*Result*: Functional characterization of a calcium-dependent bacterial-binding C-type lectin from amphioxus.

*C-type lectins (CTLs) function widely as pattern recognition receptors (PRRs) in innate immunity, mediating microbial recognition, phagocytosis, complement activation and immune regulation. In amphioxus, a basal chordate, more than 1200 CTL gene models have been predicted through genomic analysis, but only a few have been functionally characterized. Here, we describe AmphiCTL6, a novel CTL from Branchiostoma japonicum(B. japonicum), with a distinctive architecture consisting of tandem epidermal growth factor (EGF)-like domains and a C-type lectin-like domain (CTLD). In situ hybridization showed that AmphiCTL6 is highly expressed in the hepatic cecum, intestine, gills, and gonads. Single-cell RNA-seq further revealed predominant expression in gut epithelial-like and ciliated cells, pointing to a potential role in gut immune surveillance. Recombinant AmphiCTL6 proteins displayed calcium-dependent carbohydrate-binding activity and could bind and agglutinate a wide range of bacteria, including Enterococcus faecalis, Klebsiella pneumoniae, Staphylococcus pasteuri, Escherichia coli, and Vibrio anguillarum. Domain-truncation analysis demonstrated that the CTLD is responsible for bacterial interactions. Together, these findings identify AmphiCTL6 as a broad-spectrum PRR, expanding our understanding of the functional diversity of the complex CTL repertoire in basal chordates.
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*Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.*