Discovery of the shortest sequence motif for high level mucin-type O-glycosylation

The consensus primary amino acid sequence for mucin-type O-glycosylation sites has not been identified. To determine the shortest motif sequence required for high level mucin-type O-glycosylation, we prepared more than 100 synthetic peptides and assayed in vitro O-GalNAc transfer to serine or threonine in these peptides using a bovine colostrum UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyl transferase (O-GalNAcT). We chose the sequence PDAASAAP from human erythropoietin (hEPO) for further systematic substitutions because it accepted GalNAc and was a fairly simple sequence consisting only of four kinds of amino acids. Several substitutions showed that threonine is approximately 40-fold better than serine as the glycosylated amino acid and a proline at position +3 on the C-terminal side is very important. To define the effect of proline residues around the glycosylation site, we analyzed a series of peptides containing one to three proline residues in a parent peptide AAATAAA. The results clearly indicated that prolines at positions +1 and +3 had a positive effect. The O-GalNAc transfer level of AAATPAP was increased approximately 90-fold from AAATAAA. The deletion of amino acids from the N-terminal side of the glycosylation site suggested that five amino acids from position -1 to +3 were especially important for glycosylation. Moreover, the influence of all 20 amino acids at positions -1, +2, and +4 was analyzed. Uncharged amino acids were preferred at position -1, and small or positively charged amino acids were preferred at position +2. No preference was observed at position +4. We propose a mucin-type O-glycosylation motif, XTPXP, which may be suitable as a signal for protein O-glycosylation. The features observed in this study also appear to be very useful for prediction of mucin-type O-glycosylation sites in glycoproteins.