Detection of retromer assembly in Plasmodium falciparum by immunosensing coupled to Surface Plasmon Resonance

Retromer complex plays a crucial role in intracellular protein trafficking and is conserved throughout the eukaryotes including malaria parasite, Plasmodium falciparum, where it is partially conserved. The assembly of retromer complex in RBC stages of malarial parasite is extremely difficult to explore because of its complicated physiology, small size, and intra-erythrocytic location. Nonetheless, understanding of retromer assembly may pave new ways for the development of novel antimalarials targeting parasite-specific protein trafficking pathways. Here, we investigated the assembly of retromer complex in P. falciparum, by an immunosensing method through highly sensitive Surface Plasmon Resonance (SPR) technique. After taking leads from the bioinformatics search and literature, different interacting proteins were identified and specific antibodies were raised against them. The sensor chip was prepared by covalently linking antibody specific to one component and the whole cell lysate was passed through it in order to trap the interacting complex. Antibodies raised against other interacting components were used to detect them in the trapped complex on the SPR chip. We were able to detect three different components in the retromer complex trapped by the immobilized antibody specific against a different component on a sensor chip. The assay was reproduced and validated in a different two-component CD74-MIF system in mammalian cells. We, thus, illustrate the assembly of retromer complex in P. falciparum through a bio-sensing approach that combines SPR with immunosensing requiring a very small amount of sample from the native source. Schematic representation of the SPR experiments performed to probe multiple components of retromer complex bound to the immobilized antibody. The four-step procedure is shown where the complex was trapped in the first step and three different proteins in that complex were probed by their specific antibodies. Each step is associated with a jump in RU. A hypothetical sensorgram in the figure represents an increment in RU in each step that becomes the baseline for the next step. Binding responses are shown here with the components involved in each of them. A sensorgram with all responses normalized is also depicted. [Display omitted] •Retromer complex assembly in Plasmodium falciparum was detected by SPR.•SPR based immunosensor technique used to analyze protein interactions in cell lysate.•Immunodetection of proteins in complex bound o