A novel C-type lectin from the sea cucumber Apostichopus japonicus (AjCTL-2) with preferential binding of d-galactose

Abstract C-type lectins (CTLs) are Ca 2+ dependent carbohydrate-binding proteins that share structural homology in their carbohydrate-recognition domains (CRDs). In the present study, a novel CTL was identified from sea cucumber Apostichopus japonicus (named as Aj CTL-2). The deduced amino acid sequence of Aj CTL-2 was homologous to CTLs from other animals with the identities ranging from 33% to 40%. It contained a canonical signal peptide at the N-terminus, a low density lipoprotein receptor class A (LDLa), a C1r/C1s/Uegf/bone morphogenetic protein 1 (CUB), and a CRD with two motifs Glu-Pro-Asn (EPN) and Trp-Asn-Asp (WND) in Ca 2+ binding site 2. The mRNA transcripts of Aj CTL-2 were extensively expressed in all the tested tissues including respiratory tree, muscle, gut, coelomocyte, tube-foot, body wall and gonad, and the highest expression level of Aj CTL-2 in coelomocyte was about 4.2-fold ( p Aj CTL-2 in coelomocyte increased significantly after Vibrio splendidus stimulation, and dramatically peaked at 12 h, which was 206.4-fold ( p Aj CTL-2 protein was mainly detected in cytoplasm of coelomocyte by immunofluorescence. The recombinant Aj CTL-2 (r Aj CTL-2) displayed binding activity to d -galactose independent of Ca 2+ , while the binding activity to other tested pathogen-associated molecular patterns (PAMPs) including lipopolysaccharide (LPS), peptidoglycan (PGN), and mannose (Man) could not be detected. Surface plasmon resonance (SPR) analysis further revealed the high binding specificity and moderate binding affinity of r Aj CTL-2 to d -galactose (KD = 4.093 × 10 −6  M). After r Aj CTL-2 was blocked by its polyclonal antibody, the binding activity to d -galactose could not be detected by using a blocking ELISA (B-ELISA). Moreover, r Aj CTL-2 could bind various microorganisms including V. splendidus , V. anguillarum , Staphylococcus aureus , Bifidobacterium breve and Yarrowia lipolytica with the strongest binding activity to B. breve . These results collectively suggested that Aj CTL-2 was a member of CTL superfamily (CTLs) with preferential binding of d -galactose and participated in the immune response of sea cucumber.