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• W2036606278 abstract "In search of possible epigenetic regulatory mechanisms ruling the initiation of O-glycosylation by polypeptide:N-acetylgalactosaminyltransferases, we studied the influences of mono- and disaccharide substituents of glycopeptide substrates on the site-specific in vitroaddition of N-acetylgalactosamine (GalNAc) residues by recombinant GalNAc-Ts (rGalNAc-T1, -T2, and -T3). The substrates were 20-mers (HGV20) or 21-mers (AHG21) of the MUC1 tandem repeat peptide carrying GalNAcα or Galβ1–3GalNAcα at different positions. The enzymatic products were analyzed by MALDI mass spectrometry and Edman degradation for the number and sites of incorporated GalNAc. Disaccharide placed on the first position of the diad Ser-16-Thr-17 prevents glycosylation of the second, whereas disaccharide on the second position of Ser-16-Thr-17 and Thr-5-Ser-6 does not prevent GalNAc addition to the first. Multiple disaccharide substituents suppress any further glycosylation at the remaining sites. Glycosylation of Ser-16 is negatively affected by glycosylation at position −6 (Thr-10) or −10 (Ser-6) and is inhibited by disaccharide at position −11 (Thr-5), suggesting the occurrence of glycosylation-induced effects on distant acceptor sites. Kinetic studies revealed the accelerated addition of GalNAc to Ser-16 adjacent to GalNAc-substituted Thr-17, demonstrating positive regulatory effects induced by glycosylation on the monosaccharide level. These antagonistic effects of mono- and disaccharides could underlie a postulated regulatory mechanism. In search of possible epigenetic regulatory mechanisms ruling the initiation of O-glycosylation by polypeptide:N-acetylgalactosaminyltransferases, we studied the influences of mono- and disaccharide substituents of glycopeptide substrates on the site-specific in vitroaddition of N-acetylgalactosamine (GalNAc) residues by recombinant GalNAc-Ts (rGalNAc-T1, -T2, and -T3). The substrates were 20-mers (HGV20) or 21-mers (AHG21) of the MUC1 tandem repeat peptide carrying GalNAcα or Galβ1–3GalNAcα at different positions. The enzymatic products were analyzed by MALDI mass spectrometry and Edman degradation for the number and sites of incorporated GalNAc. Disaccharide placed on the first position of the diad Ser-16-Thr-17 prevents glycosylation of the second, whereas disaccharide on the second position of Ser-16-Thr-17 and Thr-5-Ser-6 does not prevent GalNAc addition to the first. Multiple disaccharide substituents suppress any further glycosylation at the remaining sites. Glycosylation of Ser-16 is negatively affected by glycosylation at position −6 (Thr-10) or −10 (Ser-6) and is inhibited by disaccharide at position −11 (Thr-5), suggesting the occurrence of glycosylation-induced effects on distant acceptor sites. Kinetic studies revealed the accelerated addition of GalNAc to Ser-16 adjacent to GalNAc-substituted Thr-17, demonstrating positive regulatory effects induced by glycosylation on the monosaccharide level. These antagonistic effects of mono- and disaccharides could underlie a postulated regulatory mechanism. N-acetylgalactosamine recombinant polypeptide:N-acetylgalactosaminyltransferase polypeptide:N-acetylgalactosaminyltransferase galactose matrix-assisted laser desorption ionization high performance liquid chromatography Although no strict sequence dependence is known for the initiation of O-glycosylation by polypeptide:N-acetylgalactosaminyltransferases (ppGalNAc-Ts),1 functionally expressed recombinant enzymes display a distinct selectivity for peptide motifs in the vicinity of putative glycosylation sites (1Sorensen T. White T. Wandall H.H. Kristensen A.K. Roepstorff P. Clausen H. J. Biol. Chem. 1995; 270: 24166-24173Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 2Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 3Wandall H.H. Hassan H. Mirgorodskaya E. Kristensen A.K. Roepstorff P. Bennett E.P. Nielsen P.A. Hollingsworth M.A. Burchell J. Taylor-Papadimitriou J. Clausen H. J. Biol. Chem. 1997; 272: 23503-23514Crossref PubMed Scopus (268) Google Scholar). Until now, the prediction of O-glycosylation sites was based on two different approaches: the analysis of in vitro or ofin vivo glycosylated peptides (4O'Connell B.C. Tabak L.A. Ramusubbu N. Biochem. Biophys. Res. Commun. 