O-GalNAc incorporation into a cluster acceptor site of three consecutive threonines Distinct specificity of GalNAc-transferase isoforms Hideyuki Takeuchi 1 , Kentaro Kato 1 , Helle Hassan 2 , Henrik Clausen 2 and Tatsuro Irimura 1 1 Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan; 2 Department of Oral Diagnostics, Faculty of Health Sciences, School of Dentistry, University of Copenhagen, Denmark O-Glycosylation of three consecutive Thr residues in a fluorescein-conjugated peptide PTTTPLK ) which mimics a portion of mucin 2 ) by four isozymes of UDP-N-ace- tylgalactosaminyltransferases (pp-GalNAc-T1, T2, T3, or T4) was investigated. Partially glycosylated versions of this peptide, PT*TTPLK, PTTT*PLK, PT*TT*PLK, PTT*T*PLK, PT*°TTPLK, and PTTT*°PLK (*, N-acetyl- galactosamine; °, galactose), were also tested. The products were separated by RP-HPLC and characterized by MALDI- TOF MS and peptide sequencing. The first and the third Thr residues act as the peptide’s initial glycosylation sites for pp-GalNAc-T4, which were different from the sites for pp-GalNAc-T1 and T2 (the first Thr residue) or T3 (the third Thr residue) shown in our previous report. All pp-GalNAc- T isozymes tested exhibited distinct specificities toward glycopeptides. The most notable findings were: (a) prior incorporation of an N-acetylgalactosamine residue at the third Thr greatly enhanced N-acetylgalactosamine incor- poration into the other Thr residues when pp-GalNAc-T2, T3, or T4 were used; (b) the enhancing effect of the N-ace- tylgalactosamine residue on the third Thr was completely abrogated by galactosylation of this N-acetylgalactosamine; (c) prior incorporation of an N-acetylgalactosamine at the first Thr did not have any enhancing effect; (d) pp-GalNAc- T2 was unique as it transferred N-acetylgalactosamine into the second Thr residue only when N-acetylgalactosamine was attached to the third one. Keywords: O-glycosylation; mucin; polypeptide N-acetylga- lactosaminyltransferase; Tn antigen; UDP-GalNAc. Biosynthesis of O-glycans is mediated by the step-wise addition of monosaccharides by a variety of glycosyl- transferases, where topology and kinetic properties of Golgi-resident glycosyltransferases are believed to generate additional diversity of carbohydrate structures [1]. The initial O-glycosylation is thought to be a highly selective process where the sequence context determines where O-glycans are attached to proteins, although the rules governing this selection are still poorly understood [2–11]. Mucins form a large family of membrane-associated or secretory glycoproteins rich in O-glycans. They are pro- duced by epithelial cells and function as a physical and biological barrier protecting mucous epithelia. There are also leukocyte and erythrocyte markers with mucin-like structures. The core polypeptides of mucins are not only rich in serines and threonines but they also contain Ser and Thr repeats, and tandem repeats of Ser/Thr-rich stretches [12,13]. Sequences with consecutive Thr and Ser residues seem to play important roles in recognition events. Trun- cated O-glycans displayed on consecutive Thr residues serve as ligands for endogenous C-type lectins on macrophages and carcinoma-specific anti-Tn antibodies [14]. Many mucin-like leukocyte markers such as CD34, CD45 and CD68 bear sequences containing consecutive Ser and Thr residues at their outermost segments [15–17]. Therefore, it is tempting to speculate that these consecutive Ser/Thr sequences with various arrangements of O-glycans are structural motifs having specific biological relevance [18]. The first step of mucin O-glycosylation is initiated by a family of UDP-N-acetyl- D -galactosamine : polypeptide UDP-N-acetylgalactosaminyltransferases (pp-GalNAc-Ts, EC 2.4.1.41) that transfer N-acetylgalactosamine(GalNAc) residues to Ser and Thr residues in a polypeptide. To date, nine members of the mammalian pp-GalNAc-T family have been cloned and characterized [19–31]. Although the kinetic properties and substrate specificities of some of these recombinant isozymes have been investigated by in vitro studies using several synthetic peptides as substrates, we are still far from understanding the regulation of O-glycosyla- tion [32–35]. When the peptide PTTTPITTTTK [that represents a portion of the mucin 2 (MUC2) tandem repeat] was used as a substrate with detergent-soluble microsome fractions from the human colon carcinoma cell line LS174T (which expresses several members of the GalNAc-Ts family), GalNAc was transferred to these Thr Correspondence to T. Irimura, Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Fax: + 81 3 5841 4879, Tel.: + 81 3 5841 4870, E-mail: irimura@mol.f.u-tokyo.ac.jp Abbreviations: pp-GalNAc-T, UDP-N-acetyl- D -galactosaminide, polypeptide N-acetylgalactosaminyltransferase. Enzymes:UDP-N-acetyl- D -galactosamine : polypeptide UDP- N-acetylgalactosaminyltransferases (EC 2.4.1.41). (Received 19 May 2002, revised 22 August 2002, accepted 28 October 2002) Eur. J. Biochem. 269, 6173–6183 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03334.x residues in a specific and distinct order [36,37]. Also, we reported that pp-GalNAc-T isoforms (GalNAc-T1, -T2, and -T3) exhibited different orders of incorporation of GalNAc residues into consecutive Thr residues of the PTTTPLK acceptor peptide [38]. These results suggest that some pp-GalNAc-T isoforms work in a cooperative fashion transferring to different acceptor sites in clusters. Evidence demonstrating negative effects of GalNAc attachments for subsequent activities of pp-GalNAc-Ts [39], suggests that the order by which GalNAc-T isoforms initiate glycosylation may lead to different pathways of biosynthesis resulting in different patterns of O-glycan occupancy. Furthermore, it has been proposed recently that some GalNAc-T isoforms function as follow-up enzymes in that they are directed by the initial action of other isoforms [21,23,28,35,40]. This latter mech- anism is not fully understood, but recent data by Hassan and coworkers indicate that the putative lectin domains of these isoforms are responsible for the unique GalNAc- glycopeptide specificities [41]. We therefore hypothesized that different subsets of pp-GalNAc-T isoforms are desig- nated to generate different arrangement of O-glycans on mucins having consecutive Thr residues. Using a simple model substrate with three consecutive Thr acceptor residues, we examined whether vicinal effects, positive as well as negative, of GalNAc and Galb1–3GalNAc residues on the efficacy and pathway of incorporation of the