Substrate search for LmxMPK4

The availability of active recombinant LmxMPK4 does not only bring the development of inhibitor screenings with LmxMPK4 as a drug target within reach, but also facilitates the search for the in vivo substrate of LmxMPK4. As LmxMPK4 has been shown to play an important, although unknown, role in promastigotes and amastigotes (Wang, Q. et al.

2005) it was assumed that protein lysates of L. mexicana promastigotes or axenic amastigotes would contain the in vivo substrate of LmxMPK4 and that this substrate could be phosphorylated by recombinant active LmxMPK4. The plasmids pJCLinkerLmxMPK4-LmxPK5 and pJCLinkerLmxMPK4KMpJCLinkerLmxMPK4-LmxPK5 were transformed into E. coli BL21 (DE3)

[pAPlaclQ] cells and the proteins expressed over night at 18°C. The His-tag fusion proteins LmxMPK4 and LmxMPK4K59M were purified on Co²⁺ sepharose and not eluted, but used in a radioactive kinase assay still bound to the resin. Parasite lysates were obtained by freeze-thaw-cycles (see 3.5.1). Twenty µg of Leishmania lysates were incubated in kinase assays with approximately 1 µg co-expressed LmxMPK4 and His-LmxMPK4K59M, as well as without the addition of protein. The assays were conducted in the presence of 1 mM ATP, containing 5 µCi [γ³²]ATP (6000 Ci/mmol), 10 mM of the phosphatase inhibitors Na-orthovanadate and sodium fluoride, respectively, and LmxMPK4 kinase buffer, containing 50 mM 3(N-morpholino)propanesulfonic acid (MOPS), pH 7.0, 10 m M MnCl2 and 0.1 M NaCl (Wang, Q. et al. 2005). There is a possibility that the in vivo substrate of LmxMPK4 is contained in the Leishmania protein lysates in its activated, phosphorylated stage. If that was the case for all contained substrate molecules, activated LmxMPK4 could not phosphorylate them any further and no phosphorylation bands would be detected in the autoradiograph. To allow for this possibility one amastigote lysate was treated for 30 minutes at 30 °C with λ-phosphatase to achieve a dephosphorylation of most proteins contained in the lysate.

Figure 18. Kinase assays with co-expressed Hi s-LmxMPK4 and L. mexicana lysate s left panels, autoradiographs of kinase assays after ex posure time of 3 mont hs (A) or 70 h (B and

C), respectively; right panels, Coomassie-stained gels; A, dephosphorylated lysate of axenic amastigotes; B, untreat ed lysate of axenic amastigotes; C, promastigote lysate; lane 1, 1’, assay in addition with His -LmxMPK4, co-ex pressed with LmxMKK5; lane 2, 2’, assay in addition with His-LmxMPK4KM, co-expressed with LmxMKK5; lane 3, 3’, assay without the addition of recombinant protein; * indicates phosphorylated protein bands in kinase assays and position of excised bands in the corresponding Coomassie-stained gels; masses of standard proteins are indicat ed in kDa.

The kinase assays in which L. mexicana protein lysates were incubated with recombinant His-LmxMPK4, resulted in autoradiographs showing a very blurred image per lane, instead of the expected clearly distinct multiple bands. Of the few distinct phosphorylation bands, however, one appeared only in the presence of active His-LmxMPK4 and not in the presence of the kinase-dead mutant His-LmxMPK4K59M or in the absence of any protein. This band at the size of about 27 kDa could be detected in both the dephosphorylated and untreated amastigote samples and also in the promastigote sample (Fig. 18). An approximately 2 mm high piece of the corresponding area of the Coomassie gel was carefully excised and sent to mass spectrometry (MS) analysis. Due to the lack of an appropriate L. mexicana protein database, the identified peptides were matched to a L. major protein database and allocated to proteins. As the phosphorylation detected in the autoradiograph (Fig. 18) occurred on a protein of the size of about 27 kDa, only proteins between 20 and 40 kDa were deemed expedient possible substrates of LmxMPK4. The rather broad range of size was chosen to allow for varying behaviour of proteins in SDS-PAGE according to their phosphorylation status or other modifications.

