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• W2092560671 abstract "Since there is no consensus sequence directing the initial GalNAc incorporation into mucin peptides,O-glycosylation sites are not reliably predictable. We have developed a mass spectrometric sequencing strategy that allows the identification of in vivo O-glycosylation sites on mucin-derived glycopeptides. Lactation-associated MUC1 was isolated from human milk and partially deglycosylated by trifluoromethanesulfonic acid to the level of core GalNAc residues. The product was fragmented by the Arg-C-specific endopeptidase clostripain to yield tandem repeat icosapeptides starting with the PAP motif. PAP20 glycopeptides were subjected to sequencing by post-source decay matrix-assisted laser desorption ionization mass spectrometry or by solid phase Edman degradation to localize the glycosylation sites. The masses of C- or N-terminal fragments registered for the mono- to pentasubstituted PAP20 indicated that GalNAc was linked to the peptide at Ser5,Thr6 (GSTA) and Thr14(VTSA) but contrary to previous in vitro glycosylation studies also at Thr19 and Ser15 located within the PDTR or VTSA motifs, respectively. Quantitative data from solid phase Edman sequencing revealed no preferential glycosylation of the threonines. These discrepancies between in vivo andin vitro glycosylation patterns may be explained by assuming that O-glycosylation of adjacent peptide positions is a dynamically regulated process that depends on changes of the substrate qualities induced by glycosylation at vicinal sites. Since there is no consensus sequence directing the initial GalNAc incorporation into mucin peptides,O-glycosylation sites are not reliably predictable. We have developed a mass spectrometric sequencing strategy that allows the identification of in vivo O-glycosylation sites on mucin-derived glycopeptides. Lactation-associated MUC1 was isolated from human milk and partially deglycosylated by trifluoromethanesulfonic acid to the level of core GalNAc residues. The product was fragmented by the Arg-C-specific endopeptidase clostripain to yield tandem repeat icosapeptides starting with the PAP motif. PAP20 glycopeptides were subjected to sequencing by post-source decay matrix-assisted laser desorption ionization mass spectrometry or by solid phase Edman degradation to localize the glycosylation sites. The masses of C- or N-terminal fragments registered for the mono- to pentasubstituted PAP20 indicated that GalNAc was linked to the peptide at Ser5,Thr6 (GSTA) and Thr14(VTSA) but contrary to previous in vitro glycosylation studies also at Thr19 and Ser15 located within the PDTR or VTSA motifs, respectively. Quantitative data from solid phase Edman sequencing revealed no preferential glycosylation of the threonines. These discrepancies between in vivo andin vitro glycosylation patterns may be explained by assuming that O-glycosylation of adjacent peptide positions is a dynamically regulated process that depends on changes of the substrate qualities induced by glycosylation at vicinal sites. Post-translational modification of proteins by glycosylation has extensively been studied in the case of N-linked glycans, and accordingly, rules for the substitution of asparagine residues by dolichol phosphate-linked glycans are well established (1Bause E. Biochem. J. 1983; 209: 331-336Crossref PubMed Scopus (520) Google Scholar). WhileN-glycosylation is directed by the consensus peptide motif Asn-X-(Ser/Thr), no strict sequence dependence is known for the initiation of O-glycosylation. Currently, there are several approaches to the identification of O-glycosylation sites: studies on in vitro O-glycosylation of synthetic peptides (2O'Connell B.C. Tabak L.A. Ramusubbu N. Biochem. Biophys. Res. Commun. 