AUTHOR=Li Jin , Liu Shumin , Zhang Yang , Huang Qiuyun , Zhang Hao , OuYang Jihua , Mao Fan , Fan Huiping , Yi Wenjie , Dong Meiling , Xu Anlong , Huang Shengfeng 

TITLE=Two novel mollusk short-form ApeC-containing proteins act as pattern recognition proteins for peptidoglycan

JOURNAL=Frontiers in Immunology

VOLUME=Volume 13 - 2022

YEAR=2022

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2022.971883

DOI=10.3389/fimmu.2022.971883

ISSN=1664-3224

ABSTRACT=The Apextrin C-terminal (ApeC) domain is a novel protein domain largely specific to marine and freshwater invertebrates. In amphioxus, a shor-form ApeC-containing protein (ACP) family has been shown to be capable of binding bacteria via its ApeC and regulating TRAF6-NF-κB signaling. However, so far in other phyla, the ACP functions remain largely unknown. Here we examined 129 ACPs from gastropods and bivalves, the first and second biggest mollusk classes. These ACPs were classified into nine groups based on phylogenetics and protein architectures, including one group of apextrins, two groups of short-form ACPs and two groups of ACPs with complex domain architectures. None of the groups have orthologs in other phyla, and only four groups have members in both gastropods and bivalves, suggesting that ACPs are not very conserved in mollusks. We investigated the molecular functions of two highly-expressed secreted short-from ACPs featuring only an ApeC domain. One (CgACP1) of them from the bivalve pacific oyster Crossostrea gigas and the other (BgACP1) from the gastropod freshwater snail Biomphalaria glabrata. We showed that recombinant CgACP1 and BgACP1 bound with yeasts and several gram-positive and negative bacteria in different affinities. Both recombinant ACPs exhibited agglutinating activities to these microbes, but failed to inhibit or kill these microbes. Further analysis showed that both CgACP1 and BgACP1 bound to peptidoglycan (PGN) but not to other microbial cell wall components including lipopolysaccharide (LPS), lipoteichoic acid (LTA), Zymosan A, Chitin, Chitosan and Cellulose. Specifically, both ACPs could interact with the Lys-type PGN from S. aureus but not with the Dap-type PGN from Bacillus subtilis. And both recombinant ACPs showed no affinities to PGN subunits examined, including N-acetylglucosamine (GlcNAc), N-acetylmuramic acid (MurNAc) and muramyl dipeptide (MDP). In line with these, the CgACP1 gene were highly expressed in the gill and mantle, and were highly up-regulated shortly after bacterial challenge. Taken together, our findings show that these two short-form mollusk ACPs could act as pattern recognition proteins for PGN and participate in anti-microbial immunity, which hence proving that PGN binding is a basic function conserved in both amphioxus and mollusk ApeC domains.