Two Amphioxus ApeC-Containing Proteins Bind to Microbes and Inhibit the TRAF6 Pathway

Area of Science:

• Molecular Biology
• Immunology
• Evolutionary Biology

Background:

• The apextrin C-terminal (ApeC) domain is a newly identified protein domain found in various invertebrates.
• The functions of ApeC-containing proteins (ACPs) and their mechanisms remain largely unknown, with prior studies suggesting roles in bacterial cell wall binding and immunity.

Purpose of the Study:

• To identify and characterize novel amphioxus ACPs (ACP3 and ACP5) and elucidate their molecular functions.
• To investigate the binding specificities and immune regulatory roles of these newly identified ACPs.

Main Methods:

• Phylogenetic analysis and sequence comparison of novel amphioxus ACPs.
• Expression analysis in response to bacterial challenge.
• Recombinant protein expression and bacterial/yeast binding assays.
• ELISA to determine interactions with cell wall components (PGN, LTA, LPS, zymosan A).
• Mammalian cell expression to study protein-protein interactions and downstream signaling (TRAF6, NF-κB).

Main Results:

• ACP3 and ACP5, distinct from ACP1/2, showed specific expression patterns and sequence divergence.
• Both ACP3 and ACP5 exhibited agglutinating activity against bacteria and yeasts, binding to Lys-type peptidoglycan (PGN) but not other cell wall components.
• ACP3 interacted with TRAF6 via a non-ApeC region, inhibiting TRAF6 ubiquitination and suppressing NF-κB activation.

Conclusions:

• A novel subfamily of ACPs (ACP3/5) with conserved structures and diversified functions was defined.
• These ACPs possess dual roles: ApeC domains act as lectins recognizing specific bacterial components, while other regions regulate signal transduction pathways.
• Findings expand the understanding of ACP functions, highlighting their roles in innate immunity and potential evolutionary significance across animal clades.