Identification and characterization of emNagII, a novel β-1,2-N-acetylglucosaminidase from Elizabethkingia meningoseptica

N-Acetylglucosamine exists in various forms and linkage patterns within organisms, playing a crucial role in numerous vital biological processes. However, research focusing on β-1,2-linked N-acetylglucosamine within complex biantennary N-glycans on the cell surface remain largely unexplored. Currently, there is a lack of efficient tools and methods capable of directly modifying terminal N-acetylglucosamine on living cells. In this study, we identified a novel β-N-acetylhexosaminidase from Elizabethkingia meningoseptica , which demonstrated favorable enzymatic stability within a pH range of 5-8 and at temperatures from 4 °C to 37 °C. This enzyme specifically cleaves non-reducing terminal β-1,2-N-acetylglucosamine, enabling direct removal of this modification from oligosaccharides and native glycoproteins. More importantly, we demonstrate for the first time that this enzyme successfully removes β-1,2-linked N-acetylglucosamine on living cell surfaces. Given its microbial origin and potential utility in living cell glycan editing, we have named it emNagII or cell-surface glycan-editing N-acetylglucosaminidase (csgeNagII). Through sequence analysis and alanine scanning mutagenesis, we identified a predicted active pocket containing the catalytic residue pair Asp317-Glu318, with Asp317 being essential for enzymatic activity. In summary, our findings provide an effective and reliable method for the targeted removal of β-1,2-N-acetylglucosamine on living cell surfaces, establishing a foundation for further functional studies and practical applications.