second and the third GalNAc residues with four pp-GalNAc-Ts were observed. EXPERIMENTAL PROCEDURES Synthesis of acceptor substrates A synthetic oligopeptide PTTTPLK, was used as the acceptor substrate for the pp-GalNAc-T isozymes. Its sequence was derived from the tandem repeat domain of the MUC2 core polypeptide (PTTTPITTTTTVTPTPTPT GTQT) [42]. It was synthesized on a Model 9020 peptide synthesizer (Milligen, Burlington, MA, USA) with a lysine as the C-terminal residue. The peptide was labelled at pH 7.5 (adjusted with 100 m M Hepes buffer) with fluores- cein isothiocyanate (FITC) at its N-terminal amino acid under conditions in which the e-amino groups of lysine residues were not modified. The lysine was added to allow further modifications to study the interaction of resultant glycopeptides with carbohydrate recognition molecules [14] but such experiments are not described in the present report. Using FITC–PTTTPLK as a substrate, glycopeptides containing GalNAc residues were prepared enzymatically. Two glycopeptides, designated FITC–PT*TTPLK or FITC–PT*TT*PLK (where * stands for a GalNAc residue), were generated with recombinant pp-GalNAc-T1. The remaining two glycopeptides, denoted FITC–PTTT*PLK and FITC–PTT*T*PLK, were prepared with recombinant pp-GalNAc-T3. Glycopeptides with Galb1–3GalNAc resi- dues were prepared enzymatically using FITC–PT*TTPLK or FITC–PTTT*PLK as acceptor substrates, UDP-Gal (final 1 m M ) as donor substrates, and detergent-soluble microsome fractions of human laryngeal carcinoma H.Ep.2 cells as the source of UDP-Gal:N-acetylgalactosaminide b1–3 galactosyltransferase(s). The incubation conditions and the preparations of microsome fractions are described in the following sections. All glycopeptides were purified by RP-HPLC on a C 18 column. Sites of GalNAc attachment were confirmed by protein sequencing using the PE Biosystems 490 Procise protein sequencing system [38]. To test the effect of the FITC residue on the acceptor specificity of pp-GalNAc-Ts, the same peptide without an FITC residue was synthesized, used as an acceptor substrate, and conjugated with FITC for the HPLC separation. In another experiment, the same peptide with additional six alanine residues at the N terminus was synthesized, conjugated with FITC, and used as an acceptor. Preparation of recombinant pp-GalNAc-Ts Soluble recombinant pp-GalNAc-T1, T2, and T3 were prepared as described previously [43]. Briefly, each of the plasmids pAcGP67-GalNAc-T1-sol, pAcGP67-GalNAc- T2-sol, and pAcGP67-GalNAc-T3-sol were cotransfected with Baculo-Gold DNA (Pharmingen) to Sf9 cells. The recombinant pp-GalNAc-T1, T2, and T3 were purified from the spent media. pp-GalNAc-T4 was prepared from the secretions of a stably transfected Chinese hamster ovary (CHO) cell line (CHO/GalNAc-T4/21 A) as described previously [22]. One unit of recombinant enzyme was defined as the amount of enzyme that transferred 1 nmol of GalNAc residues in 30 min onto FITC–PTTTPITTTTK at a final concentration of 5 l M in 50 lL-incubation mixtures. Preparation of detergent-soluble microsome fractions of H.Ep.2 cells Human laryngeal carcinoma H.Ep.2 cells were cultured in modified Eagle’s medium supplemented with 10% fetal bovine serum. Cells were homogenized in 50 m M Tris/HCl buffer pH 7.5 containing 250 m M sucrose, 1 lgÆmL )1 aprotinin (Sigma), 1 lgÆmL )1 leupeptin (Peptide Institute Inc.,Osaka,Japan),and0.5lgÆmL )1 pepstatin A (Sigma). After centrifugation at 3000 g at 4 °C for 10 min, the decanted supernatant was centrifuged at 100 000 g for 1 h. The pellet was re-suspended in the buffer used during the homogenization containing an additional 0.1% Triton X-100 (Sigma). Protein concentrations were determined using Protein Assay Kit (Bio-Rad) with BSA as a standard. The solutions were stored in aliquots at )80 °C until use. Enzymatic GalNAc incorporation into peptide and glycopeptide acceptors The standard enzyme reaction mixture consisted of 50 m M Hepes buffer pH 7.5, 5 m M MnCl 2 ,5m M 2-mercapto- ethanol, 0.1% Triton X-100, 1 m M UDP-N-acetyl- D -gal- actosamine (Sigma), 5 l M acceptor peptides or glycopeptides, and 0.2 U recombinant enzyme pp-GalNAc T1, T2, T3, or T4 (0.2268 lg, 1.098 lg, 1.365 lg, and 2.768 lg, respectively), in a final volume of 100 lL. Reactions were performed at 37 °C for 16 h and were terminated by adding 20 lL of 500 m M EDTA. Monitoring of in vitro O-glycosylation by RP-HPLC The glycosylated peptides were separated by RP-HPLC (JASCO, Tokyo, Japan). A Cosmosil column (C 18 , 10 · 250 mm; Nacalai tesque, Japan) was used. The 6174 H. Takeuchi et al. (Eur. J. Biochem. 269) Ó FEBS 2002 column was eluted with a linear gradient ranging from 0 to 50% solvent B (0.05% trifluoroacetic acid in 70% 2-pro- panol in acetonitrile) in solvent A (0.05% trifluoroacetic acid in water) at a flow rate of 2 mLÆmin )1 for 30 min. Eluates were monitored by fluorescence intensity at 520 nm. MALDI-TOF MS of glycosylated peptides Glycosylated peptides were applied on a tip and mixed with a10mgÆmL )1 solution of a-cyano-4-hydroxycinnamic acid dissolved in 0.1% trifluoroacetic acid/50% ethanol in water. All mass spectra were obtained on a Voyager Elite instrument (Nippon PerSeptive Biosystems, Tokyo, Japan) operating at an accelerating voltage of 20 kV (grid voltage 93.5%, guide wire voltage 0.05%) in the linear mode with the delayed extraction setting. Recorded data were pro- cessed by using GRAMS/386 software. Amino acid sequencing Pulsed liquid Edman degradation amino acid sequencing of glycopeptides was performed with the Applied Biosys- tems 490 Procise protein sequencing system (Perkin Elmer). With this system, a phenylthiohydantoin-deri- vative of GalNAc-attached Thr was identified as a pair of peaks eluted near the positions of phenylthiohydantoin- Ser and phenylthiohydantoin-Thr [44]. Amino acid sequencing of fully glycosylated peptide (FITC– PT*T*T*PLK) confirmed the eluting positions. The peptides used in the present study were modified at the N terminals and the amino acid (Pro) was not detected. The second amino acid (Thr2) was detected at the first cycle of Edman degradation. RESULTS Fractionation of products resulting from glycosylation of FITC–PTTTPLK peptide by pp-GalNAc-T4 An FITC-labelled oligopeptide PTTTPLK that mimicked the tandem repeat portion of MUC2 was chemically synthesized and labelled with FITC at its N-terminal amino acid residue. Theoretically, seven different products could be generated from this peptide upon incubation with a pp-GalNAc-T isozyme in the presence of UDP- N-acetyl- D -galactosamine. When