An HECT domain ubiquitin ligase CgWWP1 regulates granulocytes proliferation in oyster Crassostrea gigas.

ABSTRACT

Ubiquitination is involved in the regulation of granulocyte proliferation in vertebrate, and E3 ubiquitin ligase WWP1 has been reported to play an essential role in this process. In the present study, an HECT type E3 ubiquitin ligase (CgWWP1) was identified from oyster Crassostrea gigas, which contained a N-terminal C2 domain, four WW domains, and a C-terminal HECT domain. CgWWP1 was able to bind the activated ubiquitin (Ub) and formed CgWWP1-Ub complex in vitro. The mRNA transcripts of CgWWP1 were expressed in granulocytes, semi-granulocytes and agranulocytes, with the highest expression level in granulocytes. The expressions of potential granulocyte markers CgSOX11 (0.18-fold, p < 0.05) and CgAATase (0.2-fold, p < 0.01) in haemocytes were significantly down-regulated at 24 h after the treatment with Indole-3-carbinol (I3C), a WWP1 inhibitor. The proportions of EdU+ granulocytes reduced significantly at 12 h (8.1% ± 1.4%) and 24 h (9.7% ± 2.8%) after I3C treatment, which were significantly lower than that in the sterile seawater treatment (SW) group at 12 h (15.8% ± 4.2%) and 24 h (17.6% ± 0.8%), respectively. Meanwhile, the green EdU signals observed by confocal scanning microscopy in granulocytes of oysters treated by I3C became weaker compared to that in the SW group. These results indicated that CgWWP1 was involved in the regulation of granulocyte proliferation as a ubiquitin-protein ligase.