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: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. 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. Glycosylation is an important post-translational modification of proteins in eukaryotic cells, and is divided broadly into three categories of N-linked, O-linked, and glycosylphosphatidylinositol-anchored types (1Lis H. Sharon N. Eur. J. Biochem. 1993; 218: 1-27Crossref PubMed Scopus (802) Google Scholar). Mucin-type sugar chains are the principal structure in various O-linked oligosaccharides. The initial transfer of anN-acetylgalactosamine (GalNAc) 1The abbreviations used are: GalNAc,N-acetylgalactosamine; O-GalNAcT, UDP-N-acetylgalactosamine:polypeptideN-acetylgalactosaminyl transferase; hVF, human von Willebrand factor; hEPO, human erythropoietin; hG-CSF, human granulocyte-colony stimulating factor; Ac, acetylation; Am, amidation; HPLC, high performance liquid chromatography. from UDP-GalNAc to the hydroxyl group of serine or threonine is catalyzed by UDP-GalNAc:polypeptide α1,O-N-acetylgalactosaminyl transferase (O-GalNAcT; EC 2.4.1.41) in the cis-Golgi compartment (2Roth J. Wang Y. Eckhardt A.E. Hill R.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8935-8939Crossref PubMed Scopus (115) Google Scholar). To date, at least three isozymes for the enzyme have been found by purification (3Sugiura M. Kawasaki T. Yamashina I. J. Biol. Chem. 1982; 257: 9501-9507Abstract Full Text PDF PubMed Google Scholar, 4Takeuchi M. Yoshikawa M. Sasaki R. Chiba H. Agric. Biol. Chem. 1985; 49: 1059-1069Google Scholar, 5Elhammer A. Kornfeld S. J. Biol. Chem. 1986; 261: 5249-5255Abstract Full Text PDF PubMed Google Scholar, 6Wang Y. Abernethy J.L. Eckhardt A.E. Hill R.L. J. Biol. Chem. 1992; 267: 12709-12716Abstract Full Text PDF PubMed Google Scholar, 7Homa F.L. Hollander T. Lehman D.J. Thomsen D.R. Elhammer A.P. J. Biol. Chem. 1993; 268: 12609-12616Abstract Full Text PDF PubMed Google Scholar, 8Hagen F.K. VanWuyckhuyse B. Tabak L.A. J. Biol. Chem. 1993; 268: 18960-18965Abstract Full Text PDF PubMed Google Scholar, 11White T. Bennett E.P. Takio K. Sørensen T. Bonding N. Clausen H. J. Biol. Chem. 1995; 270: 24156-24165Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) and cDNA cloning (7Homa F.L. Hollander T. Lehman D.J. Thomsen D.R. Elhammer A.P. J. Biol. Chem. 1993; 268: 12609-12616Abstract Full Text PDF PubMed Google Scholar, 8Hagen F.K. VanWuyckhuyse B. Tabak L.A. J. Biol. Chem. 1993; 268: 18960-18965Abstract Full Text PDF PubMed Google Scholar, 9Yoshida A. Hara T. Ikenaga H. Takeuchi M. Glycoconj. J. 1995; 12: 824-828Crossref PubMed Scopus (16) Google Scholar, 10Hagen F.K. Gregoire C.A. Tabak L.A. Glycoconj. J. 1995; 12: 901-909Crossref PubMed Scopus (36) Google Scholar, 11White T. Bennett E.P. Takio K. Sørensen T. Bonding N. Clausen H. J. Biol. Chem. 1995; 270: 24156-24165Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 12Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 13Zara J. Hagen F.K. Hagen K.G. T VanWuyckhuyse B.C. Tabak L.A. Biochem. Biophys. Res. Commun. 1996; 228: 38-44Crossref PubMed Scopus (52) Google Scholar). Although the consensus amino acid sequence Asn-Xaa-Ser/Thr (Xaa ≠ Pro) is extensively known in the case of N-glycosylation sites, such a consensus primary amino acid sequence has not been identified among the mucin-type O-glycosylation sites. However, because mucin-type O-glycosylation occurs only at specific serine or threonine residues, it seems that a certain primary sequence or three-dimensional structure must be required for the recognition by O-GalNAcT. Proline residues are found with considerable frequency around glycosylation sites and may play an important role in recognition byO-GalNAcT. Statistical studies by Wilson et al.