A Single Malaria Merozoite Serine Protease Mediates Shedding of Multiple Surface Proteins by Juxtamembrane Cleavage

Abstract

Erythrocyte invasion by the malaria merozoite is accompanied by the regulated discharge of apically located secretory organelles called micronemes. Plasmodium falciparum apical membrane antigen-1 (PfAMA-1), which plays an indispensable role in invasion, translocates from micronemes onto the parasite surface and is proteolytically shed in a soluble form during invasion. We have previously proposed, on the basis of incomplete mass spectrometric mapping data, that PfAMA-1 shedding results from cleavage at two alternative positions. We now show conclusively that the PfAMA-1 ectodomain is shed from the merozoite solely as a result of cleavage at a single site, just 29 residues away from the predicted transmembrane-spanning sequence. Remarkably, this cleavage is mediated by the same membrane-bound parasite serine protease as that responsible for shedding of the merozoite surface protein-1 (MSP-1) complex, an abundant, glycosylphosphatidylinositol-anchored multiprotein complex. Processing of MSP-1 is essential for invasion. Our results indicate the presence on the merozoite surface of a multifunctional serine sheddase with a broad substrate specificity. We further demonstrate that translocation and shedding of PfAMA-1 is an actinindependent process.

A new release on life: emerging concepts in proteolysis and parasite invasion

“…Although this is some 96 residues upstream of the GPI anchor, the compact, inverted U-shaped structure of the two EGF-like modules which make up MSP-1 19 (Morgan et al, 1999;Pizarro et al, 2003) means that the cleavage site is probably spatially close to the membrane. A recent analysis of shedding of P. falciparum apical membrane antigen-1 (PfAMA1) showed that release from the parasite surface is the result of cleavage at a Thr-Ser bond just 29 residues upstream of the TMD (Howell et al, 2003). Quite unexpectedly, the protease responsible was found to be the same as that which sheds the MSP1 complex.…”

“…More to the point, there is evidence of a rhomboid-like activity at the merozoite surface. In the study of Howell et al (2003) alluded to above on shedding of PfAMA1, it was noticed that inhibition of MESH activity resulted in inefficient cleavage at an alternative, more membrane-proximal site. We have now mapped this alternative processing site to within the luminal half of the predicted TMD (S. Howell and M. Blackman, unpubl.).…”

“…1B) whereas capping is observed for apical invasion proteins. Shaving occurs continuously as the parasite penetrates and is (Dubremetz et al ., 1985;Grimwood and Smith, 1995) Yes (Mineo and Kasper, 1994;He et al ., 2002) Unknown TgMIC1/MIC4/MIC6 (Meissner et al ., 2002b) Yes (Fourmaux et al ., 1996;Brecht et al ., 2001) MPP1 TgMIC2/M2AP Yes (Carruthers et al ., 1999;Harper et al ., 2004) MPP1 Zhou et al ., 2004) TgMIC3/MIC8 (Meissner et al ., 2002b) Yes (Garcia-Reguet et al ., 2000;Meissner et al ., 2002b) MPP1 TgAMA1 (Donahue et al ., 2000;Hehl et al ., 2000) Unknown MPP1 (S. A. Howell, M. J. Blackman and V. B. Carruthers, unpublished) Plasmodium PfTRAP and PbTRAP (Bhanot et al, 2003;Silvie et al ., 2004) Yes (McCormick et al ., 1999;Akhouri et al ., 2004) Serine protease (Silvie et al .. 2004) PfCSP (Stewart and Vanderberg, 1988; Yes (Pinzon-Ortiz et al ., 2001) Unknown PfMSP1/MSP6/MSP7 (Stafford et al ., 1994) Yes (Goel et al ., 2003;Li et al ., 2004) MESH (Howell et al ., 2003) PkAMA1 and PfAMA1 (Deans et al ., 1984;Howell et al ., 2001) Yes (Fraser et al ., 2001) MESH (Howell et al ., 2003) EBL-DBP (Camus and Hadley, 1985;Haynes et al ., 1988) Yes (Camus and Hadley, 1985;Haynes et al ., 1988) Unknown Py235 (Ogun and Holder, 1996) Yes (Ogun and Holde...…”

Conditional Expression ofToxoplasma gondiiApical Membrane Antigen-1 (TgAMA1) Demonstrates That TgAMA1 Plays a Critical Role in Host Cell Invasion

“…AMA1 proteins are type I transmembrane proteins, with a short C-terminal cytoplasmic tail and a large N-terminal extracellular domain (ectodomain) containing 12-16 conserved cysteine residues (Waters et al, 1990;Hodder et al, 1996;Donahue et al, 2000;Hehl et al, 2000;Gaffar et al, 2004). Like other microneme proteins, AMA1 is secreted onto the parasite surface, where its ectodomain is proteolytically cleaved and shed (Narum and Thomas, 1994;Donahue et al, 2000;Hehl et al, 2000;Howell et al, 2003).The recently determined crystal structure of Plasmodium vivax AMA1 demonstrates that the conserved cysteines divide the ectodomain into three distinct subdomains (Pizarro et al, 2005). Domains I and II belong to the PAN module superfamily, suggesting that they may function in adhesion to protein or carbohydrate receptors (Pizarro et al, 2005).…”

Does protein phosphorylation govern host cell entry and egress by the Apicomplexa?

“…Analysis of a conditional knock-out of TgAMA1 established its important role in invasion and was found to be dispensable for gliding motility (Mital et al, 2005). In T. gondii, AMA1 is cleaved during invasion into its membranespanning domain by the rhomboid protease ROM4 (Buguliskis et al, 2010), whereas in Plasmodium a juxtamembrane cleavage is performed by the subtilisin protease (PfSUB2) (Harris et al, 2005;Howell et al, 2003;Olivieri et al, 2011). Both in T. gondii and P. falciparum, AMA1 was found to be phosphorylated ( Fig.…”