Major yolk protein and HSC70 are essential for the activation of the TLR pathway via interacting with MyD88 in Apostichopus japonicus.

The Toll cascade plays important functions in innate immunity against infectious pathogens in animals. Toll cascade as an ancient immune defender were conserved among different species. The activation of the TLR pathway between different species often involves different interacting proteins. The core members of this pathway have been well established in a wide range of organisms, including the marine invertebrate sea cucumber. However, these proteins do not function as single isolated entities but are engaged in a dynamic physical network with other proteins in the biomolecular context of a cell. To fill the knowledge gap in this context, two novel members of major yolk protein (MYP) and heat shock cognate protein 70 (HSC70) were identified as myeloid differentiation factor 88 (MyD88) interacting proteins by GST pull-down and mass spectrometry assays in Apostichopus japonicus. Their interactions were further confirmed by a co-immunoprecipitation analysis. Confocal microscopy analysis revealed that these three proteins were co-localized in the cytoplasm. A functional experiment indicated that each protein alone could suppress NF-κB translocated in the nucleus in cultured coelomocytes via a siRNA interference assay, suggesting that the three proteins functioned as a complex. To better address these interactions, we used the ZDOCK docking platform to mock the structure of the MyD88-HSC70-MYP complex. The death domain of MyD88 bound to HSC70 and MYP in separate spatial positions. The extent of interaction between MyD88 and HSC70 were K574, D591, E592 and E619 in HSC70 and E75, R76, K197 and R203 in MyD88. In the MYP-MyD88 model, K260, K452, K467 and E839 of MYP and D29, R40 and E62 of MyD88 were considered essential sites. Site-specific mutagenesis of these sites showed that most residues were key sites for their interaction with distinctly reduced binding constants relative to those of their native counterparts by biolayer interferometry assays, in which only K197 and R203 of MyD88 mutants displayed no effect on these interactions. Our results provide the first evidence of the roles of HSC70 and MYP in immune regulation via interacting with MyD88 and activating the TLR pathway in Apostichopus japonicus.