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Identification and functional characterization of AP-2 complex subunit mu-A as a new member of antimicrobial protein
1Department of Marine Biology, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China.
Abstract:
AP-2 complex subunit mu-A (AP2M1A) is a component of the adaptor complexes that link clathrin to receptors in coated vesicles. It has recently been shown to be involved in the resistance to oxidative damage, challenging the conventional role of AP2M1A. Here we demonstrated that AP2M1A was a heparin-binding protein abundantly stored in eggs and embryos of zebrafish, and its gene expression was markedly up-regulated by LPS and LTA treatment. We also showed that recombinant AP2M1A (rAP2M1A) was not only able to interact with Gram-negative and Gram-positive bacteria as well as their signature molecules LPS and LTA, but also able to inhibit the growth of the bacteria. Additionally, we found that AP2M1A354-382 that contained 2 closely positioned heparin-binding motifs could also bind to LPS and LTA, and inhibit the bacterial growth. Both rAP2M1A and AP2M1A354-382 were shown to execute antibacterial activity by a combined action of destabilization/destruction of bacterial cell wall through interaction with LPS and LTA, disturbance of the usually polarized membrane through depolarization, and apoptosis/necrosis through intracellular ROS production. Finally, we showed that AP2M1A could protect zebrafish developing embryos/larvae against attack by the potential pathogen Aeromonas hydrophila. All these demonstrate for the first time that AP2M1A is a maternal antimicrobial protein previously uncharacterized. It also establishes a correlation between antibacterial activity and heparin-binding motifs.
Insights
AP-2 complex subunit mu-A (AP2M1A) is a maternal antimicrobial protein found in zebrafish. This protein binds heparin and inhibits bacterial growth by disrupting cell walls and inducing apoptosis, protecting embryos from infection.
Area of Science:
- Molecular Biology
- Immunology
- Developmental Biology
Background:
- AP-2 complex subunit mu-A (AP2M1A) is traditionally known for its role in coated vesicle formation.
- Recent findings suggest AP2M1A is involved in oxidative stress resistance, expanding its known functions.
Purpose of the Study:
- To investigate the novel antimicrobial properties of AP2M1A.
- To explore the role of heparin-binding motifs in AP2M1A's antibacterial activity.
- To determine AP2M1A's protective effect against bacterial pathogens in zebrafish.
Main Methods:
- Characterization of AP2M1A as a heparin-binding protein in zebrafish eggs and embryos.
- Analysis of AP2M1A gene expression in response to LPS and LTA.
- In vitro studies with recombinant AP2M1A (rAP2M1A) and a specific peptide fragment (AP2M1A354-382) to assess bacterial interaction and growth inhibition.
- Investigation of the mechanisms of antibacterial action, including cell wall destabilization, membrane depolarization, and ROS production.
- In vivo assessment of AP2M1A's protective role against Aeromonas hydrophila in zebrafish embryos/larvae.
Main Results:
- AP2M1A is abundant in zebrafish eggs/embryos and its expression is upregulated by LPS and LTA.
- rAP2M1A and AP2M1A354-382 bind to bacterial molecules (LPS, LTA) and inhibit bacterial growth.
- Antibacterial activity involves cell wall destabilization, membrane depolarization, and ROS-mediated apoptosis/necrosis.
- AP2M1A protects zebrafish embryos/larvae against Aeromonas hydrophila infection.
Conclusions:
- AP2M1A functions as a previously uncharacterized maternal antimicrobial protein in zebrafish.
- Heparin-binding motifs are crucial for AP2M1A's antibacterial activity.
- AP2M1A plays a significant role in innate immunity and protection during early development.
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