1991; 180: 1024-1030Crossref PubMed Scopus (64) Google Scholar, 3Wang Y. Abernethy J.L. Eckhardt A.E. Hill R.L. J. Biol. Chem. 1992; 267: 12709-12716Abstract Full Text PDF PubMed Google Scholar, 4Wang Y. Agrwal N. Eckhardt A.E. Stevens R.D. Hill R.L. J. Biol. Chem. 1993; 268: 22979-22983Abstract Full Text PDF PubMed Google Scholar) or studies on in vivo processed mucin-type glycoproteins (5Pisano A. Redmond J.W. Williams K.L. Gooley A.A. Glycobiology. 1993; 3: 429-435Crossref PubMed Scopus (95) Google Scholar, 6Pisano A. Packer N.H. Redmond J.W. Williams K.L. Gooley A.A. Glycobiology. 1995; 4: 837-844Crossref Scopus (84) Google Scholar, 7Nehrke K. Hagen F.K. Tabak L.A. J. Biol. Chem. 1996; 271: 7061-7065Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The relative merits of both approaches have been discussed (8Gooley A.A. Williams K.L. Glycobiology. 1994; 4: 413-417Crossref PubMed Scopus (57) Google Scholar). The results obtained so far agree with the statement that there are no clear-cut motifs for the addition of GalNAc at Ser or Thr residues; however, the non-random patterns ofO-glycosylation suggest influences of flanking sequences (2O'Connell B.C. Tabak L.A. Ramusubbu N. Biochem. Biophys. Res. Commun. 1991; 180: 1024-1030Crossref PubMed Scopus (64) Google Scholar). Wang et al. (3Wang Y. Abernethy J.L. Eckhardt A.E. Hill R.L. J. Biol. Chem. 1992; 267: 12709-12716Abstract Full Text PDF PubMed Google Scholar) proposed different motifs for threonine and serine glycosylation, and the studies of O‘Connell et al. (2O'Connell B.C. Tabak L.A. Ramusubbu N. Biochem. Biophys. Res. Commun. 1991; 180: 1024-1030Crossref PubMed Scopus (64) Google Scholar) revealed critical positions in the vicinity of putativeO-glycosylation sites. The obviously less specific GalNAc addition to Ser/Thr residues compared with N-glycosylation is reflected also in the existence of at least four distinct species of UDP-GalNAc/peptide N-acetylgalactosaminyltransferase(s) (GalNAc-transferase) for which different substrate specificities have been established (9Clausen H. Bennett E.P. Glycobiology. 1996; 6: 635-646Crossref PubMed Scopus (228) Google Scholar). Accordingly, the differentiation and organ localization of a cell should determine its characteristic equipment of active GalNAc-transferases (T1–T4) and, hence, the site-specificO-glycosylation of proteins processed within this cell. Site specificity of O-glycosylation strongly affects the antigenicity of a protein as has been revealed for the highly immunogenic peptide motif PDTR within MUC1 tandem repeats (10Spencer D.I.R. Price M.R. Tendler S.J.B. DeMatteis C.I. Stadie T. Hanisch F.-G. Cancer Lett. 1996; 100: 11-15Crossref PubMed Scopus (26) Google Scholar). Most antibodies reactive to the PDTR sequence are more or less influenced by GalNAc incorporation into threonine residues of the flanking motifs VTSA and GSTA by showing a significant enhancement or decrease of their binding activities (10Spencer D.I.R. Price M.R. Tendler S.J.B. DeMatteis C.I. Stadie T. Hanisch F.-G. Cancer Lett. 1996; 100: 11-15Crossref PubMed Scopus (26) Google Scholar, 11Burchell J. Taylor-Papadimitriou J. Epithelial Cell Biol. 1993; 2: 155-162PubMed Google Scholar). In vitro studies have established that preferentially Thr residues within VTSA and GSTA are glycosylated (12Nishimori 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, 13Nishimori I. Perini F. Mountjoy K. Sanderson S.D. Johnson N. Cernay R.L. Gross M.L. Fontenot J.D. Hollingsworth M.A. Cancer Res. 1994; 54: 3738-3744PubMed Google Scholar, 14Stadie 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, 15Goletz, S., Thiede, B., Hanisch, F.