FITC–PTTTPLK was incubated with recombinant pp-GalNAc-T4 for various periods ranging up to 24 h and then subjected to RP-HPLC, six peaks, including the unaltered peptide, were observed depending on the incubation period (Fig. 1). These fractions (a–e) were collected separately and analysed by MALDI-TOF MS, which showed that the fractions corresponded to FITC–PTTTPLK bearing either one, two, or three glycosylated residues (Fig. 2). Thus, peaks (b) and (c) apparently contained two GalNAc residues and peaks (d) and (e) apparently contained a single GalNAc residue. Peak (a) appears to be the fully glycosylated peptide. The associated peaks on the MALDI-TOF MS profiles are not likely to be due to contaminating glycopeptides with smaller numbers of attached GalNAc residues, judging from the clear separ- ation of glycopeptides with given numbers of GalNAc residues on the RP-HPLC. These peaks in MALDI-TOF MS profiles should be the result of degradation during the matrix-assisted ionization of these glycopeptides. The degree of glycosylation depended on the duration of incubation. Up to 6 h, the six peaks could be detected, with the major fraction being unglycosylated peptide. At 24 h, peptides bearing one or three GalNAc residues were prominent. After the addition of fresh enzyme and UDP- GalNAc, the proportion of the peptide bearing three GalNAc residues increased (data not shown). Characterization of the pp-GalNAc-T4 glycosylation products The sites of GalNAc attachment to the peptide were analysed by amino acid sequencing. As shown in Fig. 3, peak (a) isolated by RP-HPLC, indicated that all three Thr residues are glycosylated, while peak (b) contained two GalNAc residues at Thr-3 and Thr-4. Peak (d), which constituted the major peak in the HPLC, consisted of the peptide glycosylated at Thr-2. Thus, it is clear that peak (d) is not the precursor of peak (b). Amino acid sequencing of the minor peaks corresponding to peptides with one or two GalNAc residues, namely, peaks (c) and (e), was unsuc- cessful because of their minute quantity. According to their retention times, peaks (e) and (c) are likely to contain FITC– PTTT*PLK and FITC–PT*TT*PLK, respectively, although the possibility that they are FITC–PTT*TPLK and FITC–PT*T*TPLK, cannot be excluded. The presence of the three major products in this incubation mixture can be explained by the unique acceptor specificity of pp-GalNAc-T4, which is different from the activities of pp-GalNAc-T1, T2, or T3 on FITC–PTTTPLK, as repor- ted previously [38]. Fig. 1. Elution profiles of products separated by RP-HPLC after incu- bation of FITC–PTTTPLK peptide with recombinant pp-GalNAc-T4 for the indicated periods. Ó FEBS 2002 Regulation of peptide O-glycosylation (Eur. J. Biochem. 269) 6175 Ability of partially glycosylated FITC–PTTTPLK with GalNAc to act as acceptor substrate for all four isozymes To understand further the regulation of GalNAc transfer to consecutive Thr residues in a mucin, acceptor specificities should be investigated with a glycopeptide whose Thr residues have already been partly occupied. Thus, the effects of prior attachment of GalNAc residues to this peptide on the activities of pp-GalNAc-T1, T2, T3 or T4 were examined. We enzymatically synthesized four GalNAc peptides with one or two GalNAc residues. Using these four glycopeptides and FITC–PTTTPLK as acceptors (all at a final concentration of 5 l M ), GalNAc-T assays in a 100- lL reaction mixture with 0.2 U pp-GalNAc-T1, T2, T3, or T4 were performed for 16 h. Incubation products were subjected to RP-HPLC (Fig. 4). The separated fractions were concentrated and analysed by MALDI-TOF MS and theresultsaresummarizedin 1 Table 1. As we had reported previously, a maximum of two, one or three GalNAc residues was transferred onto the ungly- cosylated FITC–PTTTPLK by pp-GalNAc-T1, T2 or T3, respectively [38]. When FITC–PT*TTPLK was used as an acceptor with pp-GalNAc-T1, T2, T3, or T4, 5.1%, 3.2%, 23.8%, and 10.8% of the products bore an additional GalNAc residue, respectively, while 0%, 3.4%, 3.7%, and 3.4% were fully glycosylated, respectively. When FITC–PT*TT*PLK was used as an acceptor substrate, incorporation of an additional GalNAc residue did not significantly occur with any of the pp-GalNAc-T isozymes. When FITC–PTTT*PLK was used as an accep- tor, glycopeptide products with an additional GalNAc constituted 65.1%, 19.0%, 16.3%, and 10.3% of the total products for pp-GalNAc-T1, T2, T3 or T4, respectively. The product resulting from the action of pp-GalNAc-T1 was FITC–PT*TT*PLK and the proportion of this product was relatively high partly because it was not converted further. pp-GalNAc-T2, T3, or T4 efficiently converted FITC–PTTT*PLK into the fully glycosylated form. Fig. 2. Representative profiles of MALDI-TOF MS of FITC– PTTTPLK peptide glycosylated by recombinant pp-GalNAc-T4 and separated by RP-HPLC. Mass indicates the (M + H) + form. The profiles a–f represent the materials retrieved from peaks a–f indicated in Fig. 1. (a) The predicted mass (1755.9) corresponds to FITC– PTTTPLK peptide with three attached GalNAc residues. (b and c) The predicted mass (1552.7) corresponds to FITC–PTTTPLK peptide with two attached GalNAc residues. (d and e) The predicted mass (1349.5) corresponds to FITC–PTTTPLK peptide with a single attached GalNAc residue. (f) The predicted mass (1146.3) corresponds to FITC–PTTTPLK peptide with no GalNAc residue. Fig. 3. Profiles of amino acid sequencing chromatograms of FITC– PTTTPLK peptide and its major derivatives glycosylated by recombin- ant pp-GalNAc-T4. (A) The profile of FITC–PTTTPLK with three GalNAc residues attached [peak (a) in Fig. 1]. (B) FITC–PTTTPLK peptide with two GalNAc residues attached [peak (b) in Fig. 1]. (C) FITC–PTTTPLK peptide with a single GalNAc residue attached [peak (d) in Fig. 1]. (D) Untreated FITC–PTTTPLK peptide [peak (f) in Fig. 1]. Asterisks indicate phenylthiohydantoin (PTH)-derivatized a-GalNAc-Thr, which was detected as a pair of peaks. Fig. 4. Elution profiles of products separated by RP-HPLC after incu- bation of FITC–PTTTPLK peptide or its glycosylated derivatives with recombinant pp-GalNAc-T1, T2, T3, or T4 for 16 h. Acceptor sub- strates were as follows: (A) FITC–PTTTPLK; (B) FITC–PT*TTPLK; (C) FITC–PT*TT*PLK; (D) FITC–PTTT*PLK; (E) FITC– PTT*T*PLK (GalNAc-Thr was indicated by T*). Broken lines indi- cate the retention time of each substrate. 