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Substrate Specificities of Three Members of the Human UDP-N-Acetyl-α-d-galactosamine:Polypeptide N-Acetylgalactosaminyltransferase Family, GalNAc-T1, -T2, and -T3

1997; Elsevier BV; Volume: 272; Issue: 38 Linguagem: Inglês

10.1074/jbc.272.38.23503

1083-351X

Hans H. Wandall, Helle Hassan, Ekaterina Mirgorodskaya, Anne K. Kristensen, Peter Roepstorff, Eric Bennett, Peter Aadal Nielsen, Michael A. Hollingsworth, Joy Burchell, Joyce Taylor‐Papadimitriou, Henrik Clausen,

Galectins and Cancer Biology

Mucin-type O-glycosylation is initiated by UDP-N-acetylgalactosamine:polypeptideN-acetylgalactosaminyltransferases (GalNAc-transferases). The role each GalNAc-transferase plays in O-glycosylation is unclear. In this report we characterized the specificity and kinetic properties of three purified recombinant GalNAc-transferases. GalNAc-T1, -T2, and -T3 were expressed as soluble proteins in insect cells and purified to near homogeneity. The enzymes have distinct but partly overlapping specificities with short peptide acceptor substrates. Peptides specifically utilized by GalNAc-T2 or -T3, or preferentially by GalNAc-T1 were identified. GalNAc-T1 and -T3 showed strict donor substrate specificities for UDP-GalNAc, whereas GalNAc-T2 also utilized UDP-Gal with one peptide acceptor substrate. Glycosylation of peptides based on MUC1 tandem repeat showed that three of five potential sites in the tandem repeat were glycosylated by all three enzymes when one or five repeat peptides were analyzed. However, analysis of enzyme kinetics by capillary electrophoresis and mass spectrometry demonstrated that the three enzymes react at different rates with individual sites in the MUC1 repeat. The results demonstrate that individual GalNAc-transferases have distinct activities and the initiation of O-glycosylation in a cell is regulated by a repertoire of GalNAc-transferases. Mucin-type O-glycosylation is initiated by UDP-N-acetylgalactosamine:polypeptideN-acetylgalactosaminyltransferases (GalNAc-transferases). The role each GalNAc-transferase plays in O-glycosylation is unclear. In this report we characterized the specificity and kinetic properties of three purified recombinant GalNAc-transferases. GalNAc-T1, -T2, and -T3 were expressed as soluble proteins in insect cells and purified to near homogeneity. The enzymes have distinct but partly overlapping specificities with short peptide acceptor substrates. Peptides specifically utilized by GalNAc-T2 or -T3, or preferentially by GalNAc-T1 were identified. GalNAc-T1 and -T3 showed strict donor substrate specificities for UDP-GalNAc, whereas GalNAc-T2 also utilized UDP-Gal with one peptide acceptor substrate. Glycosylation of peptides based on MUC1 tandem repeat showed that three of five potential sites in the tandem repeat were glycosylated by all three enzymes when one or five repeat peptides were analyzed. However, analysis of enzyme kinetics by capillary electrophoresis and mass spectrometry demonstrated that the three enzymes react at different rates with individual sites in the MUC1 repeat. The results demonstrate that individual GalNAc-transferases have distinct activities and the initiation of O-glycosylation in a cell is regulated by a repertoire of GalNAc-transferases. To date three human UDP-N-Acetylgalactosamine:polypeptideN-acetylgalactosaminyltransferases (1Homa F.L. Hollander T. Lehman D.J. Thomsen D.R. Elhammer Å.P. J. Biol. Chem. 1993; 268: 12609-12616Abstract Full Text PDF PubMed Google Scholar, 2White T. Bennett E.P. Takio K. S⊘rensen T. Bonding N. Clausen H. J. Biol. Chem. 1995; 270: 24156-24165Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 3Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) (GalNAc-transferases) 1The abbreviations used are: GalNAc-transferase, UDP-N-acetyl-α-d-galactosamine:polypeptideN-acetylgalactosaminyltransferase; MALDI-TOF, matrix-assisted laser desorption mass spectrometry; GalNAc-T1, -T2, and -T3, GalNAc-transferases cloned and expressed by Homa et al. (1Homa F.L. Hollander T. Lehman D.J. Thomsen D.R. Elhammer Å.P. J. Biol. Chem. 1993; 268: 12609-12616Abstract Full Text PDF PubMed Google Scholar), White et al. (2White T. Bennett E.P. Takio K. S⊘rensen T. Bonding N. Clausen H. J. Biol. Chem. 1995; 270: 24156-24165Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), and Bennett et al. (3Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar), respectively; HPLC, high performance liquid chromatography; CE, capillary electrophoresis; PAGE, polyacrylamide gel electrophoresis; Man transferase, Dol-P-Man:polypeptide mannosyltransferases; HIV, human immunodeficiency virus; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol. have been identified and characterized (1Homa F.L. Hollander T. Lehman D.J. Thomsen D.R. Elhammer Å.P. J. Biol. Chem. 1993; 268: 12609-12616Abstract Full Text PDF PubMed Google Scholar, 2White T. Bennett E.P. Takio K. S⊘rensen T. Bonding N. Clausen H. J. Biol. Chem. 1995; 270: 24156-24165Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 3Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 4Hagen F.K. Van Wuyckhuyse B. Tabak L.A. J. Biol. Chem. 1993; 268: 18960-18965Abstract Full Text PDF PubMed Google Scholar). Although the three GalNAc-transferases show similarities in primary structure with regard to predicted domain structures, sequence motifs, and conserved cysteine residues, the overall amino acid sequence similarity of only 45% suggests that the members of the GalNAc-transferase family have undergone significant changes during evolution. The genes encoding these enzymes are located on different chromosomes and have distinct structures, although some intron positions are conserved, suggesting an evolutionary relationship. 2E. P. Bennett, D. O. Weghuis, G. Merkx, A. G. van Kessel, H. Eiberg, and H. Clausen, submitted for publication. The genes are differentially expressed in organs as revealed by Northern analysis (1Homa F.L. Hollander T. Lehman D.J. Thomsen D.R. Elhammer Å.P. J. Biol. Chem. 1993; 268: 12609-12616Abstract Full Text PDF PubMed Google Scholar, 2White T. Bennett E.P. Takio K. S⊘rensen T. Bonding N. Clausen H. J. Biol. Chem. 1995; 270: 24156-24165Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 3Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar); in particular GalNAc-T3 exhibited a restricted expression pattern. One question addressed here is whether these three GalNAc-transferases are isoenzymes with redundant or unique functions. Hennet et al. (5Hennet T. Hagen F.K. Tabak L.A. Marth J.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12070-12074Crossref PubMed Scopus (232) Google Scholar) recently addressed this question by analyzing mice rendered deficient in a close homologue of GalNAc-T1 by gene targeting. No obvious phenotypic differences were observed and preliminary characterization of the residual GalNAc-transferase activity with a few substrates did not reveal differences in enzyme activities. There was a reduction in GalNAc-transferase activity in ES cells in which the gene was inactivated. It is difficult to assess the full significance of these findings because the enzyme deleted in these studies is not well characterized with respect to substrate specificity and tissue expression pattern. Disruption of Dol-P-Man:polypeptide mannosyltransferases