1991; 180: 1024-1030Crossref PubMed Scopus (64) Google Scholar, 5Nehrke K. Hagen F.K. Tabak L.A. J. Biol. Chem. 1996; 271: 7061-7065Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 6Gooley A.A. Williams K.L. Glycobiology. 1994; 4: 413-417Crossref PubMed Scopus (57) Google Scholar). Strikingly, these two methodological strategies revealed deviating results when the patterns of GalNAc addition to MUC1 tandem repeat peptide were analyzed (7Stadie T.R.E. Chai W. Lawson A.M. Byfield P.G.H. Hanisch F.-G. Eur. J. Biochem. 1995; 229: 140-147Crossref PubMed Scopus (64) Google Scholar, 8Müller S. Goletz S. Packer N. Gooley A.A. Lawson A.M. Hanisch F.-G. J. Biol. Chem. 1997; 272: 24780-24793Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). All five putative sites were identified as glycosylation targetsin vivo (8Müller S. Goletz S. Packer N. Gooley A.A. Lawson A.M. Hanisch F.-G. J. Biol. Chem. 1997; 272: 24780-24793Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), whereas only Thr within the VTSA motif and/or Thr and Ser within the GSTA motif was glycosylated in vitroby the enzymes from tumor cells (7Stadie T.R.E. Chai W. Lawson A.M. Byfield P.G.H. Hanisch F.-G. Eur. J. Biochem. 1995; 229: 140-147Crossref PubMed Scopus (64) Google Scholar, 9Nishimori 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) or milk (7Stadie T.R.E. Chai W. Lawson A.M. Byfield P.G.H. Hanisch F.-G. Eur. J. Biochem. 1995; 229: 140-147Crossref PubMed Scopus (64) Google Scholar) or by the recombinant GalNAc-Ts (T1–T3) (1Sorensen T. White T. Wandall H.H. Kristensen A.K. Roepstorff P. Clausen H. J. Biol. Chem. 1995; 270: 24166-24173Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 2Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 3Wandall H.H. Hassan H. Mirgorodskaya E. Kristensen A.K. Roepstorff P. Bennett E.P. Nielsen P.A. Hollingsworth M.A. Burchell J. Taylor-Papadimitriou J. Clausen H. J. Biol. Chem. 1997; 272: 23503-23514Crossref PubMed Scopus (268) Google Scholar). These differences could be explained by substrate specificities of the enzymes involved. On the other hand, the source of ppGalNAc-Ts used in the in vitrostudies and that of the in vivo processed MUC1 were the same. However, distinct enzyme species may have been lost during preparation or may not be active under the conditions used for in vitro glycosylation. It could also be assumed that there is a need for these enzymes to act spatially or temporally in specific subcellular compartments that are not retained in the in vitro system. Finally, a further explanation should be considered that assumes that initial glycosylation of a peptide substrate influences the subsequent glycosylation events at vicinal or distant Ser/Thr positions. There are two observations that would favor this concept: 1) although ppGalNAc-Ts have largely been localized to the cis-Golgi (10Roth 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), there are reports demonstrating a more diffuse distribution of these enzymes throughout the Golgi system (11Röttger S. White T. Wandall H.H. Olivo J.C. Stark A. Bennett E.P. Whitehouse C. Berger E.G. Clausen H. Nilsson T. J. Cell Sci. 1998; 111: 45-60Crossref PubMed Google Scholar); 2) the mucin core peptide can cycle between cis-Golgi and the endoplasmic reticulum (11Röttger S. White T. Wandall H.H. Olivo J.C. Stark A. Bennett E.P. Whitehouse C. Berger E.G. Clausen H. Nilsson T. J. Cell Sci. 1998; 111: 45-60Crossref PubMed Google Scholar) and GalNAc may be added successively to incompletely glycosylated substrates carrying short, core-type glycans. Positive or negative influences on the acceptor qualities of the remaining, still unglycosylated positions could be postulated to be exerted on the mono- or oligosaccharide level and could account for the partial unpredictability of the actual glycosylation sites. According to this concept, the site specificity of initial