6176 H. Takeuchi et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Two products containing two GalNAc residues, namely, FITC–PT*TT*PLK and FITC–PTT*T*PLK, were both generated by the action of pp-GalNAc-T2, T3, or T4. FITC–PTT*T*PLK was the apparent intermediate product to be converted into the fully glycosylated form because FITC–PTT*T*PLK was efficiently converted to the fully glycosylated form by pp-GalNAc-T2, T3, or T4, as shown in Table 1. Effects of galactosylation of GalNAc residues at Thr-2 or the Thr-4 Vicinal effects of attachment of a Gal residue to GalNAc at the first Thr residue (Thr-2) or the third Thr residue (Thr-4) were investigated. Using FITC–PT*TTPLK and FITC– PTTT*PLK as acceptors, two glycopeptides with Galb1– 3GalNAc at Thr-2 or Thr-4 were prepared. The structures of these glycopeptides, FITC–PT*°TTPLK and FITC– PTTT*°PLK (T*° indicates Galb1–3GalNAca-Thr), were confirmed by MALDI-TOF MS, by their binding to peanut agglutinin specific for Galb1–3GalNAc, and sensitivity to b-galactosidase from Bacillus circulans specific for 1–3 linked b-galactoside [45]. Using four glycopeptides (FITC– PT*TTPLK, FITC–PT*°TTPLK, FITC–PTTT*PLK and FITC–PTTT*°PLK) as acceptors, GalNAc-T assays were Table 1. Relative quantity of glycopeptides formed after incubation of FITC-PTTTPLK with pp-GalNAc-T1, T2, T3 or T4 and UDP-GalNAc for 16 h. Acceptor Enzyme Retention time (min) Number of GalNAc attached Percent of total products PTTTPLK T1 23.1 2 22.4 23.9 1 59.8 24.3 1 15.0 25.0 0 2.8 T2 21.8 3 3.1 23.1 2 2.1 23.8 1 71.5 24.2 1 16.5 24.9 0 6.7 T3 21.9 3 68.6 22.6 2 10.4 23.1 2 2.8 23.8 1 4.5 24.2 1 13.6 T4 21.9 3 30.2 22.7 2 5.4 23.1 2 1.9 23.8 1 29.1 24.2 1 14.6 24.9 0 18.8 PT*TTPLK T1 23.2 1 5.1 24.0 0 94.9 T2 21.8 2 3.3 23.0 1 3.2 23.7 0 93.4 T3 21.8 2 3.7 23.0 1 23.8 23.7 0 72.4 T4 21.9 2 3.3 22.8 1 7.3 23.2 1 3.4 23.9 0 85.8 PT*TT*PLK T1 23.7 0 100 T2 22.0 1 1.6 23.2 0 98.4 T3 21.9 1 2.1 23.0 0 97.9 T4 22.0 1 4.7 23.1 0 95.3 PTTT*PLK T1 23.3 1 65.1 24.1 0 34.9 T2 22.0 2 72.8 22.8 1 10.4 23.2 1 8.5 23.9 0 8.3 T3 22.0 2. 72.7 22.7 1 12.6 23.1 1 3.7 23.9 0 11.0 T4 21.8 2 82.2 22.6 1 6.3 23.0 1 3.9 23.7 0 7.5 Table 1. (Continued). Acceptor Enzyme Retention time (min) Number of GalNAc attached Percent of total products PTT*T*PLK T1 21.8 1 8.2 22.5 0 91.8 T2 21.9 1 92.8 22.6 0 7.2 T3 22.0 1 87.4 22.7 0 12.5 T4 22.0 1 93.4 22.7 0 6.6 Fig. 5. Elution profiles of products separated by RP-HPLC after incu- bation of glycosylated derivatives of FITC–PTTTPLK with recombinant pp-GalNAc-T1, T2, T3, or T4 for 16 h. Acceptor substrates were as follows: (A) FITC–PT*TTPLK; (B) FITC–PT*°TTPLK; (C) FITC– PTTT*PLK; (D) FITC–PTTT*°PLK (GalNAc-Thr and Galb1– 3GalNAc-Thr were indicated by T* and T*°, respectively). Broken lines indicate the retention time of each substrate. Ó FEBS 2002 Regulation of peptide O-glycosylation (Eur. J. Biochem. 269) 6177 performed in a 100-lL reaction mixture with 0.2 U pp-GalNAc-T1, T2, T3, or T4. After a 16-h incubation products were subjected to RP-HPLC (Fig. 5). The peak fractions were pooled, concentrated by evaporation, and analysed by MALDI-TOF MS. The results indicated that the influence of prior Gal transfer on the vicinal GalNAc transfer depended on the site and the isozyme type of pp-GalNAc-T 1 as summarized in Table 2. Gal transfer to GalNAc on Thr-2 did not increase the efficiency of GalNAc-incorporation by pp-GalNAc-T1. pp-GalNAc T2, 3, or 4, transferred GalNAc to a very low extent when Thr-2 was occupied by GalNAc or Galb1–3GalNAc. The effect of Gal transfer to the GalNAc attached to the Thr-4 was very slight as far as pp-GalNAc-T1 is concerned. As stated in the previous sections, greater proportions of Thr-2 and Thr-3 residues in FITC–PTTT*PLK received transfer of GalNAc residues with pp-GalNAc-T2, T3, or T4 than FITC–PTTTPLK. The first site of the incorporation was apparently Thr-3 then to Thr-2. This vicinal enhancing effect was abrogated by the addition of a Gal residue to the GalNAc residue in FITC–PTTT*PLK. The position of the GalNAc incorporation was Thr-2 resulting in the formation of FITC-PT*TT*°PLK in the case of the action of pp-GalNAc-T1 and T2 according to the protein sequencing analysis (Fig. 6). Effects of modification of N terminals of PTTTPLK Differences in the incorporation of GalNAc into underiva- tized and the fluorescein-labeled PTTTPLK by pp- GalNAc-T2 or T3 were compared. The products from the underivatized peptide were reacted with FITC and applied to RP-HPLC. The number of GalNAc incorporated residues was estimated by MALDI-TOF MS. A glycopep- tide with one GalNAc residue was the predominant product after 16 h incubation with pp-GalNAc-T2. Peptide sequen- cing analysis indicated that the GalNAc residue was attached to Thr-2. Four peaks of glycopeptides with three, two, two, or one GalNAc residues were identified in the reaction mixture with pp-GalNAc-T3. By peptide sequen- cing analysis, FITC–PTT*T*PLK and FITC–PT*TT*PLK were identified as indicated in Fig. 7A. These results indicated that modification of the N terminus of acceptor peptides with FITC had no significant effect on the order or maximum number of attachment of GalNAc residues. When FITC–PTTTPLK was used as an acceptor and incubated with pp-GalNAc-T3 for 16 h, a glycopeptide with three GalNAc residues was the major product, whereas PTTT*PLK was the major product from underivatized peptide incubated under the same conditions. Peptide AAAAAAPTTTPLK was synthesized and labelled with FITC. FITC–AAAAAAPTTTPLK was Table 2. Relative quantity of glycopeptides formed after incubation of FITC-PTTTPLK containing a Galb1-3GalNAca residue with pp-Gal- NAc-T1, T2, T3 or T4 and UDP-GalNAc for 16 h. Acceptor Enzyme Retention time (min) Number of GalNAc attached Percent of total products PT*TTPLK T1 23.2 1 1.5 24.0 0 98.5 T2 21.9 2 2.2 23.1 1 0.8 23.8 0 97.0 T3 21.8 2 2.5 23.0 1 19.7 23.7 0 77.8 T4 21.8 2 2.6 22.6 1 8.6 23.0 1 2.0 23.7 0 86.7 PTTT*PLK T1 23.3 1 68.6 24.0 0 31.4 T2 21.8 2 74.1 22.6 1 6.8 23.0 1 4.8 23.8 0 14.2 T3 21.9 2 71.9 22.6 1 12.1 23.0 1 3.7 23.8 0 12.3 T4 21.8 2 80.7 22.6 1 6.6 23.8 0 12.7 PT*°TTPLK T1 22.9 0 100 T2 23.0 0 100 T3 22.4 1 6.0 23.1 0 94.0 T4 21.5 1 10.6 23.1 0 89.4 PTTT*°PLK T1 22.4 1 81.0 23.4 0 19.0 T2 22.4 1 33.2 23.4 0 66.8 T3 22.5 1 8.5 23.5 0 91.4 T4 22.6 1 9.6 23.5 0 90.4 Fig. 6. Profiles of amino acid sequencing chromatograms of products after incubation of FITC–PTTT*°PLK peptides with (A) recombinant pp-GalNAc-T1 or (B) pp-GalNAc-T2 and those of untreated substrates (C) FITC–PTTT*°PLK and (D) FITC–PT*°TTPLK. Asterisks indi- cate PTH-derivatized a-GalNAc-Thr, which is detected as a pair of peaks. 6178 H. Takeuchi et al. (Eur. J. Biochem. 