-G., Peter-Katalinic, J., Schultz, M., Müller, S., Seitz, O., and Karsten, U. (1997)Glycobiology, in pressGoogle Scholar), whereas only Ser within GSTA serves as a substrate to a minor degree (14Stadie 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, 15Goletz, S., Thiede, B., Hanisch, F.-G., Peter-Katalinic, J., Schultz, M., Müller, S., Seitz, O., and Karsten, U. (1997)Glycobiology, in pressGoogle Scholar). To establish the actual sites of O-glycosylation on thein vivo processed mucin, we have applied a mass spectrometric sequencing strategy (15Goletz, S., Thiede, B., Hanisch, F.-G., Peter-Katalinic, J., Schultz, M., Müller, S., Seitz, O., and Karsten, U. (1997)Glycobiology, in pressGoogle Scholar) using the MUC1 glycoform in human milk as a model. A fragmentation of the highly glycosylated mucin by specific endopeptidases becomes possible only after a limited deglycosylation with trifluoromethanesulfonic acid (TFMSA). 1The abbreviations used are: TFMSA, trifluoromethanesulfonic acid; HexNAc, N-acetylhexosamine; HMFGM, human milk fat globule membrane; PSD-MALDI-(TOF)-MS, post-source decay matrix-assisted laser desorption ionization mass spectrometry; RP-HPLC, reversed phase-high performance liquid chromatography; PTH, phenylthiohydantoin; PBS, phosphate-buffered saline; mAb, monoclonal antibody; BSA, bovine serum albumin; ACN, acetonitrile. This treatment is reported to result in the removal of complex carbohydrates, but to leave during short reaction times a considerable portion of the core GalNAc residues intact (16Sojar H.T. Bahl O.M.P. Arch. Biochem. Biophys. 1987; 259: 52-57Crossref PubMed Scopus (79) Google Scholar, 17Gerken T.A. Gupta R. Jentoft N. Biochemistry. 1992; 31: 639-648Crossref PubMed Scopus (69) Google Scholar). This partially deglycosylated mucin (GalNAc-MUC1) can be effectively fragmented by clostripain cleaving at Arg-C (18Aitken A. Geisow M.J. Findley J.B.C. Holmes C. Yarwood A. Findley J.B.C. Geisolo M.J. Protein Sequencing: A Practical Approach. IRL Press at Oxford University Press, Oxford1989: 43-68Google Scholar). Clostripain fragmentation of the tandem repeat peptide results in a series of differentially glycosylated icosapeptides PAP20 as shown in Sequence 1:PDTR‖CPAPGSTAPPAHGVTSAPDTRPAP20‖CPAPGSTAPPASEQUENCE1As demonstrated previously for a series of in vitroglycosylated peptides corresponding to the MUC1 tandem repeats (TAP25) (15Goletz, S., Thiede, B., Hanisch, F.-G., Peter-Katalinic, J., Schultz, M., Müller, S., Seitz, O., and Karsten, U. (1997)Glycobiology, in pressGoogle Scholar), the sequencing of PAP20 peptides was possible by mass spectrometric analysis of post-source decay fragments in a MALDI-TOF instrument. Independent evidence for the localization ofO-glycosylation sites and, moreover, quantitative estimations of the degree of glycosylation at each site were obtained from solid phase Edman sequencing. The results obtained suggest thatin vitro data should be carefully reevaluated by the analysis of in vivo processed glycoprotein and that a prediction of O-glycosylation sites cannot be based solely on peptide sequence. Human milk was obtained from healthy women during days 2–5 of lactation and immediately frozen at −20 °C. Milk samples from multiple donors were thawed at 4 °C and combined, and milk fat was separated from skim milk by centrifugation at 3,000 × g (4 °C) for 1 h (Fig. 1). Secretory MUC1 from skim milk was isolated by extraction with hot phenol and subsequent gel filtration on Sephacryl S300 as described previously (19Hanisch F.-G. Uhlenbruck G. Peter-Katalinic J. Egge H. Dabrowski J. Dabrowski U. J. Biol. Chem. 1989; 264: 872-883Abstract Full Text PDF PubMed Google Scholar) (Fig. 1). This preparation is designated MUC1(skim milk). Milk fat was used for the preparation of human milk fat globule membranes (HMFGM) according to the procedure of Shimidzu and Yamauchi (20Shimidzu M. Yamauchi K. J. Biochem. ( Tokyo ). 