2Institute of Biochemistry, Medical Faculty of the University, Joseph-Stelzmann-Str. 52, 50931 Köln, Germany, 3School of Dentistry, Department of Diagnostics, Faculty of Health Sciences, Norre Alle 20, DK 2200, Denmark, and 4Institute of Organic Chemistry, University of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany
Received on February 27, 2001; revised on April 11, 2001; accepted on April 12, 2001.
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Abstract |
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Key words: glycosyltransferases/polypeptide N-acetylgalactosaminyltransferases/O-glycosylation/glycoproteins/mucins
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Introduction |
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There are currently seven recombinant GalNAc-Ts available, which can serve to simulate in vitro the site-specific O-glycosylation of in vivo processed glycoproteins (Clausen and Bennett, 1996; Ten Hagen et al., 1998
; Bennett et al., 1998
, 1999a). Although they have not yet been fully explored, each of these enzymes may have a unique site preference with some degree of overlap between the enzyme-specific target patterns (Wandall et al., 1997
; Hanisch et al., 1999
). It is expected that suitable combinations of the enzymes and appropriate incubation conditions should allow mimicking of the in vivo situation. The relevance of in vitro data for the prediction of in vivo O-glycosylation sites has conclusively been demonstrated for rGalNAc-T3 and a specific acceptor site on the V3 loop of the HIV gp120 protein (Nehrke et al., 1998
).
Calculations using neural network algorithms and sequence information from glycoprotein databases are generally based on the principal assumption that the addition of GalNAc to Ser/Thr is ruled by amino acid sequences around the potential target site. No specific motif for O-glycosylation has been identified as an acceptor site for polypeptidyl GalNAc-Ts and, in particular, for individual isoforms. On the other hand, the vicinal and proximal amino acids (positions 4 to +4), that is, the primary sequence context, play crucial roles in determining the qualities of potential substrate positions (Elhammer et al., 1999).
Recently, it has been shown that the primary peptide sequence context is not the only determining parameter of site-specific O-glycosylation. Instead, there is evidence for a dynamic epigenetic regulation of initial GalNAc addition mediated by competition of different substrate sites and by competition of ppGalNAc-Ts with the core-specific glycosyltransferases (Hanisch et al., 1999). The finding that multiple ppGalNAc-T isoforms in some cases (e.g., high-density O-glycosylation sequences from mucin tandem repeats) can act on the same substrate (albeit often with different kinetics and/or specific acceptor sites) introduces competition and order of GalNAc attachments. In this respect we and others previously showed that GalNAc substitution at certain substrate positions can influence the glycosylation at other vicinal or proximal sites. On the other hand, a substitution with core 1 disaccharides generally depressed proximal and in some cases even distant GalNAc addition, resulting in less dense glycosylation patterns. Furthermore, some isoforms appear to exhibit strict dependence on prior attachments of GalNAc by other isoforms (Bennett et al., 1998
, 1999b; Hassan et al., 2000
).
On the basis of these considerations the present study attempted to elucidate the influences of epigenetic parameters on initial O-glycosylation, with a focus on rGalNAc-T2 and -T4 and their action on differently glycosylated acceptor peptides of the MUC1, MUC2, and MUC4 repeat domains. The activity of rGalNAc-T4 with some substrates, including the tandem repeat of MUC1, has previously been shown to be dependent on the presence of GalNAc residues on the acceptor peptide, which may mediate conformational effects on the peptide or serve in the lectin-induced triggering of transferase activity (Bennett et al., 1998; Hassan et al., 2000
). On the contrary, rGalNAc-T2 acts very effectively on nonglycosylated substrates, including the MUC1 repeat peptide. All currently known rGalNAc-Ts, however, in particular rGalNAc-T2, are characterized by a putative lectin domain in their C-terminal regions. Hence, the possibility exists that also other isoforms show glycosylation-dependent effects exerted by substrate-linked GalNAc on their activities.