O-glycosylation would not merely be ruled by the peptide sequence around putative target sites. Evidence for negative effects on vicinal sites induced by mono- or disaccharide substituents has previously been reported for a series of glycopeptide substrates based on the MUC2 repeat peptide (12Brockhausen I. Toki D. Brockhausen J. Peters S. Bielefeldt T. Kleen A. Paulsen H. Meldal M. Hagen F. Tabak L.A. Glycoconj. J. 1996; 13: 849-856Crossref PubMed Scopus (49) Google Scholar). Another in vitro glycosylation study indicated that positive regulatory effects on vicinal sites could also be responsible for the accelerated GalNAc transfer (7Stadie T.R.E. Chai W. Lawson A.M. Byfield P.G.H. Hanisch F.-G. Eur. J. Biochem. 1995; 229: 140-147Crossref PubMed Scopus (64) Google Scholar). We followed this line of considerations by testing a panel of mono- and disaccharide substituted peptides corresponding to one MUC1 tandem repeat and carrying GalNAc or Galβ1–3GalNAc at single or multiple positions. The site-specific activities of three recombinant enzymes (rGalNAc-T1, -T2, and -T3) were assayed and compared with those of the soluble enzymes shed into human milk (milk GalNAc-Ts). The products were separated and quantitated by reversed phase HPLC, identified by MALDI mass spectrometry and the “terminally” glycosylated peptides with a maximum number of incorporated GalNAc were sequenced by Edman degradation to localize the sites of glycosylation. We were able to demonstrate that negative vicinal effects on glycosylation are exerted on the disaccharide level and are site-restricted. On the monosaccharide level, positive effects could be proven to occurin vitro, resulting in greatly accelerated GalNAc transfer to a vicinal position. Finally, also distant effects on initialO-glycosylation were observed that suggest that glycosylation-induced conformational changes of the peptide substrate may influence the accessibility of particular acceptor sites for ppGalNAc-Ts. The glycopeptides listed in Table I were synthesized as described previously (13Karsten U. Diotel C. Klich G. Paulsen H. Goletz S. Müller S. Hanisch F.-G. Cancer Res. 1998; 58: 2541-2549PubMed Google Scholar) and were analyzed by 1H-NMR spectroscopy (400 MHz) (14Mathieux N. Paulsen H. Meldal M. Bock K. J. Chem. Soc. Perkin Trans. 1997; 1: 2359-2368Crossref Scopus (90) Google Scholar) and by MALDI mass spectrometry (15Goletz S. Thiede B. Hanisch F.-G. Schultz M. Peter-Katalinic J. Müller S. Seitz O. Karsten U. Glycobiology. 1997; 7: 881-896Crossref PubMed Scopus (44) Google Scholar). The peptides A1 to A8 correspond to a 21-mer of the MUC1 tandem repeat domain starting with the AHG motif and carrying one to fourO-linked disaccharides Galβ1–3GalNAcα at various positions. Glycopeptides A11 and A13 carrying GalNAc in defined positions of a 20-mer (HGV20) or 21-mer (AHG21) were synthesized similarly (13Karsten U. Diotel C. Klich G. Paulsen H. Goletz S. Müller S. Hanisch F.-G. Cancer Res. 1998; 58: 2541-2549PubMed Google Scholar). Nonglycosylated peptide TAP25, corresponding to one repeat and five overlapping amino acids (starting with the TAP motif), was kindly provided by Dr. Taylor-Papadimitriou (Imperial Cancer Research Fund, London, United Kingdom).Table ISynthetic (glyco)peptides used in this study Open table in a new tab Polypeptide GalNAc-transferases were obtained as follows: secreted, soluble recombinant forms of GalNAc-T1, -T2, and -T3 were expressed in insect cells as described previously (2Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 3Wandall H.H. Hassan H. Mirgorodskaya E. Kristensen A.K. Roepstorff P. Bennett E.P. Nielsen P.A. Hollingsworth M.A. Burchell J. Taylor-Papadimitriou J. Clausen H. J. Biol. Chem. 1997; 272: 23503-23514Crossref PubMed Scopus (268) Google Scholar). Enzymes were partially purified from serum-free culture supernatant of transfected High-FiveTM (Invitrogen) cells as described previously (3Wandall H.H. Hassan H. Mirgorodskaya E. Kristensen A.K. Roepstorff P. Bennett E.P. Nielsen P.A. Hollingsworth M.A. Burchell J. Taylor-Papadimitriou J. Clausen H. J. Biol. Chem. 1997; 272: 23503-23514Crossref PubMed Scopus (268) Google Scholar). The ppGalNAc-transferases from human milk were enriched by ultracentrifugation as described previously and next mixed with an equal volume of 0.9% NaCl, 0.4% Triton X-100, containing a series of protease inhibitors (7Stadie T.R.E. Chai W. Lawson A.M. Byfield P.G.H. Hanisch F.