(14Wilson I.B.H. Gavel Y. von Heijne G. Biochem. J. 1991; 275: 529-534Crossref PubMed Scopus (240) Google Scholar), O'Connell et al. (15O'Connell B. Tabak L.A. Ramasubbu N. Biochem. Biophys. Res. Commun. 1991; 180: 1024-1030Crossref PubMed Scopus (64) Google Scholar), and Hansen et al.(16Hansen J.E. Lund O. Engelbrecht J. Bohr H. Nielsen J.O. Hansen J.-E.S. Brunak S. Biochem. J. 1995; 308: 801-813Crossref PubMed Scopus (235) Google Scholar) suggested that prolines occur most commonly at position −1 and +3 from the glycosylated residue (number with − or + refers to a position of amino acid that is N-terminal or C-terminal to a glycosylated amino acid). Elhammer et al. (17Elhammer A.P. Poorman R.A. Brown E. Maggiora L.L. Hoogerheide J.G. Kézdy F.J. J. Biol. Chem. 1993; 268: 10029-10038Abstract Full Text PDF PubMed Google Scholar) suggested that all proline residues from −4 to +4 may be acceptable forO-glycosylation. Pisano et al. (18Pisano A. Redmond J.W. Williams K.L. Gooley A.A. Glycobiology. 1993; 3: 429-435Crossref PubMed Scopus (95) Google Scholar) proposed fourO-glycosylation motifs from the identification of sites in human glycophorin A. One of them, XPXX (at least one X is a glycosylated threonine residue), indicates that prolines at position −1, −2, and +1 may be important. However, these findings have not been fully supported by biochemical studies of the effect of proline residues at each position around theO-glycosylation sites. Some attempts to analyze the influence of the primary sequence using synthetic peptides in vitro have been reported. This method is useful for identifying sequences capable of being glycosylated and for determining the efficiency of O-GalNAc transfer to a certain sequence. By using synthetic peptides derived from bovine myelin, Young et al. (19Young J.D. Tsuchiya D. Sandlin D.E. Holroyde M.J. Biochemistry. 1979; 18: 4444-4448Crossref PubMed Scopus (48) Google Scholar) showed that the sequences containing TPPP are good substrates and suggested that a triproline sequence C-terminal to threonine might be sufficient for mucin-typeO-glycosylation. However, it has also been reported that peptides not containing the triproline sequence are substrates forO-GalNAcT (15O'Connell B. Tabak L.A. Ramasubbu N. Biochem. Biophys. Res. Commun. 1991; 180: 1024-1030Crossref PubMed Scopus (64) Google Scholar, 17Elhammer A.P. Poorman R.A. Brown E. Maggiora L.L. Hoogerheide J.G. Kézdy F.J. J. Biol. Chem. 1993; 268: 10029-10038Abstract Full Text PDF PubMed Google Scholar, 20O'Connell B.C. Hagen F.K. Tabak L.A. J. Biol. Chem. 1992; 267: 25010-25018Abstract Full Text PDF PubMed Google Scholar). Tabak's group have made efforts to analyze the in vitro GalNAc transfer using more than 50 peptides derived from human von Willebrand factor (hVF) (15O'Connell B. Tabak L.A. Ramasubbu N. Biochem. Biophys. Res. Commun. 1991; 180: 1024-1030Crossref PubMed Scopus (64) Google Scholar, 20O'Connell B.C. Hagen F.K. Tabak L.A. J. Biol. Chem. 1992; 267: 25010-25018Abstract Full Text PDF PubMed Google Scholar). Although they reported that a proline at position +3 is essential and a second proline placed at −1, +1, +2, or +4 is particularly effective, single substitutions from the native sequence are not enough to understand the effect of proline residues in general (20O'Connell B.C. Hagen F.K. Tabak L.A. J. Biol. Chem. 1992; 267: 25010-25018Abstract Full Text PDF PubMed Google Scholar). Similar experiments using more than forty synthetic peptides derived from human Muc1 mucin were reported by Nishimori et al. (21Nishimori I. Johnson N.R. Sanderson S.D. Perini F. Mountjoy K. Cerny R.L. Gross M.L. Hollingsworth M.A. J. Biol. Chem. 1994; 269: 16123-16130Abstract Full Text PDF PubMed Google Scholar). Their findings suggested that a proline at position +3 enhances the glycosylation but is not sufficient and the minimal size of the peptide required for efficient glycosylation is six amino acids from −1 to +4, while Young et al. (19Young J.D. Tsuchiya D. Sandlin D.E. Holroyde M.J. Biochemistry. 