plos pathogens
Malaria Parasite Invasion of the Mosquito Salivary Gland Requires Interaction between the Plasmodium TRAP and the Anopheles Saglin Proteins
Pull-down assays reveal that SM1 binds to the salivary gland surface protein saglin
To identify salivary gland protein(s) to which SM1 bound, we used a double-derivatized SM1 peptide that had biotin at the N-terminus and an UV-crosslinker attached to the phenylalanine (F) within the 8-amino acid loop formed by the disulphide bond between cysteines 2 and 11 (Figures 1B and 3A). The derivatized SM1 was incubated with freshly dissected salivary glands followed by UV irradiation to activate peptide crosslinking to its target salivary gland receptor (Figure 3A). The glands were then solubilized and the peptide, with its crosslinked proteins, was captured on a streptavidin column. The retained proteins were then fractionated by gel electrophoresis. The four specific protein bands (Figure 3B) were excised and microsequenced. The upper two bands corresponded to the recently described mosquito salivary gland surface protein saglin [8] whereas the two lower bands corresponded to the mosquito salivary gland secreted protein SG1 of unknown function. Figure S3 illustrates the identification of saglin by LCMS/MS. Saglin has a predicted secretion signal sequence at its amino terminus and is rich in the amino acid glutamine (47/412 amino acids or 11.4%) that may be involved in protein-protein interactions via hydrogen bonds. Monoclonal antibodies recognizing saglin had previously been shown to inhibit sporozoite invasion of salivary glands[7].
Figure 3. Pull-down of salivary gland proteins that interact with SM1.
(A) Schematic diagram of the pull-down approach. A double-derivatized SM1 peptide, with a biotin residue (yellow pentagon) at the N-terminus and a UV-crosslinking residue (green star) attached to a phenylalanine residue in the loop (‘F’ in Figure 1B) was incubated in vitro with freshly dissected salivary glands and washed. After shining UV to promote crosslinking of the peptide to the protein to which it was bound, the salivary glands were lysed and the peptide with the crosslinked protein was captured on streptavidin beads. The beads were washed and the retained proteins were fractionated by SDS-PAGE (panel B). (B) Gel electrophoresis of proteins captured on the streptavidin beads. Materials recovered from a pull-down experiment illustrated in panel A were fractionated by SDS-PAGE under reducing conditions and the gel was stained with Coomassie Blue. Lane 1, Materials eluted from streptavidin beads that were not incubated with any proteins. The stained bands are presumed to be bead-derived contaminants. Lane 2, Complete experiment, except that the crosslinking step was omitted. Lane 3, complete experiment, including the crosslinking step (same number of salivary glands as in Lane 2). The four arrows point to protein bands consistently observed only in the complete experiment.
http://dx.doi.org/10.1371/journal.ppat.1000265.g003
In vitro interactions of saglin with TRAP domain-A and SM1
The results presented so far suggested that SM1 mimics the conformation of TRAP domain-A and that SM1 interacts with saglin. Further experiments were conducted to verify that these components interact directly. Figure 4A and 4B present results of far-western experiments in which bacterial lysates containing either recombinant saglin or control beta-galactosidase were fractionated by gel electrophoresis and blotted. The blot in Figure 4A was incubated with the SM1 peptide and the blot in Figure 4B was incubated with TRAP domain-A and peptide or TRAP domain-A binding was detected with their respective antibodies. Panel 3 of Figure 4Ashows that SM1 binds to saglin and panel 2 of Figure 4B shows that TRAP domain-A binds to saglin. Binding was specific and was not detected when the control beta-galactosidase was run on the gel (Figure 4A panel 6 and Figure 4B panel 5).
Figure 4. In vitro and in vivo protein interactions.
