LCCL protein complex formation in Plasmodium is critically dependent on LAP1

Abstract

Successful sporogony of Plasmodium berghei in vector mosquitoes requires expression of a family of six modular proteins named LCCL lectin domain adhesive-like proteins (LAPs). The LAPs share a subcellular localization in the crystalloid, a unique parasite organelle that forms during ookinete development. Here, LAP interactions in P. berghei were studied using a series of parasite lines stably expressing reporter-tagged LAPs combined with affinity purification and high accuracy label free quantitative mass spectrometry. Our results show that abundant complexes containing LAP1, LAP2 and LAP3 are formed in gametocytes through high avidity interactions. Following fertilization, LAP4, LAP5 and LAP6 are recruited to this complex, a process that is facilitated by LAP1 chiefly through its scavenger receptor cysteine-rich modules. These collective findings provide new insight into the temporal and molecular dynamics of protein complex formation that lead up to, and are required for, crystalloid biogenesis and downstream sporozoite transmission of malaria parasites.

Keywords: Crystalloid organelle; LC/MS/MS; LCCL; Transmission blockade.

Graphical abstract

Fig. 1

Schematic diagram of Plasmodium LAP1-LAP6 (PlasmoDB IDs shown on the right hand side). All proteins possess a predicted N-terminal ER signal peptide (red). A variety of modules are shown with significant homologies to known protein domains. Black: Limulus coagulation factor C, Coch-5b2 and Lgl1 (LCCL) domain (Pfam03815, Smart00603); Light green: Polycystin-1, Lipoxygenase, Alpha-Toxin (PLAT) domain (Pfam01477, Smart00308); Light blue: scavenger receptor cysteine-rich (SRCR) domain (Pfam00530, Smart00202); Pink: pentaxin (PTX)/Laminin-G domain (Pfam00354, Smart00159); Orange: ricin-type beta trefoil lectin domain (Pfam00161, Smart00458); Red: coagulation factor 5/8 carboxy-terminal/discoidin domain (Pfam00754, Smart00231); Yellow: fibrillar collagen (COLFI) carboxy-terminal domain (Pfam01410, Smart00038); Dark blue: Levanase-like domain; Purple: anthrax protective antigen domain (Pfam07691); Dark green: fibronectin type II domain (Pfam00040, Smart00059); Grey: apicomplexan-specific cysteine-rich domain. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Immunoaffinity pull-down of LAP complexes from P. berghei gametocytes with anti-GFP antibody-coated magnetic beads. A: Coomassie brilliant blue staining shows specific pull down of the LAP3:GFP target protein and several other proteins (arrow heads). Lanes 1 + 2: wildtype; lanes 3 + 4; LAP3/GFP; lanes 1 + 3: before pull down; lanes 2 + 4: after pull down. B: Western blot using anti-GFP antibodies shows enrichment of the LAP3:GFP target protein. Nonspecific (ns) antibody binding is indicated. Molecular weight markers in kDa are shown (M).