Figure 1.
Recombinant AMA1 C-terminal tail is phosphorylated in vitro by P. falciparum (3D7 line) parasite lysates in a calcium and cAMP dependent manner.
(A) Schematic representation of AMA1 and the GST-fusion protein used. Signal peptide (blue), prosequence (PS), ectodomains I, II & III, transmembrane domain (grey), cytoplasmic tail (C) and thrombin cleavage site are indicated. (B, C) Auto-radiographs showing phosphorylation of recombinant AMA1 tail by parasite lysates in the presence of 1.5 mM EGTA/1 mM EDTA or 2 mM CaCl2 or 1 µM cAMP. The AMA1 tail was incubated with schizont (S), merozoite (M) or red blood cell (RBC) lysates and 32[P]-γ -ATP. After washing the GST part was cleaved off with thrombin. As a loading control the membrane was probed with an anti-AMA1 antibody detecting the AMA1 tail. Molecular sizes are indicated on the left. (D) Quantitation of signal intensities in panel C with Image Gauge software. In the absence of additional EGTA/EDTA or cAMP the strength of the phosphorylation signal in untreated schizont lysate was set to 100% and all other signals are relative to that. The numbers of experimental replicates in in vitro phosphorylation assays are found in the Supplementary data (Text S1). Error bars correspond to standard deviation.
Figure 2.
PfPKA phosphorylates the AMA1 tail exclusively on residue S610.
(A) Multiple alignment of AMA1 C-terminal cytoplasmic domains including protein sequences of nine different Plasmodium species, T. gondii, B. bovis and two Theileria species. The conservation is scored and colour coded by PRALINE (www.ibi.vu.nl). Amino acids predicted to be phosphorylated in P. falciparum AMA1 by NetPhos (www.cbs.dtu.dk/services/NetPhos) are scaled up in font size according to their relative predicted probabilities above the alignment and numbered regarding the protein sequence. As shown below the alignment the GST-fusion protein of the AMA1 tail was mutated at residues S601, S610, T612, T613, Y621 and Y622, respectively. (B, C) SDS-PAGE and radiograph of phosphorylation samples. GST-fusion proteins of the AMA1 tail and mutants were incubated with schizont lysate and 32[P]- γ -ATP. As a loading control the membrane was probed with an anti-AMA1-C antibody. (C) H89 (50 µM) and KT5720 (10 µM), two inhibitors to PKA reverse the effect of cAMP (1 µM) on AMA1 tail phosphorylation. (D, E) Quantitation of signal intensities in (C) with Image Gauge software. The phosphorylation signal strength in untreated schizont lysate was set to 100% and all other signals were relative to that. The numbers of experimental replicates in in vitro phosphorylation assays are found in the Supplementary data (Text S1). Error bars correspond to standard deviation. (F) PfPKA-HA was immuno-precipitated from lysate of 3D7PfPKA-HA transgenic parasites using protein-G-sepharose. PfPKA-HA was detected by Western Blot analysis using anti-HA antibodies and parasite lysate, washed fraction or sepharose beads. (G) SDS-PAGE and radiograph of in vitro phosphorylation of AMA1-GST using either protein-G-sepharose incubated with 3D7PfPKA-HA or with wild type (WT) parasites in the presence of 32[P]- γ -ATP.
Figure 3.
Two dimensional gel analyses confirm that P. falciparum AMA1 is phosphorylated at S610 in vivo.
P. falciparum proteins were metabolically labelled with 32[P], detergent extracted and resulting 2DE blots developed using Deep Purple stain to visualize total protein (A). Autoradiography detected in vivo phosphorylated proteins (B), and Western Blotting with an anti-AMA1 antibody detected the AMA1 tail (C). Isoelectric point and molecular sizes are indicated on the top and left. 2D spots corresponding to the 83 kDa precursor (AMA183) or post-translationally processed ∼66 kDa AMA1 fragment (AMA66) are highlighted by bounding boxes, respectively. Magnification of the AMA166 region reveals a series of five discrete 32[P]-labelled (a, c, d) or -unlabelled (b, e) protein spots recognized by anti-AMA1 antibody (arrows). (D-I) all show ∼66 kDa species of AMA1 separated by 2DE and analysed by Western Blot. 3D7 parasites either expressed a transgenic TY1-tagged wild type W2mef AMA1 (AMA1-WT-TY1; D, E), or mutant forms where the W2mef transgene carried the AMA1-S610A mutation (AMA1-S610A-TY1; F, G) or mutations at all putative phosphorylation sites in the cytoplasmic tail AMA1-PM-TY1; H, I). Blots were probed with a mouse monoclonal antibody against TY1 to detect transgenic epitope-tagged AMA1 followed and by an anti-AMA1 antibody against the AMA1 tail to detect both endogenously expressed 3D7 AMA1 as well as the W2mef transgenic species.
Figure 4.
Maturation of phosphorylated AMA1 species.
Schizont, merozoite and ring-stage parental 3D7 parasites were analysed from a sequential time-course experiment by two dimensional gel electrophoresis and Western Blotting. Blots were probed with C-terminal AMA1 tail antibodies. Bands representing 83 kDa, 66 kDa and 22 kDa AMA1 species as well as presumed cross-reacting contaminating species (cont) are indicated on the right. Isoelectric points and molecular weight markers are indicated on the top and left of the autoradiographs, respectively.
Figure 5.
The PfPKA phosphorylated residue S610 is required for efficient merozoite invasion.
(A) Schematic representation of the ectopically expressed TY1-tagged AMA1. Signal peptide (blue), prosequence (PS), ectodomains I, II & III, transmembrane domain (grey), cytoplasmic tail (C) and TY1-tag (red) are indicated. (B) Mutations introduced into the cytoplasmic tail of W2mef-derived AMA1 are shown in red colour. (C) Expression of W2mef-derived AMA1-TY1 and native 3D7 AMA1 detected by Western Blot analysis using an anti-TY1 and anti-AMA1 (1F9) antibody, respectively (C, left panel). Whereas no AMA1-TY1 protein can be detected in 3D7 wild type parasites, a double band corresponding to AMA183-TY1 and processed AMA166-TY1 can be detected in AMA1-TY1-expressing parasites. (C, right panel) The endogenous 3D7 AMA1 is recognised by the anti-AMA1-1F9 antibody and shows identical proteolytic forms of AMA1 (AMA183 and AMA166). (D) Invasion inhibition assays using AMA1-TY1 expressing parasite strains. Assays were performed in the presence of 100 µg/mL R1 peptide and were performed in triplicate in three independent experiments. Error bars correspond to standard deviation. 3D7 and AMA1-WT-TY1 served as control.