2Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and 3Department of Oral Diagnostics, School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Nørre Alle 20, 2200 N, Denmark.
Received on May 7, 2001; revised on June 27, 2001; accepted on July 6, 2001.
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Abstract |
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Key words: O-glycosylation/mucin/pp-GalNAc-T/MUC2/biosynthesis
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Introduction |
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We have previously reported that all seven Thr residues of a peptide PTTTPITTTTK, a portion of the MUC2 tandem repeat having three and four consecutive Thr residues, were potentially glycosylated with GalNAc when microsome fractions of LS174T cells were used as the source of pp-GalNAc-Ts (Iida et al., 2000). Extensive attempts to fractionate all of the products of glycosylation only identified 10 different glycopeptides. These were PTTT*PITTTTK, PT*TTPITTTTK, PTTT*PITTT*TK, PTTT*PITT*T*TK, PT*TTPIT*T*TTK, PTT*T*PITT*T*TK, PT*TTPIT*T*T*TK, PTT*T*PIT*T*T*TK, PT*T*T*PIT*T*T*TK, and PT*T*T*PIT*T*T*T*K. If GalNAc incorporation into this peptide were a random process, the number of different products potentially generated by this procedure would be 128. Moreover, these 10 products identified seem to be aligned into two hypothetical pathways (Iida et al., 2000
). These hypothetical pathways are likely to be initiated from either PTTT*PITTTTK or PT*TTPITTTTK, because there were no other glycopeptide product with a single GalNAc residue despite five other potential glycosylation sites (Iida et al., 2000
). We hypothesize that these two initial sites were chosen from seven possible initial glycosylation sites by the glycosylation machinery of LS174T cells, which then govern the subsequent events. After the generation of the two initial products, further GalNAc incorporation appears to occur in a nonrandom fashion and only through these initial products. As an extreme alternative, one might assume that GalNAc incorporation into seven Thr residues in a cell-free system was a random process. If so, a maximum of 7! (1 + 1/1! + 1/2! + 1/3! + 1/4! + 1/5! + 1/6! + 1/7!), that is, 13,700 possible pathways could exist. We are intrigued as to why two pathways are chosen out of 13,700 possible pathways in this cell-free glycosylation system. In the present article, we asked whether there is a single pathway when PTTT*PITTTTK or PT*TTPITTTTK are used as the acceptor substrates and microsome fractions of LS174T cells were used as the enzyme source. Another curious finding from our previous studies is that all the products with five, six, and seven GalNAc residues appear to be derived from PTT*T*PITT*T*TK, not from PT*TTPIT*T*T*TK (Iida et al., 2000
). We therefore tested whether these glycopeptides are further glycosylated by purifying them and then used them as acceptors.
It was previously assumed that the rate of incorporation of GalNAc into Thr or Ser residues within a peptide is greatly influenced by sequence and tertiary structures. Also, GalNAc and other carbohydrate residues incorporated into the Ser or Thr vicinal to the free Ser or Thr have profound effects on the rate of GalNAc incorporation (Hanisch et al., 1999; Takeuchi et al., unpublished data). These effects may at least be in part due to the lectin-like domain of pp-GalNAc-Ts, as has been demonstrated with pp-GalNAc-T4 and T7 (Bennett et al., 1999
; Ten Hagen et al., 1999
; Hassan et al., 2000a
). Despite endeavors to clarify the specificity of each pp-GalNAc-T, the possibility of a clear understanding of the regulatory mechanism of O-glycosylation of various peptides with multiple and consecutive Thr and Ser is still remote. Our present results suggest that the attachment of each GalNAc residue to the peptide, PTTTPITTTTK, through O-glycosylation using microsome fractions of LS174T cells is the result of specific enzymatic actions of distinct pp-GalNAc-Ts at each step.