which initiate O-glycosylation in yeast showed that loss of one enzyme did not affect cell growth andO-glycosylation in a severe manner. In contrast, disruption of two or more genes affected growth or was lethal (6Gentzsch M. Tanner W. EMBO J. 1996; 15: 5752-5759Crossref PubMed Scopus (221) Google Scholar). The parameters that determine sites of O-glycan attachment to glycoproteins are poorly understood (7Sadler J.E. Ginsburg V. Robbins P.W. Biology of Carbohydrates. John Wiley & Sons, New York1984: 199-331Google Scholar, 8Gooley A.A. Williams K.L. Glycobiology. 1994; 4: 413-417Crossref PubMed Scopus (58) Google Scholar, 9Elhammer A.P. Poorman R.A. Brown E. Maggiora L.L. Hoogerheide J.G. Kezdy F.J. J. Biol. Chem. 1993; 268: 10029-10038Abstract Full Text PDF PubMed Google Scholar, 10Clausen H. Bennett E.P. Glycobiology. 1996; 6: 635-646Crossref PubMed Scopus (230) Google Scholar). UnlikeN-linked glycosylation and most other types of protein glycosylation, a consensus peptide sequence motif for acceptor sites has not emerged for either GalNAc- or Man-typeO-glycosylation (6Gentzsch M. Tanner W. EMBO J. 1996; 15: 5752-5759Crossref PubMed Scopus (221) Google Scholar, 8Gooley A.A. Williams K.L. Glycobiology. 1994; 4: 413-417Crossref PubMed Scopus (58) Google Scholar, 9Elhammer A.P. Poorman R.A. Brown E. Maggiora L.L. Hoogerheide J.G. Kezdy F.J. J. Biol. Chem. 1993; 268: 10029-10038Abstract Full Text PDF PubMed Google Scholar, 10Clausen H. Bennett E.P. Glycobiology. 1996; 6: 635-646Crossref PubMed Scopus (230) Google Scholar, 11Lis H. Sharon N. Eur. J. Biochem. 1993; 218: 1-27Crossref PubMed Scopus (813) Google Scholar) despite extensive studies of the acceptor substrate specificities of different GalNAc-transferase preparations (8Gooley A.A. Williams K.L. Glycobiology. 1994; 4: 413-417Crossref PubMed Scopus (58) Google Scholar, 12Briand J.P. Andrews Jr., S.P. Cahill E. Conway N.A. Young J.D. J. Biol. Chem. 1981; 256: 12205-12207Abstract Full Text PDF PubMed Google Scholar, 13Wilson I.B.H. Gavel Y. von Heijne G. Biochem. J. 1991; 275: 529-534Crossref PubMed Scopus (257) Google Scholar, 14O'Connell B.C. Hagen F.K. Tabak L.A. J. Biol. Chem. 1992; 267: 25010-25018Abstract Full Text PDF PubMed Google Scholar). Analysis of sequences around confirmed sitesO-glycosylated in vivo failed to reveal a simple model for prediction of glycosylation (9Elhammer A.P. Poorman R.A. Brown E. Maggiora L.L. Hoogerheide J.G. Kezdy F.J. J. Biol. Chem. 1993; 268: 10029-10038Abstract Full Text PDF PubMed Google Scholar, 15O'Connell B. Tabak L.A. Ramasubbu N. Biochem. Biophys. Res. Commun. 1991; 180: 1024-1030Crossref PubMed Scopus (73) Google Scholar, 16Hansen J.E. Lund O. Rapacki K. Clausen H. Mosekilde E. Nielsen J.O. Hansen J.-E. Bohr H. Brunak S. Protein Structure by Distance Analysis. IOS Press, Amsterdam1994: 247-254Google Scholar). Attempts to infer sequence specificity from analysis of the substrate specificities of GalNAc-transferase activities obtained from extracts is likely to be misleading due to variable expression of a number of different GalNAc-transferases, which may show distinct specificities for acceptor substrates. Some GalNAc-transferases may compete for acceptor substrate sites, even if they do not glycosylate the acceptor substrate site (17S⊘rensen 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). Mathematical models designed to predict sequence preferences ofO-glycan sites are flawed because of the limited number of sites identified to date and the selected class of glycoproteins these represent (9Elhammer A.P. Poorman R.A. Brown E. Maggiora L.L. Hoogerheide J.G. Kezdy F.J. J. Biol. Chem. 1993; 268: 10029-10038Abstract Full Text PDF PubMed Google Scholar, 16Hansen J.E. Lund O. Rapacki K. Clausen H. Mosekilde E. Nielsen J.O. Hansen J.