-G. Eur. J. Biochem. 1995; 229: 140-147Crossref PubMed Scopus (64) Google Scholar). The peptide or glycopeptide substrates (22 nmol, 430 μm) were dried from solutions in water in a speed vac and solubilized in 20 μl of imidazol-HCl (0.1 m), pH 7.2, containing MnCl2 (10 mm). Addition of the cosubstrate UDP-GalNAc (300 μm) was followed by the respective enzyme preparations (25 μl of rGalNAc-T1, 2.04 milliunits/ml; rGalNAc-T2, 4.04 milliunits/ml; rGalNAc-T3, 4.65 milliunits/ml; milk GalNAc-Ts, 1.85 milliunits/ml; specific activity measured for TAP25 as a substrate) to yield a total volume of 50 μl. The reaction mixtures were incubated for varying time periods at 37 °C (maximum, 7 days) by adding fresh aliquots of cosubstrate and enzyme(s) at 24-h intervals. The samples were diluted with incubation buffer to give a total volume of 210 μl prior to ultrafiltration through ultrafree MC membranes (Millipore, Eschborn, Germany) with a nominal cut-off of 10 kDa. Kinetic parameters were calculated by double reciprocal Lineweaver-Burk transformations at four substrate concentrations using assay conditions described by Brockhausen et al. (12Brockhausen I. Toki D. Brockhausen J. Peters S. Bielefeldt T. Kleen A. Paulsen H. Meldal M. Hagen F. Tabak L.A. Glycoconj. J. 1996; 13: 849-856Crossref PubMed Scopus (49) Google Scholar). Aliquots (50–100 μl) of the reaction mixtures were injected onto a PLRP-S column (250 × 4.6 mm, Polymer Laboratories, Shropshire, United Kingdom) or a narrow-bore ODS Ultrasphere column (150 × 2 mm, Beckman Instruments, Munich, Germany) and chromatographed on an HPLC system (System Gold Beckman Instruments, Munich, Germany) by gradient elution in a mixture of acetonitrile in water (0.1% trifluoroacetic acid) from 2% (solvent A) to 80% (solvent B) (duration, 80 min). Alternatively, gradient elution was performed starting from 0% solvent B to 6% solvent B (duration, 3 min), followed by a gradient from 6% solvent B to 16% solvent B (duration, 30 min). The glycopeptides were run at 1 ml/min and detected photometrically at 214 nm. The HPLC-purified glycopeptides were dried in a speed vac and dissolved in a mixture of water and methanol (1:1, v/v) to yield concentration of approximately 1 mg/ml. 2,6-Dihydroxyacetophenon (concentration, 10 g/liter in 50% aqueous methanol) as a MALDI matrix was co-crystallized with the analyte in a dried droplet preparation. MALDI-time-of-flight experiments were performed on a VISION 2000 prototype mass spectrometer with a 2.3 m flight tube. N2 laser at 337 nm was used. Measurements were carried out in linear mode using appropriate delay time and potential to focus the ions of interest. Glycopeptides were sequenced on a Hewlett-Packard G1000A protein sequencer with 3.1 (solid) chemistry that uses methanol rather than ethyl acetate to transfer the ATZ-amino acid (16Zachara N.E. Gooley A.A. Corfield T. Mucin Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ1998Google Scholar). The Sequelon AATM membranes were from the Perseptive Biosystems division of Perkin-Elmer. Quantitation of theO-glycosidic serine/threonine substitution was calculated using a glycopeptide derived from Dictyostelium discoideumrecombinant expressed glycoprotein PsA, which contains a 100% substituted GlcNAcα-Thr in position 4. The corrected yield for this cycle (repetitive yield for the glycopeptide ITATPAPT) was 95%. The retention time of GlcNAcα-Thr was almost identical to the corresponding GalNAc derivative on reversed phase HPLC. The glycopeptide substrates A1–A8, A11, and A13 (TableI) were incubated with the single or combined recombinant enzymes (or with the GalNAc-Ts from milk) for at least 72 h or up to 7 days in order to identify the sites and to measure the maximal number of GalNAc residues added to the Thr/Ser positions of