Major Vault Protein of the Protozoan Raphidiophrys contractilis Has a Binding Property to β-1,3-Glucan and is Involved in Food Capturing

Mousumi Bhadra, Mayumi Kobayashi, Rina Higuchi, Lin Chen, Toshinobu Suzaki

A centrohelid heliozoan Raphidiophrys contractilis was found to recognize curdlan, an insoluble β-1,3-glucan, as food. When a suspension of curdlan gel was added to the heliozoans, the gel particles were ingested into food vacuoles. By affinity purification with curdlan gel, a protein of 100 kDa was isolated as the only β-1,3-glucan-binding protein from the detergent-extracted cell homogenate of R. contractilis. The protein was subjected to mass spectrometry with a reference contig dataset obtained by transcriptome analysis. From the obtained partial nucleotide sequences and additional PCR-based sequence determination, the protein was identified as major vault protein (MVP), the main component of Vault complex. The R. contractilis MVP reacted with an antibody against human MVP, and specific binding to β-1,3-glucan was verified by a competition assay with laminarin, a soluble-type β-1,3-glucan. The heliozoans were mixed with prey flagellate Chlorogonium capillatum and extracellular fluid was collected during food-uptake. The fluid was then subjected to pull-down assay with curdlan gel, by which multiple protein species were detected including MVP as one of the major proteins, suggesting that MVP is secreted from the heliozoans during food uptake as a component of a large protein complex.  The involvement of the heliozoan MVP in prey recognition suggests a possible novel function of this protein in self/nonself discrimination.