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Results |
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Figure 1A represents HPLC chromatographs of the reaction mixture containing PTTT*PITTTTK and reagents described in Materials and methods after various incubation periods (0, 3, 6, 12, and 24 h). Each peak was separately pooled and concentrated prior to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis (Figure 2A) where the number of GalNAc attached to the peptide was assigned. The proportion of glycopeptides with a larger number of incorporated GalNAc increased using longer incubation periods (Figure 1A). The predominant products after 12 h of incubation were those with three GalNAc residues, and the most prominent product after 24 h of incubation contained five GalNAc residues. The maximum number of GalNAc incorporated into PTTT*PITTTTK was seven. Peptides with two GalNAc and four GalNAc residues were in smaller relative abundance compared to those with three GalNAc and five GalNAc residues, respectively. This would indicate that intermediate products with two and four GalNAc residues have a relatively short half-life and probably accept additional GalNAc immediately. It should be noted that the MALDI-TOF MS peaks showing smaller molecular masses are not contaminating glycopeptides but degradation during the ionization because the relative intensity changes on changing the laser intensity and because a glycopeptide with fewer GalNAc residues can easily be separated on the reverse-phase high-performance liquid chromatography (HPLC). Figure 1B shows the HPLC chromatograms of the products derived from using PT*TTPITTTTK as an initial substrate. The predominant product had three GalNAc residues, and the maximum number of attached GalNAc residues was four. The relatively short half-life of glycopeptides with two GalNAc residues has been previously reported. The eluted peak at 24.0 min appears to be a degradation product of this glycopeptide, as indicated by its molecular mass of 1107 using MALDI-TOF MS analysis (Figure 2B). A similar peak did not appear when PTTT*PITTTTK was used as an acceptor by unknown reasons. In general, the profiles derived from PTTT*PITTTTK resembled those when unglycosylated peptide was used as an acceptor (Iida et al., 2000).
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Discussion |
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According to our previous reports (Iida et al., 1999a,b, 2000; Wandall et al., 1997
), each separate pp-GalNAc-T appears to have a more specific role than engaging to achieve a high degree of glycosylation or determining the relative preference of the site of O-glycosylation. For example, we previously reported how GalNAc was incorporated into three consecutive Thr residues by pp-GalNAc-T1, -T2, and -T3 by using a peptide (PTTTPLK) that mimicked the tandem repeat portion of MUC2 (Iida et al., 1999a
). These enzymes and pp-GalNAc-T4 showed different specificity when PTTTPLK, PT*TTPLK, PTTT*PLK, PT*TT*PLK, and PTT*T*PLK were used as acceptors (Iida et al., 1999a
; Takeuchi et al., unpublished data). In these experiments, recombinant pp-GalNAc-T4 was tested for its action on fluorescein isothiocyanate (FITC)-PTTTPLK. The major products corresponding to glycopeptides with one, two, and three GalNAc residues were FITC-PT*TTPLK, FITC-PTT*T*PLK, and FITC-PT*T*T*PLK respectively and did not align on a single pathway. Thus, the products obtained after in vitro glycosylation did not always represent intermediate and final products. In the present study, it was clearly demonstrated that FITC-PT*TTPITTTTK, FITC-PTTT*PITTTTK, and FITC-PTT*T*PITT*T*TK act as precursors of glycopeptides with greater numbers of GalNAc residues.
