Genome-wide identification and characterization of mucin15 involved in bacterial binding and agglutination from...

Mucin is a highly glycosylated macromolecular protein that is widely present in the mucosal tissues of fish. MUC15 has been identified in humans and higher vertebrates as playing a significant role in maintaining the mucosal barrier and regulating various signaling pathway. However, the potential role of mucin15 in fish immune responses remains poorly understood. In this study, mucin15 derived from fat greenling (Hexagrammos otakii) was identified and characterized, designated as Homuc15. The genomic DNA full length of Homuc15 was 14110 bp with an open reading frame (ORF) (1245 bp), encoding a polypeptide of 414 amino acids. The theoretical molecular weight of the HoMUC15 protein was 43.9 kDa. Subcellular localization analysis indicated that HoMUC15 was predominantly distributed in the extracellular matrix. Sequence alignment and phylogenetic tree analysis revealed that HoMUC15 was structurally similar to MUC15 orthologs from other teleost fish and displayed a high degree of conservation. Moreover, Homuc15 exhibited widespread expression across mucosal immune tissues, including skin, intestine, and gill. Its expression level in the skin was upregulated following stimulation with Vibrio harveyi. Furthermore, recombinant HoMUC15 (rHoMUC15) specifically bound to a variety of bacteria. The rHoMUC15 was able to bind PAMPs such as lipopolysaccharide (LPS), peptidoglycan (PGN), and Polyinosinic-polycytidylic acid (Poly I:C) in a concentration-dependent manner. However, antibacterial assays demonstrated that rHoMUC15 could not directly inhibit bacterial growth, bacterial agglutination assays indicated that rHoMUC15 was capable of agglutinating Vibrio harveyi, Bacillus subtilis, and Edwardsiella tarda in a Ca²⁺-dependent manner. Meanwhile, rHoMUC15 was found to promote the clearance of Vibrio harveyi from the immune tissues of Hexagrammos otakii. Collectively, these findings suggest that HoMUC15 possesses the ability to bind and agglutinate bacteria and may act as a pattern recognition receptor to assist Hexagrammos otakii in resisting pathogenic invasion.