Enzymatic basis of branching and extension of O-Man glycans for keratan sulfate biosynthesis

O-Mannose (Man) glycans are branched specifically in the brain by a dedicated glycosyltransferase, N-acetylglucosaminyltransferase IX (GnT-IX, also known as MGAT5B). Such branching of O-Man glycans was reported to be involved in diseases, including demyelination and glioma, but the enzymatic mechanisms by which O-Man glycan is specifically recognized by GnT-IX and how branched O-Man glycans are subsequently elongated by other enzymes in the brain have remained unclear. To shed light on these issues, we here first compared the structural model of GnT-IX complexed with its O-Man substrate with the crystal structure of the homologous N-glycan branching enzyme GnT-V (also known as MGAT5). Several residues in GnT-IX were predicted to be critical to recognition of the O-Man substrate, and an enzyme assay revealed that R304 in GnT-IX is crucial for the specificity toward O-Man glycans. We further investigated the role of O-Man branching for subsequent elongation in the brain and found that the level of keratan sulfate (KS) in O-Man glycans was significantly reduced in GnT-IX-knockout (KO) mouse brain, suggesting that O-Man branching promotes KS biosynthesis. Mechanistically, our enzymatic assays of the KS biosynthetic enzymes demonstrated that B4GALT1, B4GALT4, and CHST1 exhibited significantly higher activity toward branched O-Man glycans than toward their linear counterparts. These results imply that branching of O-Man glycans by GnT-IX provides the scaffold for efficient subsequent glycan elongation. Our findings deepen our understanding of the complex biosynthetic pathway of O-Man glycans in the brain.