-E. Bohr H. Brunak S. Protein Structure by Distance Analysis. IOS Press, Amsterdam1994: 247-254Google Scholar), and the fact that the analyzed sites were obtained from a number of different organisms and cell types that probably express different repertoires of GalNAc-transferases. In the present study we investigated the in vitrospecificity and kinetic properties of purified recombinant GalNAc-transferases, GalNAc-T1, -T2, and -T3. The results demonstrate unique but partly overlapping acceptor substrate specificities among the three enzymes. Specific sites on peptides were glycosylated, however, there were differences in kinetic properties at these sites. The same sites were glycosylated in a 20-mer or a 105-mer peptide based on the MUC1 tandem repeat. Selective specificity of GalNAc-T3 for a 6-mer sequence in fibronectin was maintained with the intact fibronectin molecule. The results indicate that the acceptor substrate specificities of the GalNAc-transferases is largely dependent on the primary sequence of the acceptor substrate. Expression constructs of soluble human GalNAc-T1, -T2, and -T3 were prepared in the vector pAcGP67 as described previously (2White T. Bennett E.P. Takio K. S⊘rensen T. Bonding N. Clausen H. J. Biol. Chem. 1995; 270: 24156-24165Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 3Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). The constructs for GalNAc-T1 and -T2 were designed to correspond to the previously identified N-terminal sequence of the purified soluble enzymes (1Homa F.L. Hollander T. Lehman D.J. Thomsen D.R. Elhammer Å.P. J. Biol. Chem. 1993; 268: 12609-12616Abstract Full Text PDF PubMed Google Scholar, 2White T. Bennett E.P. Takio K. S⊘rensen T. Bonding N. Clausen H. J. Biol. Chem. 1995; 270: 24156-24165Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The N terminus of the expressed recombinant forms included the following residues (underlined) derived from the vector construct: T1, NH2-DLGSRGL; T2, NH2-DPGTLLEPKKK; and T3, NH2-DLGSSTMER-. Sf9 cells were grown at 27 °C in TMN-FH medium containing 10% fetal calf serum (Pharmingen). Plasmids pAcGP67-GalNAc-T1-sol, pAcGP67-GalNAc-T2-sol, and pAcGP67-GalNAc-T3-sol were cotransfected with Baculo-Gold™ DNA (Pharmingen) as described previously (3Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Recombinant Baculovirus was obtained after two successive amplifications in Sf9 cells grown in serum-containing medium, and titers of virus were estimated by titration in 24-well plates with monitoring of enzyme activities. Controls included soluble human blood group A GalNAc-transferase (18Bennett E.P. Steffensen R. Clausen H. Weghuis D.O. van Kessel D.G. Biochem. Biophys. Res. Commun. 1995; 206: 318-325Crossref PubMed Scopus (105) Google Scholar), and the enzymatically non-functional blood group O2 allele (19Grunnet N. Steffensen R. Bennett E.P. Clausen H. Vox Sang. 1994; 67: 210-215Crossref PubMed Scopus (85) Google Scholar). For large scale expression in serum-free medium, Sf9 cells were adapted to growth in 2.5 expanded surface area roller bottles (in vitro) in 200 ml of 30% Grace containing 10% fetal calf serum and 70% SF-900 II medium (Life Technologies) in a 27 °C roller at 0.6 rpm. Cells which could be loosened by centrifugal agitation were transferred to regular roller bottles maintained upright in a shaking bath (27 °C, 140 rpm) and 200 ml of SF-900 II medium containing 1 mm glutamine and 0.025% F-68 (Life Technologies) were added. Cells were grown for 2 days, split 1:1 with SF-900 II medium, and