1982; 91: 515-524Crossref PubMed Scopus (120) Google Scholar). For the isolation of membrane-associated MUC1, the HMFGM were homogenized and sonicated in 0.45 mm sodium phosphate, 125 mm sodium chloride, pH 7.2 (PBS), containing 4 m urea, before they were extracted with an equal volume of 90% phenol for 30 min at 65 °C. The extract was allowed to cool to room temperature; 0.5 volumes of chloroform were added, and the phases were separated by centrifugation at 30,000 × g (10 °C) for 30 min. The aqueous phase was dialyzed against several changes of demineralized water, concentrated by ultrafiltration, and applied to a column (2.5 × 100 cm) of Sephacryl S300 equilibrated in PBS. Fractions were analyzed by colorimetric microassays for hexoses (21Monsigny M. Petit C. Roche A.-C. Anal. Biochem. 1988; 175: 525-530Crossref PubMed Scopus (320) Google Scholar) and sialic acid (22Bhavanandan V.P. Sheykhnazari M. Anal. Biochem. 1993; 213: 438-440Crossref PubMed Scopus (28) Google Scholar) and in an enzyme-linked immunosorbent assay with monoclonal antibody BW835. The combined void fractions, containing the mucin, were extensively dialyzed against demineralized water, concentrated by ultrafiltration, and freeze-dried. This preparation is designated MUC1(HMFGM). SDS-polyacrylamide gel electrophoretic separations of mucin samples were carried out in the Mini Protean II gel electrophoresis apparatus (Bio-Rad, München, Germany) according to the method of Laemmli. Gradient gels (3–15%), overlaid with a 3% stacking gel, were run at 200-V constant voltage for 50 min and stained with Coomassie Brilliant Blue R-250. In addition, gels were blotted onto polyvinylidene difluoride membranes using a semi-dry blotting apparatus and the buffer system of Towbin et al. (23Towbin H. Staehelin T. Gordon T. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44924) Google Scholar). Remaining protein binding sites were blocked with 3% BSA in PBS (1 h, room temperature), and mucin was detected by an overnight incubation with mAb BW835 (Behringwerke, Marburg, Germany, 2 μg/ml in 0.5% BSA in PBS) followed by 1-h incubations with biotinylated goat anti-mouse immunoglobulin serum (E 413, Dako, 1:5,000 in 0.5% BSA in PBS) and avidin-conjugated alkaline phosphatase (Boehringer Mannheim, 1:2,500 in 0.5% BSA in PBS) using nitro blue tetrazolium/5-bromo-4-chloro-3-indoyl phosphate as substrate. Alternatively, blotted glycoproteins were detected after oxidation with sodium periodate according to the method of O'Shannessy et al. (24O‘Shannassy D.J. Voorstad P.L. Quarles R.H. Anal. Biochem. 1987; 163: 204-209Crossref PubMed Scopus (86) Google Scholar). MUC1(skim milk) or MUC1(HMFGM) were deglycosylated by treatment with anhydrous TFMSA (Sigma, Unterhaching, Germany) in the absence (16Sojar H.T. Bahl O.M.P. Arch. Biochem. Biophys. 1987; 259: 52-57Crossref PubMed Scopus (79) Google Scholar) or presence of anisole (17Gerken T.A. Gupta R. Jentoft N. Biochemistry. 1992; 31: 639-648Crossref PubMed Scopus (69) Google Scholar) (Fig. 2). Lyophilized mucin was resuspended in 0.1 m hydrochloric acid at 4 mg/ml and heated at 70 °C for 60 min to hydrolyze sialic acids. Hydrochloric acid was removed by dialysis against demineralized water. Following the method of Sojar and Bahl aliquots corresponding to 0.1–8 mg of native mucin were lyophilized in Teflon-lined screw-cap vials, flushed with argon, and precooled on ice, before 0.2–0.4 ml of TFMSA (−20 °C) were added for each milligram of mucin. The tubes were flushed with argon once more and then immediately closed and incubated on ice for the appropriate time. The reaction was stopped by cooling the tubes on dry ice in methanol and slowly adding 3 volumes of ice-cold 60% pyridine/water. Finally, the deglycosylated samples were dialyzed against several changes of buffer, appropriate for the following manipulations, and stored frozen at −20 °C. The method of Gerkenet al. (17Gerken T.A. Gupta R. Jentoft N. Biochemistry. 