1979; 18: 4444-4448Crossref PubMed Scopus (48) Google Scholar) described TPPP as the minimal substrate. These results show that the surrounding sequences of theO-glycosylated amino acid affect the GalNAc transfer remarkably; however, further experiments are to the general for mucin-type O-glycosylation. most studies were by the that was made single and or serine and threonine as the glycosylated this we the features of amino acid sequence around single mucin-type O-glycosylation sites by in transfer using a series of synthetic colostrum was to transfer GalNAc to a peptide indicates the glycosylated derived from an mucin-type O-glycosylation site of human erythropoietin substitutions of the sequence showed the minimal peptide required for high level GalNAc the preference of amino and proline residues at position +1 and results observed in this we propose is an mucin-typeO-glycosylation and were from UDP-GalNAc and bovine mucin were from and were from was from were from was prepared from bovine mucin as described by and A. Biochem. Biophys. PubMed Scopus Google Scholar). The peptides were by method using an peptide on a or from The peptides were by high performance liquid on a by with a containing a single by the on a To peptide and amino acid the peptides were containing at for and the were analyzed by a amino acid The of each peptide was analyzed on an was from of bovine colostrum by and of as described by Elhammer et al. A. Kornfeld S. J. Biol. Chem. 1986; 261: 5249-5255Abstract Full Text PDF PubMed Google Scholar). The enzyme of the was by the transfer from to an as described by al. (3Sugiura M. Kawasaki T. Yamashina I. J. Biol. Chem. 1982; 257: 9501-9507Abstract Full Text PDF PubMed Google Scholar). The 50 synthetic and enzyme in a of 50 The initial of GalNAc transfer to peptide was by the at from to on the of the The was by 50 of The glycosylated peptide was from on a with as The of was in a and with liquid The was by a for was with AAATPAP as O-GalNAc transfer was assayed using synthetic peptides derived from mucin-type mucin A.E. Abernethy J.L. Y. Hill R.L. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar), mucin M. Hill R.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar), human granulocyte-colony stimulating factor N. T. K. M. N. T. J. Biochem. PubMed Scopus Google Scholar), bovine A. Redmond J.W. Williams K.L. Gooley A.A. Glycobiology. 1994; PubMed Scopus Google Scholar), and E. T. J. F. F. T. S. Biochemistry. 1992; PubMed Scopus Google Scholar, R. Wang T. E. J. Biol. Chem. 1986; 261: Full Text PDF PubMed Google Scholar). The peptide derived from bovine myelin, which is not mucin-type (19Young J.D. Tsuchiya D. Sandlin D.E. Holroyde M.J. Biochemistry. 1979; 18: 4444-4448Crossref PubMed Scopus (48) Google Scholar), and the peptide which was to be a good from of the mucin-typeO-glycosylation sites by Elhammer et al. (17Elhammer A.P. Poorman R.A. Brown E. Maggiora L.L. Hoogerheide J.G. Kézdy F.J. J. Biol. Chem. 1993; 268: 10029-10038Abstract Full Text PDF PubMed Google Scholar), were also assayed as substrates for to the with The results are in A. The transfer of GalNAc was observed in peptides from hG-CSF, bovine hEPO, bovine myelin, and the sequence proposed by while the transfer was not in peptides from and the peptide was the the of GalNAc to each peptide was indicated in to this The peptide from was more glycosylated than peptides derived from mucin-type and the of GalNAc was of the The and as a glycosylated amino acid was using the sequences from bovine and The glycosylation level of the peptide containing was ∼40-fold than containing in This is in good with the results for the and the bovine colostrum F.K. VanWuyckhuyse B. Tabak L.A. J. Biol. Chem. 1993; 268: 