grown for another 2 days. Cell densities approached approximately 1 × 106/ml. Cells were infected as follows: cells were harvested and resuspended in 100 ml of SF-900 II medium in the original shaker bottle and infected with 1:1,000 to 1:5,000 of a stock of the second amplification of virus (3Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). After 1 h 350 ml of SF-900 II medium containing 2 mm glutamine, 0.1% glucose, 0.25% lipid mixture (Life Technologies), and 0.2% yeast extract ultrafiltrate (200 g/liter) (Sigma) were added and flasks shaken for 72–96 h (27 °C, 140 rpm). The spent medium was harvested by centrifugation at 1,000 rpm and stored at 4 °C in the presence of 0.02% NaN3. Attempts to use Sf9 cells maintained throughout in SF-900 II medium failed to yield significant quantities of recombinant proteins. Purification of recombinant enzymes from serum-free medium was performed as follows (Table I). Approximately 400 ml of medium containing 2–5 units of enzyme were harvested and processed individually. Medium was dialyzed against 25 mm Bis-Tris, pH 6.0, 10 mm NaCl, 2 mm MnCl2, and 2 mm EDTA, centrifuged at 10,000 × g, and passed through a 120-ml DEAE (Sigma) column equilibrated in dialysis buffer without EDTA. The excluded fractions were applied to a 30-ml S-Sepharose Fast-flow (Pharmacia) column equilibrated in the same buffer and GalNAc-transferases were eluted with a gradient of NaCl from 10 to 500 mm. Fractions containing enzyme were pooled and simultaneously dialyzed and concentrated using a Spectrum Dialysis Concentrator with 10,000 cut off (Spectrum). The concentrated GalNAc-T1 and -T2 were diluted 5-fold in Bis-Tris buffer with 10 mmNaCl, applied to a Mono-S column (HR 5/5, Pharmacia), and eluted with a NaCl gradient from 10 to 500 mm. GalNAc-T3 was not subjected to the second cation exchange chromatography as this step inactivated the enzyme. Mono-S fractions of GalNAc-T1 and -T2 as well as concentrated S-Sepharose fractions of GalNAc-T3 were further purified by S12 gel filtration chromatography (HR 10/30, Smart System, Pharmacia) run in phosphate buffer (pH 7.4) with 1.15 mNaCl. The purity and protein concentration of final fractions of the GalNAc-transferases were assessed by S12 gel filtration chromatography (PC3.2/30, Smart System, Pharmacia) and SDS-PAGE using bovine serum albumin as a standard.Table IPurification of recombinant GalNAc-transferasesPurification stepGalNAc-T1GalNAc-T2GalNAc-T3VolumeActivityYieldSpecific activityVolumeActivityYieldSpecific activityVolumeActivityYieldSpecific activitymlunits 1-aOne unit of enzyme is defined as the amount of enzyme that will transfer 1 μmol of GalNAc from UDP-GalNAc in 1 min using the standard reaction mixture as described under “Experimental Procedures” with 25 μg of Muc2 peptide as acceptor substrate for GalNAc-T1, 25 μg of Muc1b as acceptor substrate for GalNAc-T2, and 25 μg of Muc1a as acceptor substrate for GalNAc-T3.%units/mgmlunits%units/mgmlunits%units/mgSf9 medium3503.71003001.51003003.6100Step 2: DEAE3503.6973001.51003003.289Step 3: S-Sepharose302.464351.493552.158Step 4: dialysis concentration1.21.2321.51.1731.51.642Step 5: Mono-S0.81.1290.60.856Step 6: S-121.20.7180.61.20.5330.51.20.5140.51-a One unit of enzyme is defined as the amount of enzyme that will transfer 1 μmol of GalNAc from UDP-GalNAc in 1 min using the standard reaction mixture as described under “Experimental Procedures” with 25 μg of Muc2 peptide as acceptor substrate for GalNAc-T1, 25 μg of Muc1b as acceptor substrate for GalNAc-T2, and 25 μg of Muc1a as acceptor substrate for GalNAc-T3. Open table in a new tab Standard assays were performed in 50 μl of total reaction