Analysis of the products after incubation of PTTTPITTTTK with microsome fractions of LS174T cells and UDP-GalNAc provided interesting results. Only 10 glycopeptides were detected, and these 10 can be aligned into two hypothetical pathways. These two hypothetical pathways were based on the sequence data obtained from the glycopeptides products detected in the cell-free system. If these two pathways are the only possible routes, then the attachment site of GalNAc at each step of the glycosylation process is likely to be strictly regulated. Theoretically, there are 7!(1 + 1/1! + 1/2! + 1/3! + 1/4! + 1/5! + 1/6! + 1/7!), that is, 13,700, alternative pathways, whereas only two pathways were experimentally detected when microsome fraction of LS174T cells were used as the source of pp-GalNAc-Ts. We found this result astonishing and therefore initiated studies to determine whether such fidelity is accomplished solely by the acceptor specificity of pp-GalNAc-Ts. Our focus is on the second and the fifth steps of the predicted pathways. As the results demonstrate, the order and maximum number of glycosylations of this stretch of MUC2 core polypeptide is regulated with a high degree of fidelity. The present results have demonstrated that the initial position of the O-glycosylation determines the fate in terms of the positional orders of GalNAc incorporation. There is no apparent biological reason why high degree of fidelity is required in the order of O-glycosylation.
Many questions remain unanswered. We would eventually like to determine whether each step is really regulated in a highly specific manner. In other words, all the intermediate products should be tested for their acceptor specificity with all pp-GalNAc-Ts expressed in these cells. So far, what we have shown is that the regulatory process functions with high degree of fidelity in cell-free systems. GalNAc incorporation into peptides in a cell could be influenced by several mechanisms, including expression of different pp-GalNAc-Ts, specificity of pp-GalNAc-Ts towards the peptide motifs and glycosylated intermediates (Hennebicq et al., 1998a; Elhammer et al., 1999
; Hassan et al., 2000b
). The localization of the various pp-GalNAc-Ts within the Golgi apparatus, through which biosynthetic intermediates are known to migrate (Elliott et al., 1994
; Hansen et al., 1995
). As an example, proline residues, located in the vicinity of O-glycosylation sites might play a role in pp-GalNAc-Ts activity and initial GalNAc incorporation (Brandts et al., 1975
; Elliott et al., 1994
). However, the second and subsequent GalNAc incorporations may be regulated by entirely different mechanisms. For instance, attachment of GalNAc to a peptide may change the conformation (Live et al., 2000
). It might also be true that pp-GalNAc-Ts recognize the presence of previously incorporated GalNAc residues in the vicinity though their lectin-like domains (Imberty et al., 1997
; Hanisch et al., 1999
).
To date, at least nine different human pp-GalNAc-Ts have been described through cDNA sequence identification. It would be expected that such different enzymes would have distinct acceptor specificity and to act on distinct Ser and Thr residues. It is noteworthy that a highly strict order of glycosylation and a maximum number of O-glycosylations were achieved in the presence of at least four pp-GalNAc-Ts in the microsome fraction used in this study. As shown in Figure 6, LS174T cells contain pp-GalNAc-T1, -T2, -T3, and -T4 proteins. Although the conditions required for optimal isozyme activity might be different for each, all of them were located in the Golgi apparatus and were shown to be active under the experimental conditions used to generate PTTTPITTTTK with GalNAc residues positioned at specific Thr residues. Our preliminary results indicate that the order and the maximum number of GalNAc incorporated into PTTTPITTTTK by pp-GalNAc-T1, -T2, -T3, and -T4 is highly restricted, and each enzyme is distinct in terms of intermediate products generated (data not shown). Furthermore, mRNA for pp-GalNAc-T6, -T7, and -T8 were also identified in these cells (Figure 7). Therefore, it is not easy to predict which pp-GalNAc-T is involved in each step of the pathways of GalNAc incorporation.
