O-Linked N,N′-Diacetyllactosamine (LacdiNAc)-modified Glycans in Extracellular Matrix Glycoproteins Are Specifically Phosphorylated at Subterminal N-Acetylglucosamine

Trefoil Factor Family Domains Represent Highly Efficient Conformational Determinants for N-Linked N,N′-di-N-acetyllactosediamine (LacdiNAc) Synthesis

“…Proteins in 5-20% or 15% SDS gels were either stained with silver or blotted onto nitrocellulose (Protran BA 83, Schleicher & Schüll) in a wet blot transfer cell (Bio-Rad) for antibody detection or onto PVDF membranes in a semidry blot chamber (both from Bio-Rad) for lectin detection (16). Fusion proteins were detected with anti-strepII mouse IgG (IBA, Göt-tingen, Germany).…”

“…Supernatant was then passed through a cellulose filter and applied to the nickel-nitrilotriacetic acid column twice in a row. For detailed description of the procedure refer to Breloy et al (16). Eluted fractions containing the recombinant protein were pooled then concentrated and desalted via Amicon centrifugation units, molecular mass cutoff 10 kDa (Millipore, Darmstadt, Germany).…”

“…On glycodelin the LacdiNAc modification was claimed to have contraceptive functions. As a peripheral modification of O-linked chains, LacdiNAc modifications were found on murine zona pellucida glycoprotein ZP3 (14), POMC (15), and on the extracellular matrix glycoproteins nidogen-1, extracellular matrix protein 1, AMACO, ␣-dystroglycan, and neurofascin (16). The LacdiNAc dihexosamine on N-and O-linked chains has been shown previously to be the substrate for further modification by fucosylation, sialylation, and sulfation and, as a specific feature of O-linked chains, by phosphorylation of the subterminal GlcNAc residue (16).…”

Polysialic Acid on Neuropilin-2 Is Exclusively Synthesized by the Polysialyltransferase ST8SiaIV and Attached to Mucin-type O-Glycans Located between the b2 and c Domain

“…For both wild-type and mutant forms of NRP2, the same cell surface (5) (see the inset) were cut out individually and digested in-gel with Pronase E to generate Pronase-stable glycopeptides (42). Glycopeptides eluted from gel slices were off-gel-treated with 50 mM NaOH in the presence of sodium borohydride to liberate O-linked glycans by reductive ␤-elimination.…”

Xylose phosphorylation functions as a molecular switch to regulate proteoglycan biosynthesis

“…Kinases that specifically phosphorylate glycans have only rarely been reported and our knowledge of their physiological functions remains in its infancy (3)(4)(5). Fam20B (family with sequence similarity 20, member B) is a recently identified atypical secretory pathway kinase (6,7).…”