269) Ó FEBS 2002 incubated with pp-GalNAc-T2 or T3 for 16 h. Elution profiles of the products on the RP-HPLC were shown in Fig. 7B. Peptide sequencing analysis indicated that pp-GalNAc-T2 transferred one GalNAc residue to Thr-8, the first Thr in the Thr triad. The first GalNAc residue transferredbypp-GalNAc-T3seemedtoattachtoThr-8 and Thr-10 and a product FITC–AAAAAAPT*TTPLK did not seem to be further modified. The ratio of FITC– T*TTPLK to FITC–PT*T*T*PLK was smaller than the ratio of FITC–AAAAAAPT*TTPLK to FITC–AAAAA APT*T*T*PLK. DISCUSSION We hypothesize that the arrangement of O-glycans on consecutive Ser/Thr residues in mucins and mucin-like cell surface receptors generate structural motifs. If this really is the case, then the biosynthetic pathway of O-glycans on consecutive Ser/Thr should strictly be regulated regarding where and what order the O-glycosylation occurs. In the study presented here, the initial sites of O-glycosylation and the subsequent order of attachment of GalNAc to a sequence containing three consecutive Thr residues by four glycosyltransferase isoforms were investigated. The prefer- ential site of glycosylation in FITC–PTTTPLK and parti- ally modified peptides by the action of each pp-GalNAc-T are summarized in Fig. 8. The initial site of GalNAc attachment to FITC–PTTTPLK with pp-GalNAc-T1, T2, and T3 was predominantly Thr-2, Thr-2, and Thr-4, respectively, as described previously [38]. pp-GalNAc-T4 appears to have two preferential initial glycosylation sites, which results in the formation of FITC–PT*TTPLK [peak (d) in Fig. 2] and FITC–PTTT*PLK [putative sequence of peak (e) in Fig. 2]. Other investigators using various synthetic peptide acceptors have already reported that each pp-GalNAc-T has preference for different flanking amino acid sequences surrounding the Thr residue. It has not been demonstrated that the order of GalNAc incorporation into three Thr and/or Ser residues in the vicinity is strictly determined. Most of the previous studies focused on probability that one site was more likely to be glycosylated Fig. 7. Elution profiles of (A) PTTTPLK and (B) FITC–AAA AAAPTTTPLK peptides. (A) Elution profiles of PTTTPLK peptides incubated with pp-GalNAc-T2 (a), pp-GalNAc-T3 (b) or buffer alone (c), for 16 h prior to labelling with FITC on the RP-HPLC. The estimated structures of the glycopeptides corresponding to the peaks are depicted schematically. (B) Elution profiles of FITC–AAA AAAPTTTPLK peptides incubated with pp-GalNAc-T2 (a), pp-GalNAc-T3 (b) or buffer alone (c), for 16 h on the RP-HPLC. The estimated structures of the glycopeptides corresponding to the peaks are depicted schematically. Fig. 8. Summary of actions of four recombinant pp-GalNAc-Ts toward TTT stretch in FITC–PTTTPLK peptide and its partially glycosylated derivatives. (A) pp-GalNAc-T1 (B) pp-GalNAc-T2 (C) pp-GalNAc- T3, and (D) pp-GalNAc-T4 are shown. The products are indicated by shaded squares according to the proportion among whole products. d, Gal residues in acceptor substrates; h, GalNAc residues in acceptor substrates. The percentage of GalNAc incorporated was calculated based on the total amount of acceptor substrates. Ó FEBS 2002 Regulation of peptide O-glycosylation (Eur. J. Biochem. 269) 6179 over another site. Our present results show that the order, i.e. which Thr is glycosylated first and which Thr is second, is determined almost exclusively when a peptide sequence and a pp-GalNAc-T are fixed. The preferential pathways of O-glycosylation of a peptide containing three consecutive Thr residues (FITC–PTTTPLK) are indicated in Fig. 9. Very interestingly and importantly, the preferential order did not change when a 10-fold concentration of the acceptor substrates were used and additional components were not generated when the incubation period was extended up to 48 h with an addition of the same amounts of pp-GalNAc-Ts. Previous reports indicated that Pro residues positively influenced GalNAc incorporation into a particular Thr residue [33]. Statistical studies on various peptides contain- ing O-glycans suggested that Pro residues located at )1and +3 positions relative to the glycosylation site had positive effects, although the pp-GalNAc-T having this preference was not clear [2–5,8]. The FITC–PTTTPLK used in the present study have two Pro residues, which potentially provide positive effects on Thr-2 according to the previous reports [32]. These Pro residues may contribute to the initial glycosylation site by pp-GalNAc-T1 and T2 but obviously not by pp-GalNAc-T3. The specificity of each pp-GalNAc- T seems to be unique toward consecutive Thr residues and their partially glycosylated derivatives. For example, when partially glycosylated FITC–PTTTPLK were used as acceptor substrates, the effect of the attached GalNAc residues on the activity of pp-GalNAc-T1 was obvious. Although the initial glycosylation site for pp-GalNAc-T1 is Thr-2, this isozyme could not glycosylate Thr-2 of FITC– PTT*T*PLK. Thus, the ability of pp-GalNAc-T1 to transfer GalNAc onto a Thr immediately upstream ()1) of an existing GalNAc-Thr residue is likely to be suppressed. Neither a GalNAc residue nor a Galb1–3GalNAc residue at Thr-4 of FITC–PTTTPLK significantly influenced the activity of pp-GalNAc-T1 which could transfer one GalNAc residue to Thr-2, resulting in the formation of FITC–PT*TT*PLK or FITC–PT*TT*°PLK. pp-GalNAc-T2, T3, and T4 behaved differently from pp-GalNAc-T1 in that the presence of GalNAc-Thr at the penultimate position (+1) promoted their efficacy. Thus, FITC–PTTT*PLK could be rapidly converted to the fully glycosylated form by all of these isozymes via the interme- diate FITC–PTT*T*PLK. The preferential glycosylation of the peptide with one GalNAc residue was inhibited by the addition of a Gal residue to this GalNAc residue in FITC– PTTT*PLK. Several issues regarding the use of a relatively short peptide with fluoresceine at the N terminus as a substrate should be carefully evaluated. The kinetic parameters reported for three FITC–conjugated peptides in our previ- ous publication were not distinct from those for unmodified MUC2 peptide (PTTTPISTTTMVTPTPTPTC) reported by Wandall and coworkers [43]. We also examined the specificity of detergent-soluble microsome fraction of human colon carcinoma LS174T cells towards larger GalNAc-glycosylated peptides than FITC–PTTTPLK used in the present study [37]. RT/PCR and immunocytological analysis indicated that LS174T cells expressed at least pp-GalNAc-T1, T2, T3, and T4. In vitro GalNAc-T assays were performed using FITC–PTTT*PITTTTK, FITC– PT*TTPITTTTK, FITC–PTT*T*PITT*T*TK, and FITC–PT*TTPIT*T*T*TK