A novel epidermal growth factor-like domain protein 6 from Apostichopus japonicus mediates coelomocyte proliferation

Epidermal growth factor-like domain protein (EGFL) 6, a member of the epidermal growth factor (EGF) domain protein superfamily, has been reported to regulate various physiological processes, including cell adhesion, proliferation, migration and inflammation in mammals. Up to date, the study of invertebrate EGFL6 expression pattern and functional analysis of EGFL6 involved in proliferation is lacking. In this study, a novel EGFL6 homologue was identified from Apostichopus japonicus (designated as AjEGFL6) by rapid amplification of cDNA ends. The full-length cDNA of AjEGFL6 was 3915 bp, with a putative open reading frame of 2490 bp encoding 829 amino acid (aa) residue proteins. Structural analysis revealed that AjEGFL6 processed characteristic five EGF repeat domains and subclass of EGF_CA domain (89-338), UPF domain (504-607) and complement control protein (CCP, 755-808) domain. Multiple sequence alignment and phylogenetic analysis supported that AjEGFL6 belongs to a new member of the EGFL6 protein subfamily. Spatial expression analysis indicated that AjEGFL6 mRNA transcripts in the polian vesicle are expressed at a high level compared with that in coelomocytes. For Vibrio splendidus-challenged sea cucumbers, the peak expression of AjEGFL6 mRNAs in coelomocytes was detected at 24 h (12.29-fold), and it remained at high levels (1.52-fold) until 72 h. Functional investigation via MTT and EdU assays revealed that 60 and 120 ng of recombinant EGFL6 administration significantly upregulated cell viability by 19.72% and 46.13% respectively. Taken together, these findings suggest that AjEGFL6 may serve as a coelomocyte proliferation regulator in the sea cucumber.