca-243 tabolizing the substrates 2-phospho L-lactate and 244 4-phosphoerythronate which are products of en-245 zymatic side reactions.Activity of PbPGP on 246 4-phosphoerythronate could not be tested due to 247 non availability of the compound.2-phospho L-248 lactate, generated by phosphorylation of L-lactate 249 by pyruvate kinase, is known to inhibit phospho-250 fructokinase and 4-phosphoerythronate, which is a 251 product of GAPDH side reaction, is known to in-252 hibit 6-phosphogluconate dehydrogenase (Fig. 4) 253 (19).Due to the detrimental effect of these metabo-254 lites, it becomes essential to clear the cell of these 255 metabolic toxins.This is reflected upon by the 256 fact that phosphoglycolate phosphatase is an es-257 sential gene in mouse (20).Also, in Arabidopsis, ume on the X-axis.Gel-filtration was performed 387 with and without NaCl in the equilibration buffer.388 Synthesis of 2-phospholactate -Synthesis 389 of both D and L phospholactate was carried out fol-390 lowing available procedure (19).The details of the 391 protocol and characterization of the molecules are 392 provided in 'Supplementary information'.393 Enzyme assays -A comprehensive sub-394 strate screen comprising of various classes of 395 molecules such as, nucleoside phosphates, sugar 396 phosphates, co-enzymes, amino acid phosphates, 397 etc. was performed.The assay was carried out in 398 100 mM Tris HCl, pH 7.4, 2 mM substrate, 1 mM 399 MgCl 2 in a volume of 100 µL.The reaction mix 400 was pre-incubated at 37 ℃ for 1 minute, the assay 401 was initiated using 2 µg enzyme and the reaction 402 was allowed to proceed at 37 ℃ for 5 minutes.The 403 reaction was stopped by the addition of 20 µL of 70 404 % trichloroacetic acid (TCA) and 1 mL of freshly 405 prepared Chen's reagent (water, 6 N sulphuric acid, 406 2.5 % ammonium molybdate and 10 % L-ascorbic 407 acid mixed in the ratio 2:1:1:1) was added, mixed 408 thoroughly and incubated at 37 ℃ for 1.5 hours.409 The color developed was measured against blank 410 (reaction mix to which enzyme was added after ad-411 dition of TCA) at 820 nm.Specific activity was 412 calculated using the value of 25000 M -1 cm -1 .413 pH optimum of PbPGP was determined by 414 performing the assay in a mixed buffer containing 415 50 mM each of glycine, MES, Tris at different pH, 416 1 mM MgCl 2 and 1 mM pNPP as substrate in 100 417 µL volume .The reaction mix was pre-incubated at 418 37 ℃ for 1 minute and the assay was initiated using 419 0.2 µg enzyme and the reaction was allowed to pro-420 ceed at 37 ℃ for 2 minutes, stopped using TCA and 421 processed using Chen's reagent as described above.422 Preferred divalent metal ion was identified 423 by using 10 mM pNPP as substrate and different 424 salts such as MgCl 2 , MnCl 2 , CaCl 2 , CuCl 2 , and 425 CoCl 2 at a final concentration of 1 mM in a 250 426 µL reaction mix containing 50 mM Tris HCl, pH 427 The reaction was initiated with 0.26 µg of en-428 zyme and conversion of pNPP to p-nitrophenol was 429 continuously monitored at 405 nm at 37 ℃ temper-430 ature.Slope of the initial 20 seconds of the progress 431 curve was used to calculate specific activity using 432 an value of 18000 M -1 cm -1 .433 Kinetic studies -K m values for 2-434 phosphoglycolate, 2-phospho L-lactate and β-435 glycerophosphate was determined by measuring 436 initial velocity at varying substrate concentra-437 tions ranging from 0.5 mM to 15 mM for 2-438 phosphoglycolate and 2-phospho L-lactate and 0.25 439 mM to 30 mM for β-glycerophosphate.The con-440 centration of MgCl 2 was fixed at 5 mM with the 441 reaction buffer being 200 mM Tricine-NaOH, pH 442 The reaction in a volume of 100 µL was ini-443 tiated with 1.89 µg of enzyme, allowed to proceed 444 at 37 ℃ for 2 minutes, stopped using TCA and 445 processed using Chen's reagent as described above.446 Specific activity was plotted as a function of sub-447 strate concentration and the data points were fitted to the Michaelis-Menten equation using GraphPad prism V5 to determine the kinetic parameters (29).

