Identification Of ZRANB2 As A Novel Maternal LPS-binding Protein That Protects Embryos Of Zebrafish (Danio Rerio) Against Bacterial Infections

As an important mode animal for studying maternal immune, zebrafish has gradually became an newly favor. ZRANB2 proteins are widespread in animals, but their functions and mechanisms remain poorly defined. Here we adopt the method of high-throughput isolation from extract of zebrafish embryos to identify a LPS-binding protein-ZRANB2. Bioinformatics analysis was made, expression pattern, antibacterial activity in vivo and in vitro were studyed, and the center of the antibacterial activity was also explored, which proved that ZRANB2 is a maternal immune factors.Firstly, no matter in mRNA level or protein level, zebrafish ZRANB2 is present in all tissues, and mostly rich in the eggs/embryos, which indicates that zebrafish ZRANB2 is really a maternal immune protein. Secondly, the sequence comparison showed that zebrafish ZRANB2 shares 79.2%,80.3%,80.6% and 82.3% identity to mammalian, avian, amphibian and teleost, which indicate that ZRANB2 is conserved between species we selected. The structure prediction revealed that the 3D structures of both zebrafish and human ZRANB2 are mainly consisted of β-sheets. Thirdly, activity identification in vitro revealed that recombinant ZRANB2 (rZRANB2) acts as a pattern recognition receptor capable of identifying the bacterial signature molecule LPS (and lipid A) as well as binding the Gram-negative bacteria Escherichia coli, Vibrio anguilarum and Aeromonas hydrophila, and functions as an antibacterial effector molecule capable of directly killing the bacteria. Activity identification in vivo revealed that microinjection of rZRANB2 into early embryos significantly enhances the resistance of the embryos against pathogenic A. hydrophila challenge, and this enhanced bacterial resistance is markedly reduced by co-injection of anti-ZRANB2 antibody but not by injection of anti-actin antibody. Lastly, we found that Z1/37 or Z11/37 with in vitro antibacterial activity, when injected into the embryos, also promote the resistance of embryos against A. hydrophila, but the peptide Z38/198 without in vitro antibacterial activity does not.Collectively, these indicate that ZRANB2 is a maternal LPS-binding protein which can protect the early embryos of zebrafish against pathogenic attacks via binding and killing potential pathogens, a novel role ever assigned to ZRANB2 proteins. This work also provides a new angle for insights into the immunological functions of zinc finger proteins that are widely distributed in various animals.