The identification of SiMyD88 confirms the presence of a functional TLR-MyD88-IRAK signaling axis in the sea urchin Strongylocentrotus intermedius.

Myeloid differentiation primary response gene 88 (MyD88), a central adaptor protein in the Toll-like receptor (TLR) signaling pathway, plays a key role in the innate immune responses of animals. However, the signal transduction pathway mediated by MyD88 and the associated immune functions of the protein in echinoderms are not well characterized. In this study, a novel MyD88 gene (SiMyD88) was cloned from the sea urchin Strongylocentrotus intermedius. Bioinformatics analysis revealed that SiMyD88 contains a 1101-bp-long open reading frame encoding a polypeptide of 366 amino acids. The N-terminal region of SiMyD88 harbors a death domain followed by a Toll/interleukin-1 receptor (TIR) domain, both of which were found through multiple sequence alignment to be highly conserved across species. Phylogenetic analysis revealed that SiMyD88 clustered closely with MyD88 proteins from other echinoderm species, suggesting a high degree of evolutionary conservation within this protein family. RT-qPCR analysis showed that the esophagus expressed the highest level of SiMyD88 transcripts, whereas subcellular localization studies indicated that the protein was predominantly localized in the cytoplasm. The induction of SiMyD88 mRNA expression by lipopolysaccharide and poly(I:C) suggests that this gene plays a critical role in mediating the immune response of sea urchins to bacterial and viral stimuli. More importantly, co-immunoprecipitation experiments revealed that SiMyD88 interacted with multiple TLR proteins in sea urchins and was capable of self-association. Furthermore, SiMyD88 interacted directly with interleukin-1 receptor-associated kinase (IRAK). Dual-luciferase reporter gene assays showed that SiMyD88 overexpression in HEK293T cells significantly activated the transcriptional activities of activator protein-1, interleukin-6, and nuclear factor-kappa B (NF-κB) reporter genes, indicating its potential involvement in multiple signaling pathways. Collectively, these results suggest that SiMyD88 responds to pathogen-associated molecular patterns in TLR-MyD88-IRAK immune signaling, and potentially regulates downstream effector proteins such as the transcription factor NF-κB. These findings contribute to our understanding of innate immunity in echinoderms and provide a foundation for future research on strategies for breeding disease-resistant sea urchins.