mixtures containing 25 mm Tris (pH 7.4), 10 mmMnCl2, 0.25% Triton X-100, 50 μmUDP-[14C]GalNAc (2,000 cpm/nmol) (Amersham), 5 mm 2-mercaptoethanol (only in assays to determineK m and V max), 0.01–0.5 milliunits of GalNAc-transferase, and 25 μg of acceptor peptide (see Table II for structures). Peptides were synthesized by ourselves or by Carlbiotech (Copenhagen) and Neosystems (Strasbourg), and quality was ascertained by amino acid analysis and mass spectrometry. Products were routinely determined by scintillation counting after Dowex-1 formic acid cycle chromatography. At least once for all combinations of enzyme sources and peptides, the products were evaluated by C-18 reverse phase chromatography (PC3.2/3 or μRPC C2/C18 SC2.1/10 Pharmacia, Smart System) with scintillation counting of peptide peak fractions. Finally, peptides and products produced by in vitro glycosylation were in most cases also confirmed by mass spectrometry.Table IIAcceptor substrate specificity of recombinant GalNAc-transferasesPeptidePeptide sequenceGalNAc-T1GalNAc-T2GalNAc-T3K mV maxK mV maxK mV maxmmpmol/minmmpmol/minmmpmol/minMuc1a′AHGVTSAPDTR0.661022.13105.70.0963.3Muc1aAPPAHGVTSAPDTRPAPGC1.42139.7ND2-aND, not determined, indicates that although incorporation is observed K m was higher than 2 mm and therefore not analyzed due to required quantities of peptides.ND0.50108.9Muc1b′RPAPGSTAPPANDND1.0575.9NDNDMuc1bPDTRPAPGSTAPPACNDND0.82114.8NDNDTAP24TAPPAHGVTSAPDTRPAPGSTAPP0.4083.70.9280.50.9452.3Muc2PTTTPISTTTMVTPTPTPTC0.12102.00.0119.00.1087.2Muc5ACAc-SAPTTSTTSAPT1.3463.20.8454.20.5342.5hCG-βPRFQDSSSSKAPPPLPSPSRLPGNA2-bNA, not applicable, indicates that no incorporation is observed with this substrate even after prolonged incubations (24 hrs).NA1.2047.9NANAOSM fragmentLSESTTQLPGGGPGCA0.3088.2NANA1.6153.7ErythropoitinPPDAASAAPLRNDNDNDNDNDNDErythropoitinPPDAATAAPLRND2-cDue to substrate inhibition at concentration of 1 mm.ND2-cDue to substrate inhibition at concentration of 1 mm.0.5131.30.7237.0HIVIIIBgp120Ac-CIRIQRGPGRAFVTIGKIGNMRNANANANA0.4125.6VTHPGYAc-PFVTHPGYDNANANANANDND2-a ND, not determined, indicates that although incorporation is observed K m was higher than 2 mm and therefore not analyzed due to required quantities of peptides.2-b NA, not applicable, indicates that no incorporation is observed with this substrate even after prolonged incubations (24 hrs).2-c Due to substrate inhibition at concentration of 1 mm. Open table in a new tab GalNAc-transferase assays used for determination ofK m of acceptor substrates were modified to include 200 μm UDP-[14C]GalNAc (2,000 cpm/nmol) with peptides in varying concentrations from 0.005 to 2 mm. Assays to determine K m for UDP-GalNAc were performed with saturating concentrations of acceptor substrates (GalNAc-T1, 500 μm Muc2; GalNAc-T2, 100 μm Muc2; and GalNAc-T3, 500 μm Muc2). Analysis of specificities for other sugar nucleotides were performed in standard reaction mixtures with 500 μm UDP-GalNAc, UDP-Gal, or UDP-GlcNAc using 250 μm Muc2 and 0.25 milliunits of purified enzymes (specific activity estimated with Muc2 peptide). Assays were performed in duplicate or quadruplicate. Assays to determine the metal ion requirement were performed in standard reaction mixtures without MnCl2 using 0.25 milliunits of GalNAc-transferase (specific activity estimated with Muc5C peptide) purified by gel filtration (run in buffer phosphate-buffered saline with 1 m NaCl) in the absence of MnCl2. Analysis of GalNAc-transferase activity without addition of Mn2+ revealed no detectable activity. The activity was assessed with Muc2 peptide in the presence of 5, 10, or 20 mm CaCl2 or MgCl2 with MnCl2 as control. Preparative