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Results |
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The question arises whether the triggering effect of glycosylated substrate on rGalNAc-T4 activity is reversed by addition of galactose to the GalNAc-substituted peptides and whether similar effects can be observed with other enzyme isoforms like rGalNAc-T2. Using the corresponding couples with GalNAc- and Galß1-3GalNAc-substitution, respectively, it was revealed that rGalNAc-T2 (1) showed a consistent pattern of GalNAc incorporation into three positions, if the substrate was nonglycosylated or GalNAc substituted peptide, and (2) was inhibited with reference to distinct sites, if the substrate was Gal-GalNAc substituted (Figure 1, top). In particular, the Ser in GSTA remained unglycosylated in substrates carrying the disaccharide at Thr or Ser in the distant VTSA motif. Similar inhibition was observed in the Ser-Thr dyad of the GSTA motif, if one of these positions carried a Gal-GalNAc. rGalNAc-T4 did consistently add maximally two GalNAc residues to the monosubstituted GalNAc-peptides of the AHG21 series (Figure 1, bottom). On the other hand, the corresponding Galß1-3GalNAc-substituted peptides were not glycosylated by the enzyme, irrespective of the positions and numbers of the disaccharide (Figure 1, bottom). Exceptionally, the disaccharide-substituted peptide AHG21(D10) was an effective substrate and incorporated two GalNAc residues (Figure 1, bottom). This activity, similar to that on nonglycosylated MUC1 peptide substrates described below, could be attributed to endogenous GalNAc-Ts. Hence, it can be claimed that rGalNAc-T4 is totally inactive on all MUC1 glycopeptide substrates with Galß1-3GalNAc substitution. To demonstrate that the disaccharide-induced effect on rGalNAc-T4 can be compensated for by GalNAc in proximal positions, acceptor peptides based on the AHG21 sequence were generated that carried Gal-GalNAc (Thr5) and one GalNAc (Thr17) or two GalNAc residues (Ser16, Thr17). The substrate with a single GalNAc was not further glycosylated compared to the background control, whereas that with two GalNAc residues showed considerable incorporation during overnight incubation (Figure 1, bottom). This observation does not rule out the possibility of steric effects exerted by Gal-GalNAc in a negative and by GalNAc in a positive sense on accessibility of peptide sites. However, a lectin-mediated trigger effect on enzyme activity is in agreement with previous evidence based on lectin domain deletion mutants (Hassan et al., 2000).
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Glycosylation-induced effects on initial O-glycosylation of MUC4 repeat peptides
The peptide LPV19 corresponds to one unit and three overlapping amino acid residues of the MUC4 repeat (Figure 5). Although the peptide contains eight potential O-glycosylation sites, the number of GalNAc residues incorporated by rGalNAc-T2 is only five. Using LPV19(GalNAc)4 as a substrate for rGalNAc-T4 the enzyme incorporated one further GalNAc residue into the glycopeptide (site not identified). rGalNAc-T4 activity did not exceed background controls on nonglycosylated LPV19 and on all nonglycosylated peptides corresponding to partial sequences of the MUC4 repeat (SVS10, ATS10, PVT10). Unexpectedly, also the GalNAc-substituted decapeptides were not substrates of rGalNAc-T4 (Figure 5).
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Inhibition of rGalNAc-T2 and rGalNAc-T4 action
According to previous evidence the glycosylation-dependent action of rGalNAc-T4 appears to be mediated by a lectin-like GalNAc interaction of the enzyme with the substrate glycopeptide (Hassan et al., 2000). Such interaction has been substantiated by inhibitory effects of GalNAc, but not of other monosaccharides, on rGalNAc-T4 activity (Hassan et al., 2000
). We intended to further evaluate the nature of these effects and to demonstrate that also other isoforms of the ppGalNAc-Ts show similar inhibition of their activities. To this end we used free GalNAc and GalNAc-peptide (fully substituted at each potential site) as inhibitors of the enzyme(s). Incorporation rates in the presence and absence of inhibitor were measured for rGalNAc-T2 and -T4 and compared to those for rGalNAc-T1 as a reference (Figure 6 and Table I). Incubation of substrates, nonglycosylated TAP25 (rGalNAc-T1, and -T2 in Figure 6, bottom) and GalNAc substituted AHG21(M10) (rGalNAc-T1 and -T4 in Figure 6, top), over a period of 18 h resulted in reproducible proportions of glycosylated sites versus total potential acceptor sites on the substrate (set as 100%). In the presence of glycopeptide inhibitor HGV20(M4-5-9-15-16) the rGalNAc-T4 catalyzed addition of GalNAc was reduced to 17% of the control (Table I). On a molar basis the glycopeptide inhibitor was more effective by at least two orders of magnitude compared to the monosaccharide GalNAc (Table I). No effect was measurable with GlcNAc or with the nonglycosylated MUC1 tandem repeat peptide TAP25.
