O-Linked LacdiNAc-Modified Glycans in Extracellular Matrix Glycoproteins are Specifically Phosphorylated in a Cell Stress Independent Manner

Some glycosylation reactions are known to be controlled not only by a consensus sequence or a favourable amino acid pattern at the site of glycosylation, but also by cis-located peptidic elements. This has been shown for the initiation of O-mannosylation and for the terminal modification of N-linked chains with LacdiNAc. In searching for mammalian proteins modified with O-linked LacdiNAc we identified six positive species among nine endogenous and recombinant O-glyco proteins, which were without exception extracellular matrix, or matrix-related proteins: ZP3 and the five novel LacdiNAc-positive species ECM1, AMACO, nidogen-1, alpha-dystroglycan and neurofascin. No cis-located peptidic elements similar to those controlling N-linked LacdiNAc formation were found in any of these O-glycoproteins. 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-glycan modification that is strictly specific for LacdiNAc termini: sugar phosphorylation with formation of GalNAcbeta1-4(phospho-3)GlcNAc. The structural details of the phosphatase-labile, but sulfatase resistant compound were elucidated in MSn experiments and by methylation analysis, and in vitro synthesis confirmed LacdiNAc as the specific kinase substrate. The detection of phospho-LacdiNAc in human HEK-293 as well as human and mouse myoblast cells indicates a cell-independent expression. Different from yeast cells, where phospho rylated glycans can be formed as a stress response, the formation of phospho-LacdiNAc is apparently not related to cellular stress.