18960-18965Abstract Full Text PDF PubMed Google Scholar, 17Elhammer A.P. Poorman R.A. Brown E. Maggiora L.L. Hoogerheide J.G. Kézdy F.J. J. Biol. Chem. 1993; 268: 10029-10038Abstract Full Text PDF PubMed Google Scholar). the sequence showed glycosylation than that showed the glycosylation in it is that the surrounding sequence of theO-glycosylation site from may suitable for the glycosylation by bovine colostrum To a parent peptide for further of the amino acid preference at each position around the glycosylation site, the effect of in the sequence was analyzed substitutions and a deletion of at position −4 O-GalNAc but at position +3 to be This suggested that certain proline residues that are observed around mucin-type O-glycosylation sites are very important. The most was glycosylated in level and as the parent The positions of a proline residue were and the is in A. proline residues at position +3 and +1 increased the transfer level by and in to the parent was by at position however, proline residues at positions not glycosylation. of a second proline were by it to which was the sequence in A. in the second proline at position +1 a effect to at +3 and increased the transfer level to 90-fold of the parent the prolines at position and showed a from second proline residues not show a series of containing a proline which was to prolines at position +1 and were analyzed No from AAATPAP was observed in all and a was by at position The of peptides are in of proline of the sequence AAATPAP by while were the peptide containing the proline at position +2, has a than that of AAATPAP but also a efficiency is the as that of The GalNAc transfer to was but to determine the the of peptides for O-GalNAc in a The positive effect by proline residues at position +1 and +3 was analyzed in the case of serine as the glycosylated amino in these proline residues were on the GalNAc transfer to and the of proline at each position was to the observed in the transfer to threonine as in The influence of deletion in the N-terminal amino acids were analyzed to determine the minimal required for high level GalNAc The deletion of at position and the glycosylation but was not However, the of at −1 made a of the that the amino acid at position −1 is required for Young et al. E. Young J.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar) suggested that of the N-terminal amino group of the glycosylated threonine might for this we also analyzed the influence of of the N-terminal amino group as as of the C-terminal The results in that effect was from the of with the peptides containing at position −1, such as and showed approximately transfer of GalNAc than The of had effect. These suggested that the minimal required for high level O-glycosylation is five amino acids from −1 to The of amino acid at position −1 and +2, at which proline not show effect were analyzed. The glycosylation was by the of amino acids at position −1 and and The bovine colostrum enzyme GalNAc to peptides containing or but not to peptides containing or at position −1 at position −1, it is that amino acids were acceptable while the charged amino acids were was that and a were as as and a small side level transfer was the amino acid at was and but level transfer was it was and these we that the amino acids with a positive or small side are at position +2. an is at position +2, which showed approximately of the glycosylation to The influence of various amino acids at position +4 was By to the results of amino acids at position −1 and +2, on the transfer was observed However, the transfer level to of this have been (3Sugiura M. Kawasaki T. Yamashina I. J. Biol. Chem. 1982; 257: 9501-9507Abstract Full Text PDF PubMed Google Scholar, 4Takeuchi M. Yoshikawa M. Sasaki R. Chiba H. Agric. Biol. Chem. 1985; 49: 1059-1069Google Scholar, 5Elhammer A. Kornfeld S. J. Biol. Chem. 