American Society of Hematology

Figure 3

$Figure 3. Native MSP1-42 and MSP1-33, but not full-length MSP1, MSP1-19, or other merozoite proteins, bind to heparin. Merozoite proteins were extracted from whole schizonts (3D7 isolate) and incubated with heparin-agarose beads with and without soluble inhibitors. (A) MSP1-42 bound to immobilized heparin in the presence of control (PBS) or soluble CSC, and MSP1-42 was depleted from the supernatant after incubation. In the presence of soluble heparin, binding of MSP1-42 was inhibited and was not depleted from the supernatant. Full-length MSP1 (MSP1-full) did not bind to beads and was only present in the supernatant. (B) MSP1-33 from culture supernatant bound heparin, being present in the bead-bound fraction and depleted from the supernatant. In the presence of soluble heparin, MSP1-33 was not able to bind and was predominately found in the supernatant (top panel). MSP1-19 did not bind to beads and was only present in the supernatant fraction (bottom panel). (C) Other merozoite antigens MSP3, MSP4, AMA1, and EBA175 showed little or no binding to heparin-agarose. For MSP3 and MSP4, no protein was seen in the bound fraction. AMA1 showed nonspecific binding to heparin-agarose beads that was not inhibited by the addition of soluble heparin. For EBA175, the majority of the protein was seen in the supernatant after incubation with heparin-agarose. Binding of merozoite proteins was tested in the presence of control (PBS), soluble heparin, or CSC. (D) Heparin did not inhibit proteolytic processing of MSP1. Purified schizonts were incubated with PBS, ethylenediaminetetraacetic acid (1mM), or heparin (1 mg/mL) until complete rupture of schizonts had occurred, and merozoites were then collected. In the presence of control (PBS) and heparin, processed forms of MSP1 (MSP1-42 and MSP1-19) were observed, in addition to full-length MSP1. Heparin did not significantly reduce the appearance of MSP1-42 or MSP1-19 compared with PBS control. Ethylenediaminetetraacetic acid prevented processing of full-length MSP1 to MSP1-42 and MSP1-19 fragments, indicating inhibition of processing. For all figures, the antibody used in the Western blot is indicated at the bottom of the panel (all antibodies were raised in rabbits). Molecular weight markers are indicated to the left, and proteins are indicated with arrows. Results shown are representative examples taken from multiple assays.$

Native MSP1-42 and MSP1-33, but not full-length MSP1, MSP1-19, or other merozoite proteins, bind to heparin. Merozoite proteins were extracted from whole schizonts (3D7 isolate) and incubated with heparin-agarose beads with and without soluble inhibitors. (A) MSP1-42 bound to immobilized heparin in the presence of control (PBS) or soluble CSC, and MSP1-42 was depleted from the supernatant after incubation. In the presence of soluble heparin, binding of MSP1-42 was inhibited and was not depleted from the supernatant. Full-length MSP1 (MSP1-full) did not bind to beads and was only present in the supernatant. (B) MSP1-33 from culture supernatant bound heparin, being present in the bead-bound fraction and depleted from the supernatant. In the presence of soluble heparin, MSP1-33 was not able to bind and was predominately found in the supernatant (top panel). MSP1-19 did not bind to beads and was only present in the supernatant fraction (bottom panel). (C) Other merozoite antigens MSP3, MSP4, AMA1, and EBA175 showed little or no binding to heparin-agarose. For MSP3 and MSP4, no protein was seen in the bound fraction. AMA1 showed nonspecific binding to heparin-agarose beads that was not inhibited by the addition of soluble heparin. For EBA175, the majority of the protein was seen in the supernatant after incubation with heparin-agarose. Binding of merozoite proteins was tested in the presence of control (PBS), soluble heparin, or CSC. (D) Heparin did not inhibit proteolytic processing of MSP1. Purified schizonts were incubated with PBS, ethylenediaminetetraacetic acid (1mM), or heparin (1 mg/mL) until complete rupture of schizonts had occurred, and merozoites were then collected. In the presence of control (PBS) and heparin, processed forms of MSP1 (MSP1-42 and MSP1-19) were observed, in addition to full-length MSP1. Heparin did not significantly reduce the appearance of MSP1-42 or MSP1-19 compared with PBS control. Ethylenediaminetetraacetic acid prevented processing of full-length MSP1 to MSP1-42 and MSP1-19 fragments, indicating inhibition of processing. For all figures, the antibody used in the Western blot is indicated at the bottom of the panel (all antibodies were raised in rabbits). Molecular weight markers are indicated to the left, and proteins are indicated with arrows. Results shown are representative examples taken from multiple assays.

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