Screening and validation of Galectin3-interacting proteins from Japanese flounder (Paralichthys olivaceus)

Galectins are proteins that play a crucial role in the innate immune response against pathogenic microorganisms. Galectin3, the only chimera-type galectin found in vertebrates, have been demonstrated a variety of biological activities in vitro, such as activation of cells, modulation of cell adhesion and regulation of apoptosis. In addition, galectin3 has been shown to be associated with inflammatory responses. However, it is unknown exactly how galectin3 contributes to the immunological response. In this research, to find the interacting proteins of galectin3, TMT liquid chromatography-tandem mass spectrometry (TMT LC-MS/MS), co-immunoprecipitation and liquid chromatography-tandem mass spectrometry (Co-IP LC-MS/MS) technologies and Co-IP Western blot (Co-IP WB) were applied. Intestinal epithelial cells of Paralichthys olivaceus (PoIEC) were transfected with pcDNA3.1-Galectin3 to obtain an overexpression plasmid and validated it by Western blot. Then we searched the proteins that may interact with galectin3 by TMT LC-MS/MS and Co-IP LC-MS/MS experiments, which suggest that cathepsin B (CTSB) and heat shock cognate 71 kDa protein (HSC71) are potential interacting proteins of galectin3. Then, HSC71 was verified as an interacting protein with galectin3 by Co-IP WB. In conclusion, our findings suggest that HSC71 acts as a target protein for galectin3, helping to elucidate the mechanism of galectin3 involvement in the inflammatory response and further exploring the cellular activation of fish pathogen signaling recognition, transmission and inflammatory cascade response.