the MUC1 repeat peptide. In all cases, glycopeptide products with the maximal number of glycosylated sites were formed within 3 days and called terminally glycosylated products. These glycopeptides were separated from substrate or intermediate products by reversed phase HPLC (Fig. 1) and were analyzed by MALDI mass spectrometry (TableII). Between one and three GalNAc residues were transferred depending on the glycopeptide substrate (Table II). This was calculated on the basis of pseudomolecular ions MH+ and the mass increment of HexNAc (203.2 mass units). Edman sequencing showed that only positions Thr-5, Ser-16, and Thr-17 could be glycosylated in vitro, and it was independent from the glycopeptide substrate (Tables II andIII). Accordingly, the “isolated” Thr within PDTR and Ser within VTSA do not represent target sites in vitro for the enzymes tested in the present study as was previously shown for the unglycosylated control peptide TAP25 (7Stadie T.R.E. Chai W. Lawson A.M. Byfield P.G.H. Hanisch F.-G. Eur. J. Biochem. 1995; 229: 140-147Crossref PubMed Scopus (64) Google Scholar). The only exception from this general pattern was A11, which after glycosylation over a period of 7 days showed the incorporation of trace amounts of GalNAc into the positions Ser-6/Thr-10 (pseudomolecular ion measured at m/z 2741.5). The terminally glycosylated final products of glycopeptides A6, A7, and A8 carrying more than one disaccharide did not reveal detectable GalNAc incorporation (Table II). The qualitative data of the site specificity of terminal glycosylation agreed for the enzyme preparation from human milk and the combined recombinant GalNAc-Ts (T1–T3).Table IINumber of GalNAc residues introduced into glycopeptide substrates after 72 h of reaction timePeptideNumber of GalNAc residues introducedExpectedaMaximum numbers of GalNAc residues expected to be incorporated into MUC1 repeat peptide on the basis of previous in vitro glycosylation studies with TAP25 (7).HPLCMALDI mass spectrometryEdmanA12111A23333A32222A43333A5bTwo products were identified due to a heterogeneity of the peptide substrate A5 (Table 1).21 (2Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar)1 (2Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar)1 (2Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar)A610NDNDA700NDNDA800NDNDA11111 (2Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar)cThe intensity of the pseudomolecular ion M + H corresponding to A11-GalNAc2 at m/z 2741.5 was below 4% of the major product A11-GalNAc1 after 168 h of reaction time.1A1333NDNDTAP253333The glycopeptide substrates were incubated for 72 h with a mixture of the recombinant enzymes GalNAc-T1 to -T3 (HPLC and Edman) or with the GalNAc-T(s) from human skim milk (HPLC and MALDI) separated by reversed phase HPLC and analysed by MALDI mass spectrometry and/or Edman sequencing for the number and sites of GalNAc addition.ND, not determined.a Maximum numbers of GalNAc residues expected to be incorporated into MUC1 repeat peptide on the basis of previous in vitro glycosylation studies with TAP25 (7Stadie T.R.E. Chai W. Lawson A.M. Byfield P.G.H. Hanisch F.-G. Eur. J. Biochem. 1995; 229: 140-147Crossref PubMed Scopus (64) Google Scholar).b Two products were identified due to a heterogeneity of the peptide substrate A5 (Table 1).c The intensity of the pseudomolecular ion M + H corresponding to A11-GalNAc2 at m/z 2741.5 was below 4% of the major product A11-GalNAc1 after 168 h of reaction time. Open table in a new tab Table IIISites and rates of GalNAc addition to glycosylated MUC1 repeat peptidesView Large Image Figure ViewerDownload (PPT) Open table in a new tab The glycopeptide substrates were incubated for 72 h with a mixture of the recombinant enzymes GalNAc-T1 to -T3 (HPLC and Edman) or with the GalNAc-T(s) from human skim milk (HPLC and MALDI) separated by reversed phase HPLC and analysed by MALDI mass spectrometry and/or Edman sequencing for the number and sites of GalNAc addition. ND, not determined. The glycopeptides (A1–A8, A11, and A13) and the nonglycosylated control peptide TAP25 were analyzed for the