1992; 31: 639-648Crossref PubMed Scopus (69) Google Scholar) was followed as described. The time course of N-acetylgalactosamine (GalNAc) exposure during removal of complex glycans by TFMSA treatment was followed by kinetic analysis (Fig. 2). 0.1 mg of MUC1(HMFGM) was desialylated and treated with TFMSA for 0, 15, 30, and 60 min in the absence of anisole or for 0, 1, 2, and 6 h in the presence of anisole. After neutralization the samples were diluted with 50 mmNH4HCO3, pH 8.0, and chromatographed on Superdex 75pg (1 × 20 cm) using the same buffer as eluant and a flow rate of 0.4 ml/min. The samples were analyzed by quantitative monosaccharide composition analysis (see below) or by Helix pomatia agglutinin binding to follow the time course of the sugar removal and the immunochemical accessibility of exposed GalNAc. Samples were diluted 1:16 in 0.1 m sodium carbonate, pH 9.6, and 50 μl of a serial 1:2 dilution in the same buffer were bound to 96-well polystyrene plates by drying in a desiccator. Remaining protein binding sites were blocked with 3% BSA in PBS at 37 °C for 1 h, and the plate was incubated with 50 μl of a 2 μg/ml solution of biotinylated H. pomatia agglutinin (Sigma) in 0.5% BSA in PBS at 37 °C for 1 h. Bound lectin was detected with avidin-alkaline phosphatase (Boehringer Mannheim, Germany, 1:2,500 in 0.5% BSA in PBS) using p-nitrophenyl phosphate as substrate. Amino sugars in the deglycosylated samples were analyzed as peracetylated alditols using gas chromatography/electron impact mass spectrometry (Fison MD800) in the scan mode. 100 μl (20%) of the dialyzed samples and 2 μg ofmeso-erythritol were lyophilized in micro-reaction vials and hydrolyzed with 4 n hydrochloric acid for 18 h at 100 °C. Subsequent reduction and peracetylation of monosaccharides was performed as described elsewhere (25Merkle R.K. Poppe I. Methods Enzymol. 1994; 230: 1-15Crossref PubMed Scopus (206) Google Scholar). To analyze total monosaccharides in the deglycosylated and gel-filtrated samples per-O-trimethylsilyl,1-O-methyl esters were prepared according to the method of Chaplin and Kennedy (26Chaplin M.F. Kennedy J.F. Carbohydrate Analysis: A Practical Approach. IRL Press at Oxford University Press, Oxford1986Google Scholar). Gas chromatography of peracetylated alditols was performed on a DB5ms capillary (J & W Scientific, 0.25 mm × 15 m). Splitless sample injection (200 °C) was used. The capillary temperature was held constant at 50 °C for 1 min, raised to 120 °C with 30 °C/min, and then to 260 °C with 7.5 °C/min. The same capillary was used for the analysis of per-O-trimethylsilyl,1-O-methyl esters. The temperature was kept at 100 °C for 1 min and then raised to 280 °C with 7.5 °C/min. On-line mass spectrometry was performed with electron impact ionization at 70 eV in the positive mode. Mass spectra were recorded in the range from 50 to 500 mass units. A synthetic glycopeptide GPT-(GlcNAcβ1–6GalNAcα)-TTPITT (kindly provided by Prof. H. Paulsen, Institute of Organic Chemistry, University of Hamburg) or partially deglycosylated MUC1 were treated with 50 mm NaOH, 1 m NaBH4 for 16 h at 50 °C, and after destruction of the excess borohydride with dilute acetic acid the reductively β-eliminated alditols were desalted on Dowex 50WX8, followed by removal of borate as its methyl ester, extensive drying and permethylation as described (19Hanisch F.