Molecular Cloning and Functional Analysis of Secretory Phospholipase A2 from Apostichopus japonicus

Abstract

Secretory phospholipase A₂ (sPLA₂) plays important roles in phospholipid metabolism, skin barrier maintenance, immune response and other processes in organisms. sPLA₂ of sea cucumber A. japonicus (AjPLA₂) has not yet been reported. This study successfully amplified the AjPLA₂ sequence. The total cDNA of AjPLA₂ is 931 bp, including a 480 bp ORF that encodes 159 amino acids. The AjPLA₂ protein includes a 16-aa signal peptide, a 5-aa precursor peptide and a 138-aa mature peptide. Homologous alignment showed that AjPLA₂ and the sPLA₂s from starfish have the typical domains of the Group IB sPLA₂. And additional amino acid sequences were found around the β-Wing, which is different from the Group IB sPLA₂. These results showed that AjPLA₂ and sPLA₂s from starfish all belong to a new group in the Group I sPLA₂ family. AjPLA₂ is widely distributed in sea cucumber tissues. The functional analysis also showed that AjPLA₂ was upregulated in the intestine by feeding. When the body wall was damaged, it was significantly upregulated around the wound. And the expression levels of AjPLA₂ were significantly increased in V. splendens-infected sea cucumbers. The results indicated that AjPLA₂ plays roles in the sea cucumber immunologic process. Combined with the upregulation of unsaturated fatty acids (PUFAs) content in A. japonicus, it demonstrated that AjPLA₂ could participate in the immune of A. japonicus by hydrolyzing phospholipid and releasing PUFAs. This study had a solid foundation for the further research of AjPLA₂ gene function in vivo, development and application of AjPLA₂ protein.

Keywords: A. japonicus secretory phospholipase A2; Digestion; Epidermal barrier; Function; Immune; Phylogenetic tree.