1986; 261: 5249-5255Abstract Full Text PDF PubMed Google Scholar, 6Wang Y. Abernethy J.L. Eckhardt A.E. Hill R.L. J. Biol. Chem. 1992; 267: 12709-12716Abstract Full Text PDF PubMed Google Scholar, 7Homa F.L. Hollander T. Lehman D.J. Thomsen D.R. Elhammer A.P. J. Biol. Chem. 1993; 268: 12609-12616Abstract Full Text PDF PubMed Google Scholar, 8Hagen F.K. VanWuyckhuyse B. Tabak L.A. J. Biol. Chem. 1993; 268: 18960-18965Abstract Full Text PDF PubMed Google Scholar, 11White T. Bennett E.P. Takio K. Sørensen T. Bonding N. Clausen H. J. Biol. Chem. 1995; 270: 24156-24165Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) and (7Homa F.L. Hollander T. Lehman D.J. Thomsen D.R. Elhammer A.P. J. Biol. Chem. 1993; 268: 12609-12616Abstract Full Text PDF PubMed Google Scholar, 8Hagen F.K. VanWuyckhuyse B. Tabak L.A. J. Biol. Chem. 1993; 268: 18960-18965Abstract Full Text PDF PubMed Google Scholar, 9Yoshida A. Hara T. Ikenaga H. Takeuchi M. Glycoconj. J. 1995; 12: 824-828Crossref PubMed Scopus (16) Google Scholar, 10Hagen F.K. Gregoire C.A. Tabak L.A. Glycoconj. J. 1995; 12: 901-909Crossref PubMed Scopus (36) Google Scholar, 11White T. Bennett E.P. Takio K. Sørensen T. Bonding N. Clausen H. J. Biol. Chem. 1995; 270: 24156-24165Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 12Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 13Zara J. Hagen F.K. Hagen K.G. T VanWuyckhuyse B.C. Tabak L.A. Biochem. Biophys. Res. Commun. 1996; 228: 38-44Crossref PubMed Scopus (52) Google Scholar). The cDNA for bovine colostrum which is used in this was from bovine small (7Homa F.L. Hollander T. Lehman D.J. Thomsen D.R. Elhammer A.P. J. Biol. Chem. 1993; 268: 12609-12616Abstract Full Text PDF PubMed Google Scholar) and as cDNA E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). with were also from various containing bovine F.K. VanWuyckhuyse B. Tabak L.A. J. Biol. Chem. 1993; 268: 18960-18965Abstract Full Text PDF PubMed Google Scholar), A. Hara T. Ikenaga H. Takeuchi M. Glycoconj. J. 1995; 12: 824-828Crossref PubMed Scopus (16) Google Scholar), and F.K. Gregoire C.A. Tabak L.A. Glycoconj. J. 1995; 12: 901-909Crossref PubMed Scopus (36) Google Scholar), is to and the of and have also been from human T. Bennett E.P. Takio K. Sørensen T. Bonding N. Clausen H. J. Biol. Chem. 1995; 270: 24156-24165Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) and human E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar), is among human and was from a containing of T. Bennett E.P. Takio K. Sørensen T. Bonding N. Clausen H. J. Biol. Chem. 1995; 270: 24156-24165Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar), and showed that is only in such as and E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). is that these have on and be We reported the features of primary peptide sequences required for high level O-glycosylation by a bovine in These results appear to be very useful for the of mucin-typeO-glycosylation efficiency and prediction of the glycosylation sites in glycoproteins. we propose the motifs for high level and to single mucin-type O-glycosylation sites by is the motif among The of sequence on GalNAc transfer suggested that of the amino acid sequence around the site might the glycosylation efficiency of in et al. S. T. E. R. T. K. Biochemistry. 1994; PubMed Scopus Google Scholar) reported that of and were in cells, while was not This indicates that the results vitro GalNAc transfer using peptides be used to an to a fully glycosylated glycoproteins. results showed that bovine colostrum transfer GalNAc to serine at a level while the GalNAc transfer to threonine of this enzyme was than that to serine by to in the surrounding sequences and This preference of threonine to serine was in good with the results of using sequences from hEPO, bovine myelin, and mucin F.K. VanWuyckhuyse B. Tabak L.A. J. Biol. Chem. 1993; 268: 18960-18965Abstract Full Text PDF PubMed Google Scholar, 17Elhammer A.P. Poorman R.A. Brown E. Maggiora L.L. Hoogerheide J.G. Kézdy F.J. J. Biol. Chem. 1993; 268: 10029-10038Abstract Full Text PDF PubMed Google Scholar, Y. N. Eckhardt A.E. Hill R.L. