Molecular characterization, microbial recognition and immunomodulatory functions of a novel ladderlectin-like lectin...

Ladderlectin, a member of the C-type lectin (CTL) family, has historically been identified only in fish and shrimp, where it is primarily involved in innate immune defense. In teleosts, ladderlectin plays a crucial dual role in both innate immunity against pathogens and reproductive regulation. However, the molecular characterization and immunomodulatory functions of ladderlectin homolog (ladderlectin-like) in Hexagrammos otakii remain poorly understood. In this study, a novel ladderlectin-like gene (HoLL-like) was identified and cloned from Hexagrammos otakii based on genomic data. Sequence analysis and domain prediction revealed that HoLL-like contains a 534-bp open reading frame encoding a 177-aa polypeptide, with an N-terminal signal peptide and a single C-type lectin-like domain (CTLD). The CTLD retains the WSD, WND, and WIGG motifs and is rich in cysteine residues. Phylogenetic analysis showed that HoLL-like forms a distinct, relatively independent clade within the teleost ladderlectin-like family. Quantitative RT-PCR analysis demonstrated that Holl-like is expressed in multiple tissues, with the highest transcript levels in the liver. Besides, it exhibited a time-dependent transcriptional response in the liver, spleen, skin, intestine, and kidney following Vibrio harveyi infection. After heterologous expression, purification, and refolding, the recombinant protein rHoLL-like displayed characteristic ladder-like oligomeric bands in immunoblotting. Functional assays revealed that rHoLL-like binds and agglutinates multiple Gram-positive and Gram-negative bacteria in a Ca²⁺-independent manner and binds pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), peptidoglycan (PGN), and poly(I:C) in a dose-dependent manner, showing higher affinity for LPS. Further analyses indicated that rHoLL-like did not directly agglutinate or lyse erythrocytes, but enhanced serum hemolytic and bactericidal activities under Ca²⁺-containing conditions. These effects were sensitive to EDTA and serum heat inactivation, suggesting that rHoLL-like may modulate heat-labile, Ca²⁺-dependent serum effector mechanisms, potentially associated with complement activity. Collectively, these findings suggest that HoLL-like plays a crucial role in the innate immune response of H. otakii against bacterial and other pathogen infections. This study expands our understanding of the functional repertoire of teleost ladderlectin-like proteins and provides new insights into the molecular strategies to prevent and control bacterial diseases in marine aquaculture, as well as for the development of immunoactive agents.