Table 2 lists all proteins that were considered possible substrates of LmxMPK4. A full table of peptides detected by MS analysis is included in the appendix. All peptides detected by matching with the L. major protein database were manually compared with the respective L. mexicana protein homologous and are marked in Table 2. Sequence alignments of the homologous L. major and L. mexicana proteins from Table 2 are displayed in the appendix, including the highlighted identified peptides. Only a small number of proteins that were detected by MS analysis in the excised gel band matched the expected size range. Only peptides that equally matched the respective homologous protein in L. mexicana were considered relevant for substrate analysis. No protein could be identified in all three samples, but two proteins were independently identified in two different samples. The first protein, the γ-subunit of ATP synthase F1 of 34.42 kDa, was identified in the dephosphorylated lysate of axenic amastigotes, as well as in the sample incubated with promastigote protein lysate. The other protein identified in two different samples was glycosomal malate dehydrogenase with a size of 33.63 kDa. This protein was identified in the untreated amastigote sample as well as in the promastigote sample.

Notably here is also that in the sample incubated with untreated amastigote lysate a set of four different peptides, two of which also matched the L. mexicana homologue, were additionally identified as belonging to the malate dehydrogenase LmjF34.0140 or LmxM33.0140, respectively. Two of these peptides also matched the mitochondrial malate dehydrogenase LmjF34.0160, but not the L. mexicana homologue LmxM33.0160. Three proteins were each identified in only one of the analysed samples. These were the hypothetical proteins LmjF34.3830 in untreated amastigote sample, the hypothetical protein LmjF36.2480 in the dephosphorylated amastigote sample and tryparedoxin peroxidase in the sample incubated with promastigote protein lysate. As the peptide

identified for LmjF36.2480 did not match the homologous L. mexicana sequence, this protein was not considered a potential LmxMPK4 substrate.

Table 2. Results of MS analysi s of excised gel bands of possible LmxMPK4 in vivo substrate; all peptides that also match L. mexicana genes are underlined in bold with the respective gene displayed in brackets and underlined in bold in the identification column

Sample Peptides Identification of protein Size of

protein Band A

(dephosphorylat ed amastigote s)

VIDSVASSR

ATP syntha se F1 subunit gamma protein, LmjF21. 1770

(LmxM21.1770)

34.42 kDa

TNGGELPR

Hypothetical protein, conserved

LmjF36. 2480

27.93 kDa

LLEAFQFVEK

tryparedoxin peroxidase, LmjF15. 1120 (LmxM15.1160)

22.12 kDa

Band B

(amastigote s) LLGVSLLDGLR

glycosomal malate dehydrogenase, LmjF19. 0710 (LmxM19.0710)

33.63 kDa

DDLFNTNAS IVR AVENADVVVIPAGIP R VAVLGAAGGIGQPLS LLLK AIVGIITNPVNSTVPV AAEALK

malate dehydrogenase, LmjF34. 0140

(LmxM33.0140)

33.36 kDa

AIVGIITNPV NS TVPVAAEALK VAVLGAAGGIGQP LSLLLK

Mitochondrial malate dehydrogenase, LmjF34. 0160 (LmxM33.0160)

34.15 kDa

DAEAAARTSR

hypothetical protein, conserved,

LmjF34. 3830 (LmxM33.3830)

26.47 kDa

Band C

(proma stigotes) LLGVSLLDGLR

glycosomal malate dehydrogenase, LmjF19. 0710 (LmxM19.0710)

33.63 kDa

K VIDSVASSR

ATP syntha se F1

subunit gamma protein, putative,

LmjF21. 1770 (LmxM21.1770)

34.42 kDa

4.1.2 Characterisation of an inhibitor-sensitised LmxMPK4