Functional characterisation and comparative analysis of two C-type lectins with different key motifs from mud crab

C-type lectins are pattern recognition receptors (PRRs) involved in innate immunity. They possess one or more carbohydrate recognition domains (CRDs) belonging to Ca2+-dependent carbohydrate binding proteins. Herein, two C-type lectins were identified from the mud crab Scylla paramamosain. The open reading frame (ORF) of SpCTL-E encodes 160 amino acids including a CRD and a signal peptide, while the SpCTL-F ORF encodes a 244 amino acid polypeptide with a signal peptide and a CRD, and the key motifs for carbohydrate binding specificity are Asp-Pro-Asn (DPN) and Gln-Pro-Asp (QPD), respectively. DPN is a newly discovered motif, while QPD is typical of C-type lectins. The binding specificity toward different pathogen-associated molecular patterns (PAMPs) was analysed, and both bound three PAMPs: lipopolysaccharide (LPS), glucan (GLU) and peptidoglycan (PGN). SpCTL-F bound more strongly than SpCTL-E. SpCTL-E and SpCTL-F bound to five and seven microbial species, respectively, both agglutinated Gram-negative bacteria and fungi, and both promoted the phagocytosis activity of hemocytes toward Escherichia coli, and both enhanced the encapsulation activity of hemocytes towards Ni-NTA beads. SpCTL-E inhibited the growth of Micrococcus luteus and E. coli, while SpCTL-F also inhibited Vibrio parahaemolyticus. Thus, SpCTL-E and SpCTL-F are important PRRs with a broad nonself-recognition spectrum and play a key role in pathogen elimination. •SpCTL-E with novel DPN motif functioned as PRR to recognise PAMPs and microbes.•SpCTL-F with typical QPD motif had broader nonself recognition spectrum.•Both of them functioned as opsonin to eliminate pathogens.•They possessed antimicrobial activity towards different microbes.