The strict regulation of GalNAc incorporation into peptides that we have observed is reminiscent of the regulation of N-glycan processing (Kornfeld and Kornfeld, 1985). The strict order of O-glycosylation as well as N-glycosylation might be necessary if all the intermediate glycosylation products need to be regulated. The biological significance and meaning of this tight regulation is not yet clear. Our present studies strongly suggest that the various pp-GalNAc-Ts have unique specificity and that they determine the order and maximum number of GalNAc incorporated into peptides containing multiple Thr arranged in a consecutive manner. Thus, the lectin-like domains of pp-GalNAc-Ts other than pp-GalNAc-T4 (Hassan et al., 2000a
) should also influence their acceptor specificity. Influence of the acceptor amino acid sequence on pp-GalNAc-T activity has been reported previously in biosynthetic (Hennebicq-Reig et al., 1997
; Wandall et al., 1997
; de Haan et al., 1998
; Hennebicq et al., 1998b
; Ten Hagen et al., 1999
) and structural studies (Gerken et al., 1997
, 1998; Hanisch et al., 1999
). However, the order of GalNAc incorporation into peptides has not been previously defined except in a few reports with MUC1 (Nishimori et al., 1994
; Muller et al., 1997
, 1999). Our present work is a unique attempt to determine the possible sequential events during the formation of mucins with consecutive Thr residues. Our understanding of the system is still rudimentary in many respects. For example, we found that the number and site of GalNAc incorporation was influenced by the transfer of galactose to existing GalNAc residues (Hanisch et al., 1999
; Takeuchi et al., unpublished data). Whether the findings in these studies, which are based on the use of a cell-free system, can be extended to events in vivo should be critically assessed. Thus it is important to answer to questions whether there is any evidence that the two pathways identified are also active for the full tandem repeat sequence and whether this regulation also functions in a complete MUC2. The initial attachment sites and subsequent glycosylation events on MUC2 should eventually be predictable based on the pp-GalNAc-Ts expressed in the cell.
LS174T cells used in the present study have been widely applied as models of cancer metastasis (Sternberg et al., 1999) and infectious diseases (Belley et al., 1996
). In these models, MUC2 with carbohydrate chains have been demonstrated to play regulatory roles in cell division and resistance to infection (Chadel et al., 1988
; Sternberg et al., 1999
). Whether altered arrangements of O-linked carbohydrate chains on MUC2 influence metastatic and other biological behaviors of carcinoma cells remains to be elucidated.
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Materials and methods |
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Preparation of microsome fractions as a source pp-GalNAc-Ts
Human colon carcinoma LS174T cells were cultured in a 1:1 mixture of Dulbeccos minimum essential medium and Hams F12 medium supplemented with 10% fetal bovine serum. Cells were homogenized in 50 mM TrisHCl buffer (pH 7.5) containing 250 mM sucrose, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 0.5 µg/ml pepstatin A. After low-speed centrifugation at 3000 x g for 10 min at 4°C, the supernatant was then centrifuged at 100,000 x g for 1 h. The pellets were then resuspended in homogenization buffer containing 0.1% Triton X-100 (Sigma, St. Louis, MO). Protein concentrations were determined using a BCA protein assay kit (Pierce Chemical Co., Rockford, IL) using bovine serum albumin as a standard. The solutions were then stored at 80°C prior to use.
Enzymatic incorporation of GalNAc into glycopeptides
Glycopeptides with O-linked threonine residues, PTTT*PITTTTK, PT*TTPITTTTK, PTT*T*PITT*T*TK, and PT*TTPIT*T*T*TK, were tested for their acceptor specificity for pp-GalNAc-Ts using microsome fractions of LS174T cells. The standard reaction mixture (50 µl) consisted of 50 mM HEPES buffer (pH 7.5), 5 mM 2-mercaptoethanol, 20 mM MnCl2, 2 mM phenylmethylsulfonyl fluoride, protease inhibitors (1 µg/ml aprotinin, 1 µg/ml leupeptin, 0.5 µg/ml pepstatin A), 1 mM UDP-GalNAc (Sigma), 5 µM synthetic oligopeptide or purified glycopeptides, and LS174T of cell microsome fraction corresponding to 700 µg/ml of protein (see previous paragraph for details). Reactions were performed at 37°C for up to 24 h, and were terminated at various times by adding a 1/5 volume of 0.5 M EDTA (pH 8.0). The amount of enzyme used and the incubation time for the reactions were based on conditions used for the incorporation of GalNAc into PTTTPITTTTK, as previously described (Iida et al., 2000).