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Discussion |
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This study has revealed further evidence for the previously postulated epigenetic regulation of glycosylation density by competition between initial GalNAc introduction and core-specific enzymes (Hanisch et al., 1999). The dependency of rGalNAc-T4 on previously introduced GalNAc residues leads to a situation where the fill-up reactions of partially glycosylated MUC1 peptides can be inhibited by ß3-galactosylation of GalNAc-peptides catalyzed by the core 1specific C1GT1. Assuming colocalization of both enzymes their relative activities in the cis-Golgi compartments may finally determine to which degree the MUC1 repeat peptide is actually glycosylated at each of the five putative sites.
In the instance of MUC1 the different glycoforms found in human milk and on carcinoma cells were demonstrated to show an inverse relationship between glycosylation density and the lengths and complexity of the glycans (Hanisch et al., 1989; Müller et al., 1997
, 1999; Hanisch and Müller, 2000
). Though 50% of the putative sites in MUC1 are substituted with polylactosamine-type chains during lactation (Müller et al., 1997
), nearly full glycosylation of the repeat peptides with core-type chains is reached in the breast cancer cell line T47D (Müller et al., 1999
). Similar results were obtained with recombinantly expressed MUC1 glycosylation probes after transfection into a variety of established breast cancer cell lines (Müller and Hanisch, unpublished data).
Not only expression of a repertoire of glycosyltransferases but also their localization in the Golgi, their relative activities in each subcompartment, and their spatial arrangements in the Golgi membrane will influence the site-specific O-glycosylation state of a mucin. The impact of the last on antigenicity and immunogenicity of MUC1, which represents a primary target in the worldwide development of cancer vaccines, has been recently reviewed (Hanisch and Müller, 2000).
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Materials and methods |
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MUC2
The glycopeptide EPT9 is structurally based on a section of the MUC2 tandem repeat peptide carrying an additional Glu residue at the N-terminus and a GlcNAcß1-6GalNAc (core 6) substitution at Thr4 EPT9(d4). Its synthesis has been described in a previous publication (Mathieux et al., 1997). The peptide is modified N-terminally by acetylation and C-terminally by amidylation. The corresponding GalNAc substituted EPT9(M4) peptide was generated by enzymatic cleavage of the GlcNAc residue using ß-hexosaminidase (jack bean, Glyco, Novato). The nonglycosylated peptide PTT15 corresponding to a section of the MUC2 repeat was synthesized by a local service facility.
MUC4
The peptide LPV19 covering one tandem repeat unit of MUC4 was synthesized in-house by a local service facility. The glycopeptides corresponding to sections of the MUC4 tandem repeat peptide, SVS10, ATS10, and PVT10, were synthesized as previously described (Mathieux et al., 1997). These glycopeptides terminate with an additional Gly residue and are modified by acetylation (N-terminal) and amidylation (C-terminal).
In vitro glycosylation
The enzyme sources used were semipurified as previously described by successive sequential ion-exchange chromatographies on Amberlite (IRA95, Sigma) or DEAE Sephacel (Pharmacia), S-Sepharose Fast-Flow (Pharmacia), and Mini-STM (PC3.2/3, Pharmacia) using the Smart system (Pharmacia) (Bennett et al., 1998). Secreted rGalNAc-T4 was obtained from stably transfected Chinese hamster ovary (CHO) line (CHO/GalNAc-T4/21A) (Hassan et al., 2000
).
Peptide or glycopeptide substrates (100500 µM) were solubilized in 25 mM cacodylate buffer, pH 7.4, containing 10 mM MnCl2 and 0.25% TritonX-100 and mixed with UDP-GalNAc (200 µM) and rGalNAc-Ts in a total volume of 20 µl. In case of rGalNAc-T1 and -T2 (obtained from pAc-GP67-GalNAc-T1-sol and pAc-GP67-GalNAc-T2-sol baculovirus-infected High Five cells) enzyme activities were used, which yielded complete incorporation of GalNAc within 18h into the sites Thr9, Ser20, and Thr21 of TAP25. For all other substrates the reaction mixture was incubated at 37°C for 18 h to reach a state where even poor substrate positions were at least partially glycosylated (Hassan et al., 2000). Insect cells as well as CHO cells produce and secrete endogenous GalNAc-transferases. These endogenous activities are merely directed to Thr5 and Thr17 in the MUC1 repeat peptide starting with the AHG motif. Enzymatic background controls included a mutant rGalNAc-T4 (459D/H) with a defect lectin domain (Hassan et al., 2000
), a truncated catalytically inactive enzyme (T2C-sol), and a preparation of ß4-galactosyltransferase ß4-Gal-T2 (Almeida et al., 1999
). All enzymes were purified from High Five media as reported previously (Bennett et al., 1998
).