-G. Uhlenbruck G. Peter-Katalinic J. Egge H. Dabrowski J. Dabrowski U. J. Biol. Chem. 1989; 264: 872-883Abstract Full Text PDF PubMed Google Scholar). Gas-chromatography/mass spectrometry of methylated dihexosamine alditols was performed on an MD800 (Fisons). The samples were solubilized in chloroform and injected onto a DB5 capillary column (15 m), which was heated from 100 to 300 °C at 10 °C/min followed by isothermic elution at 300 °C for 10 min. Mass spectra were recorded in cyclic scans from m/z 100 to 800. Three aliquots corresponding to 8 mg of native mucin were desialylated and treated with TFMSA according to Sojar and Bahl (16Sojar H.T. Bahl O.M.P. Arch. Biochem. Biophys. 1987; 259: 52-57Crossref PubMed Scopus (79) Google Scholar) for 20 min (P1), 30 min (P3), or 60 min (P2) (Fig. 1). The neutralized samples were dialyzed into 25 mm sodium phosphate, pH 7.5; 0.2 mm calcium acetate and concentrated by ultrafiltration. 0.5 ml (50%) of the concentrated samples (8 mg/ml) were supplemented with 2.5 mm dithiothreitol, and proteolytic digestion was performed by adding 120 μg of clostripain (1 mg/ml in 2.5 mm dithiothreitol, 2 mm calcium acetate, activated at room temperature for 3 h) and incubating at 37 °C for 24 h. An aliquot of the digest was chromatographed on Superdex 75pg (1 × 20 cm) in 50 mm sodium phosphate, 500 mm NaCl, pH 7.0. Elution of mucin/glycopeptides was registered at 214 nm, and aliquots of the fractionated eluate were measured for MUC1 peptide epitopes (see below). The column had been calibrated with a mixture of synthetic MUC1 tandem repeat oligomers (kindly provided by Dr. J. Hilgers, Free University Hospital, Amsterdam). Digested partially deglycosylated mucin samples were cleared by centrifugation for 5 min at 15,000 × g and separated by reversed phase-HPLC on a 250 × 4.6 mm PLRP-S column (Polymer Laboratories) (Fig. 1 and Fig.3). The samples were injected as 5 × 100-μl aliquots, and the column was eluted at a flow rate of 1 ml/min using a linear gradient from 2% (v/v) acetonitrile (ACN) in 0.1%(v/v) trifluoroacetic acid (solvent A) to 80% (v/v) ACN in 0.09% (v/v) (solvent B) during 80 min. Peaks were detected at 214 nm, and 1-ml fractions were collected. 10 μl of each fraction were used for immunochemical detection with a mixture of the MUC1-specific mAbs BC3 and VA1 (see “Immunochemical Procedures”). Immunoreactive fractions (19–25 and 26–30 min) were combined, lyophilized in a speed vac, and rechromatographed on a 250 × 2.1 mm C18 column (Vydac) using a flow rate of 0.25 ml/min and a gradient of 0–100% solvent B during 100 min (P1, P2). Rechromatography of P3 glycopeptides was performed with a flow rate of 0.3 ml/min and a gradient from 0 to 100% during 80 min. 0.3-ml fractions were collected, and 5 μl were used for immunochemical detection as described above. Immunoreactive fractions were lyophilized in a speed vac and used for matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) or Edman sequencing (Fig. 1). The monoclonal antibodies BC3 and VA1 are directed against peptide epitopes within the MUC1 tandem repeat sequence and were kindly provided by Dr. Ian McKenzie (Austin Research Institute, Heidelberg, Australia). BW835 (Behringwerke, Marburg, Germany) is specific for exposed core 1 disaccharide within a peptide motif of the MUC1 tandem repeat sequence (27Hanisch F.-G. Stadie T. Boβlet K. Cancer Res. 1995; 55: 4036-4040PubMed Google Scholar). Samples were immobilized onto 96-well polystyrene plates (Nunc, Wiesbaden, Germany), and bound antibodies were quantified by alternating incubations with polyclonal anti-mouse immunoglobulin (Z 259, Dako, Hamburg, Germany) and a complex of alkaline phosphatase/anti-alkaline phosphatase, as described previously (27Hanisch F.