glycosylation of peptides was performed with 10–50 nmol of peptide, 0.5–2.5 mmol of UDP-[14C]GalNAc (10-fold excess of potential Ser/Thr acceptor sites), and 0.25–5 milliunits of GalNAc-transferase (specific activity determined using the relevant acceptor peptide to be glycosylated) in a final volume of 200 μl. Reactions were allowed to incubate for 24–48 h at 37 °C, and at 18–24 h additional enzyme and UDP-GalNAc (50% of originally added) were added. A peptide was considered terminally glycosylated by a GalNAc-transferase when addition of enzyme and UDP-GalNAc did not result in further incorporation over 4 h as estimated by [14C]GalNAc incorporation, CE, HPLC, or MALDI-TOF.In vitro glycosylation of plasma fibronectin (Sigma) was performed using the standard reaction mixture with 0.5 milliunits of GalNAc-T3 or a mixture of 0.25 milliunits of GalNAc-T1 and 0.25 milliunits of GalNAc-T2 for 6 h at 37 °C. Controls included heat-inactivated (5 min 95 °C) enzyme. SDS-PAGE Western blotting using monoclonal antibodies FDZ to fibronectin and FDC-6 and 5C10, which specifically detect the oncofetal fibronectin epitope, as well as enzyme-linked immunoadsorbent assay using the same antibodies. A reaction mixture for preparative glycosylation was used with cold 1–2 mm UDP-GalNAc and 0.5–1 mm acceptor peptides in a total volume of 50–100 μl. The assay was incubated in the sample carousel at 30 °C and injections performed at 30–60-min intervals. Capillary zone electrophoresis was performed on a Applied Biosystems model HT270 (Perkin-Elmer). Coated fused silica capillaries, 72 cm × 75 μm, with 35-cm length between sample injection and optical cell were used. Electrophoresis were performed at 30 °C using 50 mm phosphate buffer (pH 2.5). Voltage across the capillary was 20 KV in the positive mode with the anode at the injection side, and the runs were monitored at 210 nm. At the beginning of each cycle the capillary was flushed with 0.1 m NaOH for 2 min, followed by flushing with 50 mm phosphate buffer (pH 2.5) for 4 min. Composition of products separated were assessed in two ways: (i) parallel reactions were stopped at time points with maximum peak height for each component, and then glycopeptides were purified by C-18 HPLC, and analyzed by MALDI-TOF and amino acid sequence analysis. (ii) Purified standard glycopeptides were co-injected with reaction mixtures to assess co-migration of products. All mass spectra, except for the Muc1 105-mer peptide, were acquired on a Voyager-Elite MALDI time of flight mass spectrometer (Perseptive Biosystem Inc., Framingham, MA), equipped with delayed extraction. The MALDI matrix was a 9:1 mixture of 25 g/liter 2,5-dihydroxybenzoic acid and 25 g/liter 2-hydroxy-5-methoxy benzoic acid (Aldrich) dissolved in a 2:1 mixture of 0.1% trifluoroacetic acid in water and acetonitrile. Samples dissolved in 0.1% trifluoroacetic acid to a concentration of approximately 2 pmol/μl were prepared for analysis by placing 1 μl of sample solution on a probe tip followed by 1 μl of matrix. All spectra were obtained in the linear mode and calibrated using external calibration. Data processing were carried out using GRAMS/386 software. All mass spectra for the Muc1 105-mer peptide were obtained on a Bruker reflex time of flight mass spectrometer (Bruker-Franzen Analytik, Bremen, Germany). Data were acquired by a LeCroy 9450A 400 megasamples/s digital storage oscilloscope (LeCroy Corp., Chestnut Ridge, NY) from which single shot spectra were transferred to a Macintosh Quadra 950 computer (Apple Computer Inc., Cupertino, CA) via a National Instruments NI DAQ GPIB controller board (National Instruments, Austin, TX). Samples were dissolved