as substrates. Similar results on the specificity to that of our present results were also observed in these assays, although the microsome fraction contained more than two pp-GalNAc-Ts. FITC– PTTT*PITTTTK were efficiently glycosylated and conver- ted to FITC–PT*T*T*PIT*T*T*T*K. When FITC– PTTT*PITTTTK was used as a substrate, the order of incorporation of GalNAc residues was restricted in the formation of PT*T*T*P. Within this motif, PTTT*P, a GalNAc residue was incorporated at Thr-3 at first, and after that, one more GalNAc residue was incorporated at Thr-2. Similarly, FITC–PTT*T*PITT*T*TK were converted to fully glycosylated FITC–PT*T*T*PIT*T*T*T*K. Thus, the presence of extra C-terminal sequence did not seem to influence the order of GalNAc incorporation. We did not examine the effect of two Pro residues on specificity of pp-GalNAc-Ts by mutation analysis, although Pro residues in a flanking sequence may influence the initial GalNAc- attachment site in a polypeptide as mentioned above. There are few previous reports regarding the acceptor specificity of GalNAc transfer by pp-GalNAc-T isozymes on unglycosylated and partially glycosylated sequences. Hanisch and coworkers reported that the addition of a GalNAc residue by pp-GalNAc-T isozymes, in particular pp-GalNAc-T2, to Ser-16 in the tandem repeat of the MUC1 mucin was accelerated when the adjacent Thr-17 Fig. 9. Putative pathways of GalNAc incorporation into FITC– PTTTPLK by the action of pp-GalNAc-T1 (A), pp-GalNAc-T2 (B), pp- GalNAc-T3 (C), and pp-GalNAc-T4 (D). *, GalNAc residues; s,Gal residues; bold arrows, reactions in which > 50% GalNAc was incor- porated; broken arrows, the reactions in which < 50% GalNAc was incorporated; shaded letters, hypothetical glycosylation products which were not detected in the present investigations. 6180 H. Takeuchi et al. (Eur. J. Biochem. 269) Ó FEBS 2002 residue was glycosylated [39,40]. Bennett and coworkers also reported that the catalytic activity of pp-GalNAc-T4 with a peptide corresponding to a MUC2 sequence was enhanced fivefold by prior incorporation of 1–2 mole of GalNAc by pp-GalNAc-T2 [23]. However, the structural characteristics responsible for this effect were not elucidated. In the study by Bennett and coworkers with a MUC1 peptide, pp-GalNAc-T4 preferentially transferred GalNAc onto a Ser residue adjacent to a glycosylated Thr [21]. Thus, our findings are consistent with prior reports. In addition, we are also able now to delineate the structural basis that regulates GalNAc incorporation into three consecutive Thr residues. The present work indicates that GalNAc attachment to one of three consecutive Thr residues is an important factor that negatively or positively affects subsequent transfers of GalNAc residues. The mechanisms behind this remain to be explored in detail, but factors should include sequence context, influence of GalNAc residues to conformation and recognition of acceptor and modulation of kinetic proper- ties potentially through the lectin domain. GalNAc residues attached to the peptides via the lectin motifs contained within their sequences, as has been postulated previously [46]. Hagen and coworkers showed that mutations in the C-terminal ricin-like lectin motif of murine pp-GalNAc-T1 did not alter its catalytic properties [27]. Attachment of Gal to GalNAc at Thr-4 of FITC– PTTTPLK inhibited the transfer of GalNAc to Thr-3 by pp-GalNAc-T2, T3, and T4. This suggests that pp-Gal NAc-T isozymes may recognize directly GalNAc residues in the vicinity. It is an interesting possibility that GalNAc-Ts compete with glycosyltransferases responsible for the extension of O-glycans. Brockhausen and coworkers showed that galactose incorporation by UDP-Gal:glyco- protein-GalNAc 3-b- D -galactosyltransferase (core 1 b3-Gal-T) purified from rat liver became less efficient when acceptor peptides were heavily converted with GalNAc [47,48]. From results to determine the glycosylation pattern of porcine submaxillary mucin tandem repeats, Gerken and coworkers suggested that local glycopeptide structures, such as GalNAc density, regulate the in vivo elongation of the O-glycan by the porcine core 1 b3-Gal-T [44,49,50]. Although many factors potentially modulate attachment and elongation of O-glycans remain unknown, coordinated actions of pp-GalNAc-Ts and Gal-Ts should play a major role in generating a variety of structural motifs on consecu- tive Thr residues. The present study suggests that a decrease in galactosylation of GalNAc residues in consecutive Thr residues in mucins does not only expose GalNAc residues but also promotes the formation of GalNAc clusters. This should result in an efficient binding to parasitic protozoa such as Entamoeba histolytica through their lectins specific for clusters of O-linked GalNAc residues [51]. The O-glycan structures of a mucin-like molecule, CD43, were shown to be modulated upon the exposure of epithelial cells to bac- terial lipopolysaccharides [52], which appeared to be similar to the change observed in T cells [53]. However, the present report is one of very few to show that glycan extension directly affects the glycosylation of backbone peptides. In conclusion, we show that a peptide mimicking a portion of MUC2 containing three consecutive Thr residues (FITC–PTTTPLK) can be glycosylated by pp-GalNAc-T1, T2, T3, T4, or combinations of these isozymes, into a variety of differently glycosylated peptides through their unique acceptor specificities. Each isozyme was unique in the specificity not only to this peptide but also to the peptides with one or two GalNAc residues or Galb1–3GalNAc residues at different positions. ACKNOWLEDGEMENT This work was supported by grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (07407063, 09254101, 11557180, and 11672162), the Research Association for Biotechnology, the Program for the Promotion of Basic Research Activities for Innovative Biosciences, and the Danish Cancer Society. We thank C. Hiraiwa for her assistance in preparing this manuscript. REFERENCES 1. Brockhausen, I. (1999) Pathways of O-glycan biosynthesis in cancer cells. Biochim. Biophy. Acta 1473, 67–95. 2. Wilson, I.B., Gavel, Y. & von Heijne, G. (1991) Amino acid dis- tributions around O-linked glycosylation sites. Biochem. J. 275, 529–534. 3. O’Connell,B.C.,Hagen,F.K.&Tabak,L.A.(1992)Theinfluence of flanking sequence on the O-glycosylation of threonine in vitro. J.Biol.Chem.267, 25010–25018. 4. O’Connell,B.,Tabak,L.A.&Ramasubbu,N.(1991)Theinflu- ence of flanking sequences on O-glycosylation. Biochem. Biophys. Res. Commun. 