O-Linked LacdiNAc-modified glycans in extracellular matrix glycoproteins are glycan-specifically phosphorylated in a cell stress independent manner

Protein-O-mannosylation and the terminal modification of N-glycans with LacdiNAc are initiated by cis located peptide elements, while it is unclear which signal triggers a LacdiNAc termination of O-glycans. In searching for mammalian proteins modified with O-linked LacdiNAc we identified six endogenous and recombinant O-glycoproteins, which were all extracellular matrix, or matrix-related proteins: ZP3 and the five novel LacdiNAc-positive species ECM1, AMACO, nidogen-1, a-dystroglycan and neurofascin.

The mass spectrometric analyses revealed a core 2-based tetrasaccharide as the common structural element that could be further modified, similar to the type 2 LacNAc termini, which form substrates for further modification with fucose, sialic acid or sulfate. We here provide structural evidence for a novel type of mucin-type O-glycans that is strictly specific for LacdiNAc termini: sugar phosphorylation with formation of GalNAcb1-4(phospho-3)GlcNAc. The structural details of the phosphatase-labile, but sulfatase resistant compound were elucidated by MSn measurements of the permethylated glycan alditols and methylation analysis of in vitro synthesized phospho-LacdiNAc. The detection of phospho-LacdiNAc in human HEK-293 as well as human and mouse myoblast cells indicates cell-independent expression. Different from yeast cells, where phosphorylated glycans can be formed as a stress response, the formation of phospho-LacdiNAc is apparently not related to cellular stress.