Biochemical and functional characterization of recombinant fungal immunomodulatory proteins (rFIPs)

In this study, two novel FIPs have been identified and characterized. The first is FIP-nha, identified in the ascomycete Nectria haematococca, and as such, FIP-nha would be the first FIP to be identified outside the order of Basidiomycota. The second is LZ-9, an LZ-8 like protein identified in Ganoderma lucidum. Recombinant FIPs (rFIPs) were produced in Pichia pastoris and purified using His-affinity magnetic beads. The bioactive characteristics of FIP-nha and LZ-9 were compared to the well-known FIPs, LZ-8 from G. lucidum and FIP-fve from Flammulina velutipes, which were produced and purified using the same method. The produced rFIPs: rLZ-8, rLZ-9, rFIP-fve and rFIP-nha were investigated for their hemagglutinating activity which revealed that rLZ-8, rLZ-9 and rFIP-nha were able to agglutinate rabbit, mouse and sheep red blood cells while rFIP-fve only agglutinated rabbit red blood cells. None of the rFIPs were able to agglutinate human red blood cells unless the cells were trypsinized. In addition, all rFIPs were studied and compared to several lectins for their effect on Caco-2 intestinal cell layer integrity using transepithelial electrical resistance (TEER) measurement. rLZ-9 appeared to have the highest effect in lowering TEER, similar to one of the tested lectins. Testing of rFIPs for their activation of inflammation-related genes of THP-1 macrophages showed rFIP-fve to be the strongest inducer of pro-inflammatory cytokine transcription. These results indicate that each rFIP has a unique bioactive profile as well as each lectin, creating the basis for further studies to relate structure to biological activity.