sites of glycosylation after in vitro transfer of the maximal number of GalNAc residues (Table III). On the basis of these qualitative data, several site-specific effects of substrate glycosylation on vicinal, proximal, and distant target sites were revealed. The terminally glycosylated product of glycopeptide A1 did not carry GalNAc at Ser-16 (Table III). By contrast, the nonglycosylated control peptide TAP25 and the glycopeptide substrates, including glycopeptide A4, were glycosylated at this position. This finding indicates that a disaccharide substituted at Thr-5 can negatively affect a distant target site with respect to its acceptor qualities. The long range effect, which is exerted over a peptide stretch of 11 amino acids, is site-specific, because a disaccharide substitution at the adjacent Ser-6 (glycopeptide A4) had no influence on Ser-16 glycosylation (Table III). Exertion of the distance effect is also strikingly dependent on the glycan substituted at Thr-5,i.e. glycopeptide A11 with GalNAc at Thr-5 exhibited Ser-16 glycosylation. Results obtained for another group of glycopeptide substrates demonstrated that besides long range effects disaccharide substituents can also exert negative effects on vicinal target sites. In glycopeptide A5 (Table I), the disaccharide at Ser-16 is located adjacent to the acceptor position Thr-17 and may exert a negative influence, possibly mediated by steric hindrance, on the binding of ppGalNAc-transferases and hence prevents glycosylation of Thr-17 (Table III). A sequence variant of A5, glycopeptide A5b (TableI), with an additional Thr in position 18 was found to be glycosylated at this site (Table III). It can, therefore, be argued that the negative influence of the disaccharide in Ser-16 is solely exerted vicinal and not proximal. The relatively low amount of this minor glycopeptide (below 20%) renders unlikely substrate competition as an explanation for nonglycosylation of A5, the major glycopeptide in the substrate mixture. Interestingly, a similar negative effect with complete inhibition of vicinal glycosylation was not observed for GalNAc addition to Ser-16 in the substrates A3 (disaccharide at Thr-17) and A4 (disaccharide at Ser-6) (Table III). It may be suggested, accordingly, that disaccharides placed on the first position of an ST diad (here, Ser-16) could prevent the glycosylation of the second position, whereas disaccharide on the second position of ST or TS diads (here, Thr-17 or Ser-6) may not necessarily inhibit addition to the first. Ser-16 glycosylation was not affected by disaccharide substitution at the proximal position −6 in glycopeptide substrate A2 (Table III). The same holds true for glycopeptide A13 exhibiting monosaccharide substitution of Thr-10 (Table III). Although the pattern of site-specific GalNAc addition on glycopeptides A2 and A13 agrees with that on the nonglycosylated control peptide TAP25, the rate of Ser-16 glycosylation in A2 and A13 was found to be affected negatively by glycans at Thr-10 (see kinetic analyses and Fig.2). Remarkably, substrate peptides substituted with more than one disaccharide (A6, A7, and A8) could not serve as acceptors for rGalNAc-Ts or milk GalNAc-Ts at any of the remaining possible positions (Table III). With respect to positions Ser-6 and Thr-10, which are not in vitro target sites for rGalNAc-T1 to -T3 or milk GalNAc-Ts, this finding excludes the possible existence of positive regulatory mechanisms mediated by core1-disaccharides. On the other hand, the negative effect on Ser-16 glycosylation in A6 induced by disaccharide substitution at Thr-5 and Thr-17 is solely exerted on the disaccharide level, because the structural analogue of the glycopeptide, the monosaccharide substituted glycopeptide A11, was a substrate of ppGalNAc-Ts (Table III). Most likely, the prevention of Ser-16 glycosylation in A6 is caused by disaccharide substitution of Thr-5 and is exerted in the same way as the negative long range effect on Ser-16 glycosylation in glycopeptide A1. It should be recalled at this point that vicinal substitution with disaccharide at Thr-17 did not