Identification of the Zinc Finger Protein ZRANB2 as a Novel Maternal Lipopolysaccharide-binding Protein That Protects Embryos of Zebrafish against Gram-negative Bacterial Infections

Abstract

Zinc finger ZRANB2 proteins are widespread in animals, but their functions and mechanisms remain poorly defined. Here we clearly demonstrate that ZRANB2 is a newly identified LPSbinding protein present abundantly in the eggs/embryos of zebrafish. We also show that recombinant ZRANB2 (rZRANB2) acts as a pattern recognition receptor capable of identifying the bacterial signature molecule LPS as well as binding the Gramnegative bacteria Escherichia coli, Vibrio anguilarum, and Aeromonas hydrophila and functions as an antibacterial effector molecule capable of directly killing the bacteria. Furthermore, we reveal that N-terminal residues 11-37 consisting of the first ZnF_RBZ domain are indispensable for ZRANB2 antimicrobial activity. Importantly, microinjection of rZRANB2 into early embryos significantly enhanced the resistance of the embryos against pathogenic A. hydrophila challenge, and this enhanced bacterial resistance was markedly reduced by co-injection of anti-ZRANB2 antibody. Moreover, precipitation of ZRANB2 in the embryo extracts by preincubation with anti-ZRANB2 antibody caused a marked decrease in the antibacterial activity of the extracts against the bacteria tested. In addition, the N-terminal peptide Z 1/37 or Z 11/37 with in vitro antibacterial activity also promoted the resistance of embryos against A. hydrophila, but the peptide Z 38/198 without in vitro antibacterial activity did not. Collectively, these results indicate that ZRANB2 is a maternal LPS-binding protein that can protect the early embryos of zebrafish against pathogenic attacks, a novel role ever assigned to ZRANB2 proteins. This work also provides new insights into the immunological function of the zinc finger proteins that are widely distributed in various animals.

Zinc finger protein 365 is a new maternal LPS‐binding protein that defends zebrafish embryos against gram‐negative bacterial infections

“…As reported previously by Wang et al (21), band a on SDS‐PAGE ( Fig . 1 A ) of the proteins eluted from the LPS‐conjugated Sepharose CL‐4B column was ZNF365 (GenBank accession number XP_001339691.1), consisting of 345 aa.…”

“…The cloning of znf365 was conducted as described by Wang et al (21). Primer pair P1 and P2 ( Table 1 ), specific for znf365 , was designed using Primer Premier 5.0 (Premier Biosoft, Palo Alto, CA, USA) on the basis of the sequence of zebrafish znf365 (XM_001339655.4) in the National Center for Biotechnology Information database.…”

“…Western blot analysis was used to detect the binding of rZNF365 to the gram‐negative bacteria E. coli, Vibrio anguillarum , and Aeromonas hydrophila and the gram‐positive bacteria Staphylococcus aureus, Bacillus subtilis, and Micrococcus luteus (1 × 10 8 cells/ml) as described by Wang et al (21). TRX‐His‐tag peptide was used as control.…”

“…We previously discovered ZNF365 to be an LPS-binding protein (21). Briefly, fertilized eggs were collected, homogenized, and centrifuged at 5000 g at 4°C for 10 min.…”

Identification of Ly2 members as antimicrobial peptides from zebrafish Danio rerio

“…These data hint a clue that Ly2.1–3 may function as pattern recognition molecules capable of identifying the Gram-negative bacteria. Recently, Wang et al [29] identified the zinc finger protein ZRANB2 as a novel maternal LPS-binding protein that protects embryos of zebrafish against Gram-negative bacterial infections and they also found rZRANB2 can bind to lipid A, a core component of LPS, and the affinity of rZRANB2 to LPS was not inhibited by any of the sugars examined. We do not know the mechanism why Ly2.1–3 only targets Gram-negative bacteria, whereas rZRANB2 and rLy2.1–3 all can bind to LPS but not to LTA.…”