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). was that the peptide derived from was more glycosylated than peptides from bovine and hG-CSF, the sequence a serine as the glycosylated amino This suggested that the surrounding sequence of the glycosylation site is more suitable than of bovine and of the proline residues surrounding the glycosylation site clearly showed that specific proline residues at position +3 and +1 the but prolines not have such These were observed serine was as the glycosylated amino acid in of threonine The studies showed that the effect of these proline residues is than This indicates that proline residues not the and O-GalNAcT. The of a proline at +3 in the surrounding sequence of was with in and mucin (15O'Connell B. Tabak L.A. Ramasubbu N. Biochem. Biophys. Res. Commun. 1991; 180: 1024-1030Crossref PubMed Scopus (64) Google Scholar, 20O'Connell B.C. Hagen F.K. Tabak L.A. J. Biol. Chem. 1992; 267: 25010-25018Abstract Full Text PDF PubMed Google Scholar, I. Johnson N.R. Sanderson S.D. Perini F. Mountjoy K. Cerny R.L. Gross M.L. Hollingsworth M.A. J. Biol. Chem. 1994; 269: 16123-16130Abstract Full Text PDF PubMed Google Scholar). However, the influence of proline at −1 was not by have suggested that at −1 and +3 might be important I.B.H. Gavel Y. von Heijne G. Biochem. J. 1991; 275: 529-534Crossref PubMed Scopus (240) Google Scholar, B. Tabak L.A. Ramasubbu N. Biochem. Biophys. Res. Commun. 1991; 180: 1024-1030Crossref PubMed Scopus (64) Google Scholar, J.E. Lund O. Engelbrecht J. Bohr H. Nielsen J.O. Hansen J.-E.S. Brunak S. Biochem. J. 1995; 308: 801-813Crossref PubMed Scopus (235) Google Scholar). Although O'Connell et al. (20O'Connell B.C. Hagen F.K. Tabak L.A. J. Biol. Chem. 1992; 267: 25010-25018Abstract Full Text PDF PubMed Google Scholar) reported that a second proline placed at −1, +1, +2, or +4 is particularly effective, indicated the second prolines at −1 and have effect. The positive of at −1 and observed in results to be by the of an amino acid to a from to at position peptide showed an from AAATPAP in the GalNAc the is not it might be from the by the with the proline at position The minimal required for the GalNAc transfer by bovine was five amino acids from position −1 to Young et al. E. Young J.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar) indicated that an amino acid at position −1 might not be required for the in from the of and results clearly showed that at −1 has a effect than the N-terminal or at −1 and deletion of N-terminal amino acids indicated that not only the amino acid at position −1 but also at and may affect the O-GalNAc transfer of the glycosylation level was observed in the from to at position −2, and −1 The in vitro glycosylation of peptides was by the of amino acids at position −1 and +2, and a preference was observed at each position position −1, amino acids were good for the glycosylation and the size of side was not The amino acids at position were with a positive or small side The positive to be very in this because the side chains of and are not at showed level side is The for this may be from the specific made by to positions −1 and +2, of the glycosylation was observed in the of amino acid at position +4 that this position is not important in the recognition by bovine We proposed motifs for mucin-type O-glycosylation vitro using a series of systematic synthetic mucin-type on have various and the of a site in a protein seems to be very useful to and We are these motifs as a signal for mucin-typeO-glycosylation they are into a We for bovine We M. D. for of the