suppress Ser-16 glycosylation in A3 (Table III). The rates of GalNAc incorporation into the three actual in vitro glycosylation sites of MUC1 repeat peptide (Thr-5, Ser-16, and Thr-17) were different for each position and ppGalNAc-T used. Kinetic studies were performed by quantitative HPLC/Edman sequencing and by radiometric measurement of [14C]GalNAc incorporation to determine the apparent kinetic constants. This approach allowed the identification and quantitation of each intermediate or final glycopeptide product. It was also possible to assign for some of the glycopeptide substrates apparent K m and V max values to distinct glycosylated sites. The ppGalNAc-Ts from milk glycosylated Thr-5 at higher rates than Thr-17 or Ser-16. This is evidenced from previous HPLC/Edman analysis of TAP25 products (7Stadie T.R.E. Chai W. Lawson A.M. Byfield P.G.H. Hanisch F.-G. Eur. J. Biochem. 1995; 229: 140-147Crossref PubMed Scopus (64) Google Scholar), but also from the apparent kinetic constants (Table III) obtained for glycopeptide substrates A1 (glycosylation of Thr-17; see also Fig.2 C), A3 (mainly glycosylation of Thr-5; see also Fig. 2,A and C), and A5 (glycosylation of Thr-5). The lowest relative reaction rates were measured for the Ser-16 position, which made glycosylation of this site the rate-limiting step in the glycosylation of peptides A2, A3, A4, A13, and TAP25 (Fig. 2,A and B). Accordingly, no Ser-16 glycosylation was generally found in HPLC fractions corresponding to intermediate products. The same relative rates of GalNAc incorporation into the three positions were found when using the recombinant enzymes in a mixture. The single rGalNAc-T species exhibited distinct preferences for the in vitro glycosylation sites within the VTSA and GSTA motifs, respectively (Tables IV andV). As demonstrated previously for rGalNAc-T1 and -T3 (1Sorensen T. White T. Wandall H.H. Kristensen A.K. Roepstorff P. Clausen H. J. Biol. Chem. 1995; 270: 24166-24173Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), the best substrates for these enzymes are characterized by a nonglycosylated Thr-5 (compare A3 to A1 in TableIV), whereas rGalNAc-T2 prefers the nonglycosylated GSTA motif (compare A1 to A3 and A5 in Table IV). rGalNAc-T2 is the enzyme species that adds GalNAc most efficiently to Ser-16 (glycosylation of A11). This is also evident from HPLC analysis of the final product of A3 glycosylation (Table V). Whereas the formation of A3(Thr-5, Ser-16)-GalNAc2 catalyzed by the rGalNAc-T1 and rGalNAc-T3 did not exceed 7% of the total glycopeptides (substrate and products) after reaction times of 72 h, the same product represented 28% of total glycopeptide using rGalNAc-T2 (Table V).Table IVGlycosylation-induced effects on the activities of rGalNAc-T1 to -T3 as revealed by the apparent kinetic constants for glycopeptide substratesSubstrate (preferential target site)Enzyme GalNAc-Tpmol h−1K MV maxmmnmol h−1A1 (Thr-17)T164NDNDT216961.44.00T3109NDNDA3 (Thr-5)T14320.40.63T22032.50.63T313170.31.82A11 (Ser-16)T1<50NDNDT21280.50.19T3<50NDNDA2 (Thr-17)T216820.73.03A4 (Thr-17)T26010.20.85A5 (Thr-5)T23092.01.00The reaction conditions were identical to those given in Table 3 except for the replacement of milk enzymes by the individual rGalNAc-Ts. In case of substrates A1, A5, and A11, where only one GalNAc is added to a defined peptide position, the apparent kinetic constants correspond to the individual site constants: Thr-17 (A1), Thr-5 (A5), and Ser-16 (A11). Considering the neglegible rates of Ser-16 glycosylation, an estimation of the individual site constants for GalNAc addition to Thr-5 (A3) is possible. Because the transferase rGalNAc-T2 glycosylates preferentially sites within the GSTA motif and Ser-16 glycosylation occurs at very low rates compared to Thr-17 glycosylation, the apparent constants for A2 and A4 primarily reflect glycosylation of Thr-17. Errors of rate determination were estimated to be in the range of 5%. ND, not determined. Open table in a new tab Table VExtent of product formation by rGalNAc-T1 to -T" @default.
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