Juxtamembrane Shedding of Plasmodium falciparum AMA1 Is Sequence Independent and Essential, and Helps Evade Invasion-Inhibitory Antibodies
Figure 2
Mutation of the intramembrane cleavage site in PfAMA1 inhibits shedding by intramembrane processing.
Western blot analysis of culture supernatants from transgenic P. falciparum clones 3D7_AMA_C_E9 (control) and 3D7_AMA_R_D4 (Ala550Tyr), following schizont rupture in the presence or absence of 10 mM EGTA. Samples were electrophoresed under reducing conditions and probed with anti-PfAMA1 polyclonal serum R5 (top) or monoclonal antibody 24C6.1F1 specific for the parasitophorous vacuole protein SERA5, which is released in the form of the P50 fragment (bottom – loading control). For both clones, shedding of PfAMA1 by PfSUB2 gives rise to the expected products of PfAMA148 plus PfAMA144 ([12] and Figure 1). In addition, PfAMA1 is shed from the surface of 3D7_AMA_C_E9 parasites via intra-membrane cleavage, giving rise to PfAMA152. This species is absent from 3D7_AMA_R_D4 culture supernatants, confirming that the introduced Ala550Tyr substitution blocks intramembrane processing of PfAMA1. Because PfSUB2-mediated shedding is calcium-dependent, virtually all PfAMA1 shedding is ablated from the 3D7_AMA_R_D4 parasites in the presence of EGTA.