Abstract
A novel method was validated for the efficient distinction between malaria parasite-derived and host cell proteins in mass spectrometry analyses. This method was applied to a ghost fraction from Plasmodium falciparum-infected erythrocytes containing the red blood cell plasma membrane, the erythrocyte submembrane skeleton, and the Maurer's clefts, a Golgi-like apparatus linked to and addressing parasite proteins to the host cell surface. This method allowed the identification of 78 parasite proteins. Among these we identified seven novel proteins of the Maurer's clefts based on immunofluorescence studies and proteinase K digestion assays. The products of six contiguous genes located on chromosome 5 were identified, and the location within the Maurer's clefts was established for two of them. This suggests a clustering of genes encoding Maurer's cleft proteins. Our study sheds new light on the biological function of the Maurer's clefts, which are central to the pathogenesis and to the intraerythrocytic development of P. falciparum.
Highlights
A novel method was validated for the efficient distinction between malaria parasite-derived and host cell proteins in mass spectrometry analyses
Mass Spectrometry Analysis of Deuterium-labeled P. falciparum-infected Red Blood Cell Ghosts—Ring stage P. falciparum-infected erythrocytes were matured for 24 h to late trophozoites and young schizonts in the presence of deuterated lysine
With two-thirds of the lysine in the medium being labeled and taking into consideration that hemoglobin contains nearly 10% lysine, we estimated that ϳ50% of the lysines that were incorporated into parasite proteins during the labeling were deuterated
Summary
5Ј-CgCAAggATCCAATTTAggAgATTCATTg 3Ј-CACTgAATTCTTgTTCATATAACTTACg 5Ј-gATATgggATCCTTAAATCCTTATggTTC 3Ј-ggATTATTgAATTCCACTTCAACTg 5Ј-gAAggATCCgATCAATTTTTACAAACg 3Ј-CTgggAATTCTTgACCTACCATTTgAgg 5Ј-gCATggATCCgATAgAACAAAAACATTTTC 3Ј-gAACgAATTCCCATgCTTgTTCAgATTC 5Ј-gATATgggATCCTTAAATCCTTATggTTC 3Ј-ggTgAATTCTCCACTTgATCTTCCT 5Ј-CACAggATCCAAACCATTCggAAATACC 3Ј-CATTTgAATTCATTTTgCATACgAAgATgC 5Ј-gTgAAggATCCAAgAATgTAAAAgATgg 3Ј-gTTCTCgAATTCTTgTACTATTCTTgTCC. Asn108–Gln193 Asp97–Glu191 His103–Gln256 Asp371–Trp495 Leu244–Gly345 Lys53–Asn143 Lys70–Gln167 proteomic analysis confirmed that ghost preparations are valuable to study Maurer’s clefts and enabled us, together with localization and topological studies, to identify new Maurer’s clefts proteins, providing insight into the biology of these interesting organelles
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