Discovery of the Shortest Sequence Motif for High Level Mucin-type O-Glycosylation

• Published in: The Journal of biological chemistry Vol. 272; no. 27; pp. 16884 - 16888
• Main Authors: Yoshida, Aruto, Suzuki, Misa, Ikenaga, Hiroshi, Takeuchi, Makoto
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04-07-1997; American Society for Biochemistry and Molecular Biology
• Subjects: Acetylgalactosamine - metabolism; Amino Acid Sequence; Animals; Binding Sites; Cattle; Colostrum - chemistry; Erythropoietin - chemistry; Glycosylation; Humans; Kinetics; Molecular Sequence Data; Mucins - chemistry; Mucins - metabolism; Peptide Fragments - metabolism; Peptide Mapping; Proline - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.27.16884

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:polypeptideN-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 ∼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-typeO-glycosylation sites in glycoproteins.