180, 1024–1030. 5. Hansen, J.E., Lund, O., Tolstrup, N., Gooley, A.A., Williams, K.L. & Brunak, S. (1998) NetOglyc: prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility. Glycoconjugate J. 15, 115–130. 6. Chou, K.C., Zhang, C.T., Kezdy, F.J. & Poorman, R.A. (1995) A vector projection method for predicting the specificity of Gal- NAc-transferase. Proteins 21, 118–126. 7.Elhammer,A.P.,Poorman,R.A.,Brown,E.,Maggiora,L.L., Hoogerheide, J.G. & Kezdy, F.J. (1993) The specificity of UDP- GalNAc: polypeptide N-acetylgalactosaminyltransferase as in- ferred from a database of in vivo substrates and from the in vitro glycosylation of proteins and peptides. J. Biol. Chem. 268, 10029– 10038. 8. Hansen, J.E., Lund, O., Engelbrecht, J., Bohr, H. & Nielsen, J.O. (1995) Prediction of O-glycosylation of mammalian proteins: specificity patterns of UDP-GalNAc: polypeptide N-acet- ylgalactosaminyltransferase. Biochem. J. 308, 801–813. 9. Stadie, T.R., Chai, W., Lawson, A.M., Byfield, P.G. & Hanisch, F.G. (1995) Studies on the order and site specificity of GalNAc transfer to MUC1 tandem repeats by UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase from milk or mammary carci- noma cells. Eur. J. Biochem. 229, 140–147. 10. Nishimori, I., Johnson, N.R., Sanderson, S.D., Perini, F., Mountjoy, K., Cerny, R.L., Gross, M.L. & Hollingsworth, M.A. (1994) Influence of acceptor substrate primary amino acid sequence on the activity of human UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyltransferase. Studies with the MUC1 tandem repeat. J. Biol. Chem. 269, 16123–16130. 11. Nishimori, I., Perini, F., Mountjoy, K.P., Sanderson, S.D., Johnson, N., Cerny, R.L., Gross, M.L., Fontenot, J.D. & Hollingsworth, M.A. (1994) N-acetylgalactosamine glycosylation of MUC1 tandem repeat peptides by pancreatic tumor cell extracts. Cancer Res. 54, 3738–3744. 12. Gendler, S.J. & Spicer, A.P. (1995) Epithelial mucin genes. Annu. Rev. Physiol. 57, 607–634. 13. Kim, Y.S., J.G. Jr & Brockhausen, I. (1996) Mucin glycoproteins in neoplasia. Glycoconjugate J. 13, 693–707. 14. Iida, S., Yamamoto, K. & Irimura, T. (1999) Interaction of human macrophage C-type lectin with O-linked N-acetylgalactosamine Ó FEBS 2002 Regulation of peptide O-glycosylation (Eur. J. Biochem. 269) 6181 residues on mucin glycopeptides. J.Biol.Chem.274, 10697– 10705. 15. Simmons, D.L., Satterthwaite, A.B., Tenen, D.G. & Seed, B. (1992) Molecular cloning of a cDNA encoding CD34, a sialo- mucin of human hematopoietic stem cells. J. Immunol. 148,267– 271. 16. Streuli, M., Hall, L.R., Saga, Y., Schlossman, S.F. & Saito, H. (1987) Differential usage of three exons generates at least five different mRNAs encoding human leukocyte common antigens. J.Exp.Med.166, 1548–1566. 17. Holness, C.L. & Simmons, D.L. (1993) Molecular cloning of CD68, a human macrophage marker related to lysosomal glyco- proteins. Blood 81, 1607–1613. 18.Irimura,T.,Denda,K.,Iida,S.,Takeuchi,H.&Kato,K. (1999) Diverse glycosylation of MUC1 and MUC2: potential significance in tumor immunity. J. Biochem. (Tokyo) 126,975– 985. 19. White, T., Bennett, E.P., Takio, K., Sorensen, T., Bonding, N. & Clausen, H. (1995) Purification and cDNA cloning of a human UDP-N-acetyl-a-D-galactosamine: polypeptide N-acetylgalactos- aminyltransferase. J.Biol.Chem.270, 24156–24165. 20. Zara, J., Hagen, F.K., Ten Hagen, K.G., Van Wuyckhuyse, B.C. & Tabak, L.A. (1996) Cloning and expression of mouse UDP- GalNAc: polypeptide N-acetylgalactosaminyltransferase-T3. Biochem. Biophys. Res. Commun. 228, 38–44. 21. Bennett, E.P., Hassan, H., Mandel, U., Mirgorodskaya, E., Roepstorff, P., Burchell, J., Taylor-Papadimitriou, J., Hollings- worth,M.A.,Merkx,G.,vanKessel,A.G.,Eiberg,H.,Steffensen, R. & Clausen, H. (1998) Cloning of a human UDP-N-acetyl-a-D- Galactosamine: polypeptide N-acetylgalactosaminyltransferase that complements other GalNAc-transferases in complete O-gly- cosylation of the MUC1 tandem repeat. J. Biol. Chem. 273, 30472–30481. 22. Bennett, E.P., Hassan, H., Mandel, U., Hollingsworth, M.A., Akisawa, N., Ikematsu, Y., Merkx, G., van Kessel, A.G., Olofsson, S. & Clausen, H. (1999) Cloning and characterization of a close homologue of human UDP-N-acetyl-a- D -galactosamine: Polypeptide N-acetylgalactosaminyltransferase-T3, designated GalNAc-T6. Evidence for genetic but not functional redundancy. J. Biol. Chem. 274, 25362–25370. 23. Bennett, E.P., Hassan, H., Hollingsworth, M.A. & Clausen, H. (1999) A novel human UDP-N-acetyl- D -galactosamine: polypep- tide N-acetylgalactosaminyltransferase, GalNAc-T7, with specifi- city for partial GalNAc-glycosylated acceptor substrates. FEBS Lett. 460, 226–230. 24. Bennett, E.P., Hassan, H. & Clausen, H. (1996) cDNA cloning and expression of a novel human UDP-N-acetyl-a-D-galactos- amine. Polypeptide N-acetylgalactosaminyltransferase, GalNAc- T3. J.Biol.Chem.271, 17006–17012. 25. Hagen, F.K., Ten Hagen, K.G., Beres, T.M., Balys, M.M., VanWuyckhuyse, B.C. & Tabak, L.A. (1997) cDNA cloning and expression of a novel UDP-N-acetyl-D-galactosamine: polypep- tide N-acetylgalactosaminyltransferase. J.Biol.Chem.272, 13843– 13848. 26. Ten Hagen, K.G., Hagen, F.K., Balys, M.M., Beres, T.M., Van Wuyckhuyse, B. & Tabak, L.A. (1998) Cloning and expression of a novel, tissue specifically expressed member of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase family. J. Biol. Chem. 273, 27749–27754. 27. Hagen, F.K., Hazes, B., de Raffo, R., Sa, D. & Tabak, L.A. (1999) Structure-function analysis of the UDP-N-acetyl-D-galacto- samine: polypeptide N-acetylgalactosaminyltransferase. Essential residues lie in a predicted active site cleft resembling a lactose repressor fold. J. Biol. Chem. 274, 6797–6803. 28. Ten Hagen, K.G., Tetaert, D., Hagen, F.K., Richet, C., Beres, T.M., Gagnon, J., Balys, M.M., Van Wuyckhuyse, B., Bedi, G.S., Degand, P. & Tabak, L.A. (1999) Characterization of a UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase that displays glycopeptide N-acetylgalactosaminyltransferase activity. J.Biol.Chem.274, 27867–27874. 29. White, K.E., Lorenz, B., Evans, W.E., Meitinger, T., Strom, T.M. & Econs, M.J. (2000) Molecular cloning of a novel human UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase, GalNAc-T8, and analysis as a candidate autosomal dominant hypophosphatemic rickets (ADHR) gene. Gene 246, 347–356. 30. Simmons, A.D., Musy, M.M., Lopes, C.S., Hwang, L.Y., Yang, Y.P. & Lovett, M. (1999) A direct interaction between EXT proteins and glycosyltransferases is defective in hereditary multiple exostoses. Hum. Mol. Genet. 8, 2155–2164. 