Analysis of reaction products
In initial experiments the reaction times and enzyme amounts were optimized for each rGalNAc-T preparation to achieve a maximum of incorporated GalNAc residues into the various peptide and glycopeptide substrates. Generally amounts of enzyme were used, which allowed glycosylation of all acceptor sites accessible for a particular isoform of GalNAc-Ts. In most cases reaction times of 18 h were sufficient to achieve exhaustive glycosylation in terms of incorporation of the maximal number of GalNAc residues. A prolongation of the reaction times up to 48 h did not increase this number. Only these "endpoint" products were used for site identification experiments after HPLC purification.
Aliquots of the reaction mixtures were diluted 1:10 in 0.1% aqueous trifluoroacetic acid (TFA) and mixed with an equal volume of -cyano-4-hydroxy-cinnamic acid matrix (saturated solution in 0.1% TFA/acetonitrile, 1:2, v/v) on the target. The samples were analyzed by matrix-assisted laser desorption and ionization mass spectrometry (MALDI MS) on a Bruker Reflex III using nitrogen laser ionization (
= 337 nm), and positive ion detection in the reflectron mode as previously described (Müller et al., 1997
). Pseudomolecular ions MH+ were registered and the number of GalNAc residues added to the substrate peptides was calculated on the basis of incremental mass increase (1 HexNAc residue corresponding to 203.2 mass units). The remaining reaction mixtures were chromatographed by RP-HPLC on a semipreparative scale and the eluting products were quantitated on the basis of their UV spectroscopic profiles as described previously (Hanisch et al., 1999
; Müller et al., 1997
). Aliquots were also run on a capillary electrophoresis apparatus (MR5000, Beckman Coulter, Munich) using uncoated 25 cm fused silica capillaries (75 µm) and 50 mM phosphate, pH 2.5, as electrophoresis buffer with normal polarity. Detection was at 200 nm and the thermostat for the cooling fluid was set at 25°C.
Localization of O-glycosylation sites
To identify the actual acceptor sites utilized by a particular enzyme, we chose reaction times which allowed at least the partial conversion into a final product with the maximum number of GalNAc residues. Prolonging the reaction times exceeding 18 h did not increase the number of substituted sites, and hence all structural studies were performed on those species exhibiting a final state of O-glycosylation.
O-glycosylated Ser/Thr residues were identified by a variety of complementary methods. HPLC-purified glycopeptides were sequenced by Edman degradation on an ABI 473A (PE Biosystems, Foster City, CA) with microcartridge under standardized conditions (Zachara and Gooley, 1998). Alternatively, partial sequences were obtained by analysis of postsource-decay fragments in MALDI MS (Müller et al., 1997
). In cases where MS fragmentation yielded insufficient sequence information, the glycosylation sites of HPLC-pure glycopeptides (10 µg) were localized by partial acid hydrolysis in a gas-phase reaction using pentafluoropropionic acid (20% v/v in water containing 0.5% dithiothreitol), which was incubated with the dry sample in a Mininert (Pierce) under vacuum at 90°C for 1 h (Mirgorodskaya et al., 1999
). The fragments were taken up in 0.1% aqueous TFA and analyzed by MALDI MS as above. Partial information on glycosylation sites was also obtained by enzymatic fragmentation of the glycopeptides with the endopeptidases from Flavobacterium meningosepticum (Pro-C specific, 100 mU in 0.1 M phoshate buffer, pH 7.2, 2 h at 30°C) or from Papaya (Gly-C specific, 100 mU, 0.1 M phosphate buffer, 1 mM EDTA, pH 6.8, 18 h at 37°C). Both enzymes were purchased from ICN Biomedicals (Eschwege, Germany).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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