-G. Stadie T. Boβlet K. Cancer Res. 1995; 55: 4036-4040PubMed Google Scholar). The matrix CCA (α-cyano-4-hydroxycinnamic acid; saturated solution in 2/3 aqueous 0.1% trifluoroacetic acid, 1/3 acetonitrile) was mixed 1:1 (v/v) with the probe in 50% methanol, placed (0.7–0.8 μl) onto a polished stainless steel target, and air-dried. Linear, reflectron, and PSD mass spectrometric analyses were performed with a MALDI-TOF instrument (Bruker-Reflex, Bruker-Franzen Analytic, Bremen, Germany) (15Goletz, S., Thiede, B., Hanisch, F.-G., Peter-Katalinic, J., Schultz, M., Müller, S., Seitz, O., and Karsten, U. (1997)Glycobiology, in pressGoogle Scholar) using a pulsed UV laser beam (nitrogen laser, λ = 337 nm). Irradiance was slightly above the threshold of ion detection. Residual gas pressure was at 0.8 × 10−7 × 10−7 mbar. Ion spectra were obtained in the positive ion mode. Acceleration and reflector voltages were set to 28.5 and 30 kV, respectively. The molecular parent ion was isolated with a pulsed field by deflection of all other ions (precursor selection). Complete PSD fragment ion spectra were obtained by stepwise reducing the reflector voltage to produce overlapping mass ranges, combination of the part spectra, and calibration according to the manufacturer's recommendations. No smoothing or filtering was applied to the shown spectra. Glycopeptides were sequenced on a HPJ000G with 3.1 chemistry and a biphasic sequencing column or after covalent attachment to Sequelon AATM membranes on a Beckman GlycoSite sequenator according to previously published procedures (6Pisano A. Packer N.H. Redmond J.W. Williams K.L. Gooley A.A. Glycobiology. 1995; 4: 837-844Crossref Scopus (84) Google Scholar,28Pisano A. Jadine D.R. Packer N.H. Redmond J.W. Williams K.L. Gooley A.A. Farnsworth V. Carson W. Cartier P.K. Townsend R. Hotchkiss A. Techniques in Glycobiology. Marcel Dekker, Inc., New York1997: 299-320Google Scholar). Quantitation of the serine/threonine HexNAc substitution was calculated using a glycopeptide derived from the Dictyostelium discoideum recombinant expressed glycoprotein PsA, which contains a 100% substituted GlcNAcβ-Thr in position 4. The corrected yield for this cycle (repetitive yield for the glycopeptide ITATPAPT) was 95%. The migration behavior of GlcNAcβ-Thr is almost identical to the corresponding GalNAcα derivative on reversed phase-HPLC (see also Refs. 6Pisano A. Packer N.H. Redmond J.W. Williams K.L. Gooley A.A. Glycobiology. 1995; 4: 837-844Crossref Scopus (84) Google Scholar and 28Pisano A. Jadine D.R. Packer N.H. Redmond J.W. Williams K.L. Gooley A.A. Farnsworth V. Carson W. Cartier P.K. Townsend R. Hotchkiss A. Techniques in Glycobiology. Marcel Dekker, Inc., New York1997: 299-320Google Scholar). A detailed description of the purification procedure and the chemical characterization of MUC1 from human skim milk is given in Refs. 19Hanisch F.-G. Uhlenbruck G. Peter-Katalinic J. Egge H. Dabrowski J. Dabrowski U. J. Biol. Chem. 1989; 264: 872-883Abstract Full Text PDF PubMed Google Scholar and 29Hanisch F.-G. Uhlenbruck G. Dienst C. Stottrop M. Hippauf E. Eur. J. Biochem. 