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J. Biol. Chem. 286 (25): 22653-22664

© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Phosvitin Plays a Critical Role in the Immunity of Zebrafish Embryos via Acting as a Pattern Recognition Receptor and an Antimicrobial Effector*Formula

Shaohui Wang{ddagger}1, Yuan Wang{ddagger}1, Jie Ma{ddagger}, Yunchao Ding§, , and Shicui Zhang{ddagger}§2

From the {ddagger}Institute of Evolution and Marine Biodiversity and
§Laboratory of Evolution and Development, Department of Marine Biology, Ocean University of China, Qingdao 266003, China

ABSTRACT Back to Top

Abstract: How fish embryos that develop externally survive microbial attacks is poorly understood. Here, we clearly demonstrated that the embryo extract of zebrafish and its early embryo both displayed antimicrobial activity against microbes, including pathogenic Aeromonas hydrophila, and phosvitin (Pv), a nutritional protein abundant in eggs, was related to this antimicrobial activity. We also showed that recombinant Pv (rPv) acted as a pattern recognition receptor capable of recognizing the microbial signature molecules LPS, lipoteichoic acid, and peptidoglycan, as well as binding the Gram-negative and -positive microbes Escherichia coli, A. hydrophila, and Staphylococcus aureus and functioned as an antimicrobial agent capable of killing the microbes. Furthermore, we revealed that its C-terminal 55 residues (Pt5) with the functional sites Arg242 and Ala201/Ile203 were indispensable for Pv antimicrobial activity. Importantly, microinjection of rPv or Pt5 into early embryos significantly enhanced their resistance to A. hydrophila challenge, and this enhanced bacterial resistance was markedly reduced by co-injection of anti-Pv antibody plus rPv (or Pt5) but not by injection of anti-actin antibody plus rPv. Moreover, the generated mutants with in vitro antimicrobial activity, when injected into the embryos, could also promote their resistance to A. hydrophila, but those without in vitro antimicrobial activity could not. It is thus proposed that Pv participates in the protection of early embryos against pathogenic attacks via binding and disrupting potential pathogens. This work also opens a new way for the study of the immunological roles of yolk proteins in oviparous animals that rely on yolk proteins for embryonic development.

Key Words: Antimicrobial Peptides • Embryo • Mutant • Protein Purification • Zebrafish • Antimicrobial Activity • Embryogenesis • Phosvitin • Vitellogenin • Zebrafish

Received for publication April 4, 2011. Revision received April 24, 2011.

FOOTNOTES Back to Top

1 Both authors contributed equally to this work.

2 To whom correspondence should be addressed: Ocean University of China, Rm. 205, Ke Xue Guan, 5 Yushan Rd., Qingdao 266003, China. E-mail: sczhang{at}ouc.edu.cn.

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