31.Toba,S.,Tenno,M.,Konishi,M.,Mikami,T.,Itoh,N.& Kurosaka, A. (2000) Brain-specific expression of a novel human UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase (GalNAc-T9). Biochim. Biophys. Acta 1493, 264–268. 32. Yoshida, A., Suzuki, M., Ikenaga, H. & Takeuchi, M. (1997) Discovery of the shortest sequence motif for high level mucin-type O-glycosylation. J. Biol. Chem. 272, 16884–16888. 33. Hennebicq, S., Tetaert, D., Soudan, B., Boersma, A., Briand, G., Richet, C., Gagnon, J. & Degand, P. (1998) Influence of the amino acid sequence on the MUC5AC motif peptide O-glycosylation by human gastric UDP-GalNAc: polypeptide N-acetylgalacto- saminyltransferase(s). Glycoconjugate J. 15, 275–282. 34. Tetaert, D., Richet, C., Gagnon, J., Boersma, A. & Degand, P. (2001) Studies of acceptor site specificities for three members of UDP-GalNAc: N-acetylgalactosaminyltransferases by using a synthetic peptide mimicking the tandem repeat of MUC5AC. Carbohydr. Res. 333, 165–171. 35. Tetaert, D., Ten Hagen, K.G., Richet, C., Boersma, A., Gagnon, J. & Degand, P. (2001) Glycopeptide N-acetylgalactosaminyl- transferase specificities for O-glycosylated sites on MUC5AC mucin motif peptides. Biochem. J. 357, 313–320. 36. Iida,S.,Takeuchi,H.,Kato,K.,Yamamoto,K.&Irimura,T. (2000) Order and maximum incorporation of N-acet- ylgalactosamine into threonine residues of MUC2 core peptide with microsome fraction of human colon carcinoma LS174T cells. Biochem. J. 347, 535–542. 37. Kato, K., Takeuchi, H., Miyahara, N., Kanoh, A., Hassan, H., Clausen, H. & Irimura, T. (2001) Distinct orders of GalNAc incorporation into a peptide with consecutive threonines. Biochem. Biophys. Res., Commun. 287, 110–115. 38. Iida, S., Takeuchi, H., Hassan, H., Clausen, H. & Irimura, T. (1999) Incorporation of N-acetylgalactosamine into consecutive threonine residues in MUC2 tandem repeat by recombinant human N-acetyl-D-galactosamine transferase-T1, T2 and T3. FEBS Lett. 449, 230–234. 39. Hanisch, F.G., Muller, S., Hassan, H., Clausen, H., Zachara, N., Gooley, A.A., Paulsen, H., Alving, K. & Peter-Katalinic, J. (1999) Dynamic epigenetic regulation of initial O-glycosylation by UDP-N-acetylgalactosamine: peptide N-acetylgalactosaminyl- transferases. Site-specific glycosylation of MUC1 repeat peptide influences the substrate qualities at adjacent or distant Ser/Thr positions. J.Biol.Chem.274, 9946–9954. 40.Hanisch,F.G.,Reis,C.A.,Clausen,H.&Paulsen,H.(2001) Evidence for glycosylation-dependent activities of polypeptide N-acetylgalactosaminyltransferases rGalNAc-T2 and -T4 on mucin glycopeptides. Glycobiology 11, 731–740. 41. Hassan, H., Reis, C.A., Bennett, E.P., Mirgorodskaya, E., Roepstorff, P., Hollingsworth, M.A., Burchell, J., Taylor-Papad- imitriou, J. & Clausen, H. (2000) The lectin domain of UDP-N- acetyl- D -galactosamine: polypeptide N-acetylgalactosaminyl- transferase-T4 directs its glycopeptide specificities. J. Biol. Chem. 275, 38197–38205. 42. Gum,J.R.Jr,Hicks,J.W.,Toribara,N.W.,Siddiki,B.&Kim, Y.S. (1994) Molecular cloning of human intestinal mucin (MUC2) cDNA. Identification of the amino terminus and overall sequence 6182 H. Takeuchi et al. (Eur. J. Biochem. 269) Ó FEBS 2002 [...]... specificity and polyvalent carbohydrate recognition by the Entamoeba histolytica and rat hepatic N-acetylgalactosamine/galactose lectins Glycobiology 8, 1037–1043 52 Amano, J., Morimoto, C & Irimura, T (2001) Intestinal epithelial cells express and secrete the CD43 glycoform that contains core 2 O-glycans Microbe Infect 3, 723–728 53 Fukuda, M (1991) Leukosialin, a major O-glycan-containing sialoglycoproetin... glycoprotein-N-acetyl-D-galactosamine 3-beta-D-galactosyltransferase activity synthesizing O-glycan core 1 is controlled by the amino acid sequence and glycosylation of glycopeptide substrates Eur J Biochem 221, 1039–1046 49 Gerken, T .A. , Owens, C.L & Pasumarthy, M (1998) Site- specific core 1 O-glycosylation pattern of the porcine submaxillary gland mucin tandem repeat Evidence for the modulation of glycan length by peptide... UDPN-acetyl -a- D-galactosamine: Polypeptide N-acetylgalactosaminyltransferase family, GalNAc-T1-T2, and -T3 J Biol Chem 272, 23503–23514 Gerken, T .A. , Owens, C.L & Pasumarthy, M (1997) Determination of the site- specific O-glycosylation pattern of the porcine submaxillary mucin tandem repeat glycopeptide Model proposed for the polypeptide: galnac transferase peptide binding site J Biol Chem 272, 9709–9719 Fujimoto, H., Miyasato,... & Paulsen, H (1990) Control of mucin synthesis: the peptide portion of synthetic O-glycopeptide substrates influences the activity of O-glycan core 1 UDPgalactose: N-acetyl-alpha-galactosaminylR-b 3-galactosyltransferase Biochemistry 29, 10206–10212 48 Granovsky, M., Bielfeldt, T., Peters, S., Paulsen, H., Meldal, M., Brockhausen, J & Brockhausen, I (1994) UDPgalactose: glycoprotein-N-acetyl-D-galactosamine... Ito, Y. , Sasaki, T & Ajisaka, K (1998) Purification and properties of recombinant b-galactosidase from Bacillus circulans Glycoconjugate J 15, 155–160 Imberty, A. , Piller, V., Piller, F & Breton, C (1997) Fold recognition and molecular modeling of a lectin-like domain in UDP-GalNac: polypeptide N-acetylgalactosaminyltransferases Protein Eng 10, 1353–1356 Brockhausen, I., Moller, G., Merz, G., Adermann,... Regulation of peptide O-glycosylation (Eur J Biochem 269) 6183 similarity to prepro-von Willebrand factor J Biol Chem 269, 2440–2446 Wandall, 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 (1997) Substrate specificities of three members of the human UDPN-acetyl -a- D-galactosamine:... Gerken, T .A. , Gilmore, M & Zhang, J (2002) Determination of the site- specific oligosaccharide distribution of the O-glycans attached to the porcine submaxillary mucin tandem repeat Further evidence for the modulation of O-glycans side chain structures by peptide sequence J Biol Chem 277, 7736–7751 51 Yi, D., Lee, R.T., Longo, P., Boger, E.T., Lee, Y. C., Petri,W .A Jr & Schnaar, R.L (1998) Substructural specificity. .. secrete the CD43 glycoform that contains core 2 O-glycans Microbe Infect 3, 723–728 53 Fukuda, M (1991) Leukosialin, a major O-glycan-containing sialoglycoproetin defining leukocyte differentialtion and malignancy Glycobiology 1, 347–356 . O-GalNAc incorporation into a cluster acceptor site of three consecutive threonines Distinct specificity of GalNAc-transferase isoforms Hideyuki Takeuchi 1 ,. conditions. Peptide AAAAAAPTTTPLK was synthesized and labelled with FITC. FITC–AAAAAAPTTTPLK was Table 2. Relative quantity of glycopeptides formed after incubation of FITC-PTTTPLK