(A) SM1 binding to saglin on protein blots. Blots from bacteria carrying a plasmid encoding either histidine-tagged saglin (experimental; panels 1–4) or histidine-tagged ß-galactosidase (control; panels 5–6) were produced from proteins recovered either before (U) or after (I) induction of recombinant protein synthesis. Panel 1: Experimental blot incubated with anti-histidine antibody. Panel 2: Experimental blot incubated with anti-saglin monoclonal antibody. Panel 3: Experimental blot first incubated with the SM1 peptide (10 µg/ml) and then probed with the anti-SM1 antibody followed by incubation with an alkaline phosphatase-tagged secondary antibody. Panel 4: Experimental blot incubated with a linear SM1 peptide and probed with anti-SM1 antibody. In the linear peptide the two cysteines were substituted by alanines. In separate experiments the linear peptide was unable to bind to salivary glands (not shown). Panel 5: Control blot probed with anti-histidine antibody. Panel 6: Control blot first incubated with the SM1 peptide (10 µg/ml) and then probed with the anti-SM1 antibody. The mobility of recombinant saglin is indicated by an arrow in Panel 1. (B) TRAP domain-A binding to saglin on protein blots. Blots from bacteria carrying a plasmid encoding either histidine-tagged saglin (experimental; panels 1–3) or histidine-tagged ß-galactosidase (control; panels 4–5) were produced from proteins recovered either before (U) or after (I) induction of recombinant protein synthesis. Panel 1: Experimental blot incubated with anti-histidine antibody; Panel 2: Experimental blot first incubated with recombinant TRAP domain-A protein (2.5 µg/ml) and then probed with anti-TRAP domain-A antibody, followed by incubation with an alkaline phosphatase-tagged secondary antibody; Panel 3: Same as Panel 2, except that incubation with anti-TRAP domain-A antibody was omitted; Panel 4: Control blot probed with anti-histidine antibody; Panel 5: Control blot subjected to the same treatment as described for Panel 2. The mobility of recombinant saglin is indicated by an arrow in panel 1. (C) In vitro interaction between saglin and TRAP domain-A. Recombinant TRAP domain-A was incubated in wells of an ELISA plate that had been previously coated with recombinant histidine-tagged saglin. The wells were washed and TRAP binding was quantified using a TRAP antibody followed by incubation with an alkaline phosphatase-tagged secondary antibody. Results of the colorimetric alkaline phosphatase reaction are reported. (1) Well not coated with recombinant saglin. (2) Complete protocol. (3) Incubation with TRAP omitted. (4) Incubation with anti-TRAP antibody omitted. (5) Incubation with secondary antibody omitted. (D) Sporozoites bind to saglin in vitro. Purified midgut sporozoites were incubated in wells of an ELISA plate that had been previously coated with recombinant histidine-tagged saglin. The wells were washed and sporozoite binding was quantified by use of an anti-CS antibody followed by incubation with an alkaline phosphatase-tagged secondary antibody. Results of the colorimetric alkaline phosphatase reaction are reported. (1) Well not coated with recombinant saglin. (2) Complete protocol. (3) Incubation with sporozoites omitted. (4) Incubation with anti-CS antibody omitted. (5) Incubation with secondary antibody omitted. (E) SM1 competes with TRAP domain-A for binding to salivary glands. Fixed salivary glands were first incubated with no (a), 0.5 (b), 5 µM (c) or 50 µM (d) of non-biotinylated SM1 peptide followed by incubation with 50 nM of recombinant TRAP domain-A. TRAP domain-A binding was detected by incubation with an anti-TRAP antibody followed by incubation with a FITC-labeled secondary antibody. Note that the SM1 peptide effectively competes with TRAP domain-A for binding to salivary glands. (F) Reverse competition: TRAP domain-A competes with SM1 for binding to salivary glands. Fixed salivary glands were first incubated with no (a), 20 nM (b), 200 nM (c) or 2 µM (d) of recombinant TRAP domain-A protein followed by incubation with biotinylated SM1 peptide (5 µM). Peptide binding was detected by incubation with FITC-labeled streptavidin. Note that the protein effectively competes with the SM1 peptide for binding to salivary glands.
Additional evidence was obtained for the direct interaction between saglin and TRAP domain-A in the following experiment. Recombinant saglin was captured on wells of a microtiter plate followed by incubation with recombinant TRAP domain-A. As seen in Figure 4C, interaction between saglin and TRAP domain-A was detected only in the complete experiment (bar 2) and not when one of the steps was omitted, attesting to the specificity of the interaction. These results confirmed that TRAP domain-A directly interacts with saglin. In separate experiments, we trapped salivary gland membranes (and ovary membranes as a control) on wells of a microtiter plate that had been pre-coated with anti-saglin antibodies. TRAP domain-A bound to salivary gland but not to ovary membranes (Figure S5), again suggesting tissue specificity of interaction.
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