Characterization of a Unique Carbohydrate-Binding Module from Akkermansia muciniphila that Binds Ganglioside-Associated Glycans

Abstract

Abstract Akkermansia muciniphila is a mucin-degrading gut symbiont that promotes host metabolic health through its ability to utilize host-derived glycans. Central to this microbe is its diverse set of glycoside hydrolases (GHs), specifically GHs involved in degrading host glycans like mucin. Here, we characterize an A. muciniphila GH from family 123 (AmGH123), that has β-N-acetyl-d-galactosaminidase activity, removing terminal β-linked GalNAc from ganglioside-associated glycans similar to other GH123s. In addition, we identify and characterize a unique carbohydrate-binding module from family 32 associated with AmGH123 (AmGH123CBM32), representing the first CBM characterized from A. muciniphila. AmGH123CBM32 selectively binds GalNAc-terminated ganglioside-associated glycans, revealing previously unrecognized ligand specificity for CBM32 members. Structural analysis and site-directed mutagenesis reveal a distinct ligand-binding pocket in AmGH123CBM32, highlighting diverse strategies for glycan recognition within this family.