1985; 149: 323-330Crossref PubMed Scopus (90) Google Scholar. The yield of MUC1(skim milk) we obtained in the present study was 50 mg/1 liter of defatted milk. For MUC1(HMFGM) the total yield was 7 mg calculated for 100 g of wet milk fat. Quality and homogeneity of both preparations were checked by SDS-polyacrylamide gel electrophoresis with 10 μg of mucin/lane and subsequent electroblotting, followed by the glycoprotein staining procedure of O'Shannessy or immunochemical detection with the MUC1-specific mAb BW835. For both mucin preparations the glycoprotein stain revealed a single, broad band that hardly entered the 3–15% gel and that was isographic with the single band obtained by staining Western blots with mAb BW835. These data confirm that MUC1 is the major component in both mucin preparations and demonstrate the absence of detectable amounts of contaminating glycoproteins. In addition, there were no visible bands after staining the gels (100 μg mucin/lane) with Coomassie R-250, indicating that MUC1(skim milk) as well as MUC1(HMFGM) were almost free of non-glycosylated protein. Although treatment with anhydrous TFMSA is generally regarded as an efficient method for the deglycosylation of proteins (16Sojar H.T. Bahl O.M.P. Arch. Biochem. Biophys. 1987; 259: 52-57Crossref PubMed Scopus (79) Google Scholar, 17Gerken T.A. Gupta R. Jentoft N. Biochemistry. 1992; 31: 639-648Crossref PubMed Scopus (69) Google Scholar), it leaves a significant amount of the peptide-linked GalNAc uncleaved, when used for limited incubation times at 0 °C (16Sojar H.T. Bahl O.M.P. Arch. Biochem. Biophys. 1987; 259: 52-57Crossref PubMed Scopus (79) Google Scholar, 17Gerken T.A. Gupta R. Jentoft N. Biochemistry. 1992; 31: 639-648Crossref PubMed Scopus (69) Google Scholar). We took advantage of this fact by using residual core GalNAc as a marker forO-glycosylation sites. Kinetic studies were performed to find conditions where removal of peripheral saccharides was quantitative and the loss of peptide-linked GalNAc could be neglected. Because the presence of GalNAc in MUC1-linked O-glycans is restricted to peptide-linked positions in core 1 (minor) or core 2 (major) structures (19Hanisch F.-G. Uhlenbruck G. Peter-Katalinic J. Egge H. Dabrowski J. Dabrowski U. J. Biol. Chem. 1989; 264: 872-883Abstract Full Text PDF PubMed Google Scholar), 2W. Chai, A. M. Lawson, and F.-G. Hanisch, unpublished results., the amount of GalNAc residues and the ratio of GlcNAc/GalNAc, as measured by quantitative monosaccharide analysis, should give a good estimation of the proceeding deglycosylation reaction. In the absence of anisole the removal of complex glycans and the chain truncation to the level of core GalNAc proceeded within 60 min (Fig. 2 A). Extension of the incubation times over 60 min resulted in substantial losses of the core GalNAc residues which was also reflected in a decreased H. pomatia agglutinin-binding rate (data not shown). On the contrary, deglycosylation in the presence of anisole resulted in decreased reaction rates, and even after prolonged incubation times up to 6 h the residual GlcNAc was detected at 60% relative to the untreated sample (Fig. 2 B). The length of the polylactosamine-type chains on lactation-associated MUC1 and evidence that indicates a stepwise deglycosylation process from the non-reducing terminus of the glycans (17Gerken T.A. Gupta R. Jentoft N. Biochemistry. 1992; 31: 639-648Crossref PubMed Scopus (69) Google Scholar) could explain this observation. On the basis of these quantitative kinetic data the method of Sojar and Bahl (16Sojar H.T. Bahl O.M.P. Arch. Biochem. Biophys. 1987; 259: 52-57Crossref PubMed Scopus (79) Google Scholar) was preferred for the preparation of MUC1-derived glycopeptides. As measured by quantitative hexosamine analysis, the content of GalNAc during TFMSA treatments up to 30 min decreased only to 86%, while the GlcNAc content during the same period dropped below 2% (Fig. 2 A). The low amounts of GlcNAc relative" @default.
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• W2092560671 title "Localization of O-Glycosylation Sites on Glycopeptide Fragments from Lactation-associated MUC1" @default.
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