Figure 3 .
Figure 3. Conditional knockdown of PbPGP and localization in P. berghei.(A-D) Schematic representation of PbPGP parental, intermediate and final RFA tagging constructs and PbPGP loci after integration.Oligonucleotide primers are indicated by vertical bars and expected PCRproduct size is represented by line between specific primer pairs.(E) PCR confirmation of parental, intermediate and final RFA-tagging construct.(F) Genotyping of the strain for integration of cassette in the correct loci.Primer pairs used are mentioned on top of the panel.(G) Western blot analysis of cell lysates of RFA-tagged parasites from mice fed with/without trimethoprim (TMP) (30 mg in 100 mL) for 6 days.The experiment was performed twice (Exp1 and Exp2) and blot from one experimental replicate is shown.Top panel is the blot probed with anti-HA antibody and the arrow mark indicates the RFA tagged PbPGP protein.

Figure 3 :
Figure 3: Conditional knockdown of PbPGP and localization in P. berghei.(A-D) Schematic representation of PbPGP parental, intermediate and final RFA tagging constructs and PbPGP loci after integration.Oligonucleotide primers are indicated by vertical bars and expected PCR-product size is represented by line between specific primer pairs.(E) PCR confirmation of parental, intermediate and final RFA-tagging construct.(F) Genotyping of the strain for integration of cassette in the correct loci.Primer pairs used are mentioned on top of the panel.(G) Western blot analysis of cell lysates of RFA-tagged parasites from mice fed with/without trimethoprim (TMP) (30 mg in 100 mL) for 6 days.The experiment was performed twice (Exp1 and Exp2) and blot from one experimental replicate is shown.Top panel is the blot probed with anti-HA antibody and the arrow mark indicates the RFA tagged PbPGP protein.The bottom panel was probed with anti-PfHGPRT antibody.(H) Ratio of the intensity of RFA tagged PbPGP to the intensity of control HGPRT.(I) Reduction in PbPGP levels upon removal of TMP relative to the levels in the presence of TMP.Protein levels under '+ TMP' condition was taken as 100 %. (J) Localization of PbPGP using RFA-tagged parasites grown in '+ TMP' condition.The erythrocyte boundary is indicated by white dotted line in the merge panel.

Figure 4 :
Figure 4: Metabolites that are substrates for phosphoglycolate phosphatase (A) and their inhibition of key metabolic pathways (B).

Table 1 :
Data represents mean ± S.E.M (N=2) V max was calculated using k cat values from Collard et al., 2016.Molecular mass values of 34540.68Da and 34624.58Da for murine PGP and Pho13, respectively was used in the calculation. * PEP, phosphoenolpyruvate. (E-G) Substrate concentration vs. specific activity plots fit to Michaelis-Menten equation for β-glycerophosphate, 2-phospho L-lactate and 2-phosphoglycolate.Substrate titration experiment was conducted in two technical replicates containing two biological replicates each.Plots from one technical replicate are shown.Each data point represents mean specific activity value and error bars represent SD (n=2).

Table 1 :
Kinetic parameters of P. berghei PGP compared with that of homologs from yeast and mouse.Data represents mean ± S.E.M (N=2) # Values taken from Collard et al., 2016 V max was calculated using k cat values from Collard et al., 2016.Molecular mass values of 34540.68Da and 34624.58Da for murine PGP and Pho13, respectively was used in the calculation. *