Glycopeptide N-acetylgalactosaminyltransferase specificities for O-glycosylated sites on MUC5AC mucin motif peptides

The recombinant proteins of the two novel UDP-N-acetyl-galactosamine (Ga1NA c) glycopeptide, N-acetylgalactosaminyl-transferases (designated gpGaNTase- T7 and gpGaNTase-T9) were assayed with O-glycosylated products obtained fro m the prior action of the ubiquitous transferases (GaNTase-T1 and GaNTase-T 2) towards MUC5AC mucin motif peptides (GTT PSPVPTTSTTSAP and peptides with single amino acid substitutions, GTTPSAVPTTSTTSVP and GTTPSPVPTTSITSVP. th at are a reflection of mucin molecule polymorphism). gpGaNTase-T9 is known to be expressed differentially and more abundantly than gpGaNTase-T7 in som e tissues; the results of in vitro glycosylation also indicates a differenc e in acceptor substrate specificities between the gpGaNTase isoforms, With the use of capillary electrophoresis, MS and Edman degradation, our study s uggests that, in the O-glycosylation of mucin-type proteins, approach and r ecognition signalling by gpGaNTase-T7 and gpGaNTase-T9 depend largely on th e peptide's primary structure (for example the presence of multiple cluster s of hydroxy amino acids and the number of Ga1NAc residues attached to the peptide backbone). O-glycosylation in terms of sites of attachment seems to be less random than previously described and, if sequential reactions are ordered throughout the Golgi slack, the complete O-glycosylation of the muc in molecules seems to be finely tuned to respond to specific damage to, or attack on, epithelia.