in 0.1% trifluoroacetic acid to a concentration of approximately 2 pmol/μl. One μl of sample solution was placed on a stainless steel probe tip followed by 1 μl of matrix solution (α-cyano-4-hydroxycinnamic acid dissolved in 70% acetonitrile, 15 g/liter). All mass spectra were obtained in the linear mode and calibrated using a singly charged matrix ion, which provided a mass accuracy of approximately 0.1%. Data processing were carried out using the computer program LaserOne, which was written in ThinkC (Symantec Corporation, Cupertino, CA) by M. Mann and P. Mortensen, EMBL, Heidelberg, Germany. Reactions containing Muc1 105-mer (2 nmol) in 100 μl 0.1 m sodium phosphate (pH 8.0) with 0.2 mg of Asp-N endoproteinase were incubated for 18 h at 37 °C. The digest was injected directly into a reverse phase HPLC column and cleaved peptides eluted by a gradient of 0–90% acetonitrile in 0.1% trifluoroacetic acid. Automated Edman degradation was performed on a Knauer 910 pulsed-liquid gas-phase sequencer using polyvinylidene difluoride membranes as immobilizing support. The peptide samples were dissolved in 0.1% trifluoroacetic acid and spotted onto Polybrene-coated polyvinylidene difluoride membranes. The PTH derivatives were separated on-line on a 2 × 250-mm column packed with 5 ml of SuperSpher C18 (Merck) using a narrow bore HPLC. Expression of recombinant GalNAc-transferases in serum-free media yielded 2–5 units/400 ml in shaker flasks harvested 72–96 h post-infection. The combination of anion and cation exchange chromatography resulted in concentration and partial purification of the enzymes (Table I, Fig.1). A gel filtration step yielded enzyme preparations in high yields with few contaminants as estimated by Coomassie staining of SDS-PAGE gels. The specific activities of transferase preparations were estimated to 0.5–0.6 units/mg. GalNAc-T3 consistently produced two faster migrating bands, and we believe that these are different glycoforms and/or proteolytic products because all three components were recognized by a monoclonal antibody to the enzyme as detected by Western blotting (not shown). Furthermore, immunoprecipitation of an expressed full-length GalNAc-T3 enzyme with a C-terminal myc-tag using an antibody to themyc-tag also produced the same banding pattern. 3T. Nilsson, personal communication. The kinetic parameters of the three GalNAc-transferases in this study are potentially affected by the following factors: (i) the enzymes are derived from human cDNA sequences; (ii) the enzymes are soluble constructs with GalNAc-T1 and -T2 having a N-terminal sequence similar to the forms originally purified and GalNAc-T3 having a longer N-terminal sequence designed to exclude the hydrophobic retention signal and 12 residues from the predicted stem region (3Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar); and (iii) the enzymes are expressed as secreted products in insect cells grown in serum-free medium. Comparisons of the kinetic parameters to purified human enzymes in membrane bound and/or secreted forms would determine whether or not these factors influence the results; however, this is currently not possible. The following observations suggest that the data obtained is not significantly affected by the design of the expression constructs and the expression system. The kinetic parameters of purified, recombinant GalNAc-T2 were found to be similar to those of the originally purified enzyme (2White T. Bennett E.P. Takio K. S⊘rensen T. Bonding N. Clausen H. J. Biol. Chem. 1995; 270: 24156-24165Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 17S⊘rensen T. White T. Wandall H.H. Kristensen A.K. Roepstorff P. C