Characterization of the glycosylated peptides
The glycosylated peptides were separated by reverse-phase HPLC (JASCO, Tokyo) using a Cosmosil (5C18-AR, 10 x 250 mm, Nacalai Tesque, Kyoto, Japan) column. The column was eluted using a linear gradient (0 to 50%) of solvent B (0.05% trifluoroacetic acid/2-propanol) in solvent A (0.05% trifluoroacetic acid/H2O) at a flow rate of 2 ml/min for 30 min. Eluates were monitored by fluorescence intensity at 520 nm (ex: 492 nm). MALDI-TOF MS analysis of glycosylated peptides was performed after HPLC separation as previously described (Iida et al., 1999b). Briefly, concentrated glycosylated peptides were mixed on a stainless steel plate in a 1:1 ethanol/water solution containing 10 mg/ml
-cyano-4-hydroxycinnamic acid and 0.1% trifluoroacetic acid. All mass spectra were obtained on a VoyagerTM Elite in linear mode set for delayed extraction. Voltages were set at 20,000 (acceleration), 93.5 (grid), and 0.05% (guide wire). Laser intensity was chosen for optimum resolution. Relative molecular mass numbers were determined using angiotensin I as a standard.
Amino acid sequencing
Pulsed-liquid Edman-degradation amino acid sequencing of glycopeptides was performed using an Applied Biosystems 492 Procise protein sequencing system (Perkin Elmer, Norwalk, CT) as previously described (Iida et al., 1999a,b). Using this system, a phenylthiohydantoin (PTH) derivative of GalNAc-attached Thr was identified. It eluted as a pair of peaks close to the peak positions of PTH-Ser and PTH-Thr (Gerken et al., 1997
). Amino acid sequencing of a fully glycosylated peptide (PT*T*T*PLK) confirmed this observation (data not shown).
Immunocytochemistry
Human colon carcinoma cell line LS174T was grown to subconfluency on small cover slips in the media described above. Cells were fixed in ice-cold acetone for 10 min and then dried for at least 30 min before use. The cells were incubated with mouse hybridoma culture supernatants for 112 h at 4°C as described previously (Bennett et al., 1998; Mandel et al., 1999
). After washing, bound mAbs were detected with FITC-conjugated rabbit anti-mouse immunoglobulin absorbed with human serum (code F-261, Dako, Denmark). Slides were mounted in glycerol containing p-phenylene-diamine and examined on a Zeiss fluorescence microscope using epi-illumination. The microscope was equipped with FITC interference filters and a 75 W xenon lamp.
RT-PCR analysis for pp-GalNAc-T expression
Expression of seven pp-GalNAc-Ts was determined by RT-PCR. Total RNA was isolated from LS174T cells using UltraspecTM RNA kit (Biotecx Lab, Houston, TX). Five micrograms of total RNA were reverse-transcribed with 200 U of SuperscriptTM II RNase H-free Reverse Transcriptase (Gibco-BRL, Rockville, MD) in a buffer solution containing 50 mM TrisHCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 0.5 mM dNTP, 1 U/µl RNase inhibitor (Ambion, Austin, TX), and 50 ng/µl oligo(dT)1218 primer (Pharmacia Biotech, Buckinghamshire, UK) in a final volume of 20 µl at 42°C for 50 min. After reverse transcription, the reaction mixture was treated with 2 U of Ribonuclease H (Gibco-BRL) at 37°C for 20 min. Two percents of the obtained cDNA was amplified by PCR using 0.02 U/µl Ampli Taq GoldTM polymerase (Applied Biosystems, Tokyo) in 10 mM TrisHCl buffer (pH 8.3), containing 50 mM KCl, 1.5mM MgCl2, 0.2 mM dNTP, 5% dimethylsulfoxide, and 0.2 µM of the respective 5' and 3' external primers. The following sequences of primers were used in this study:
pp-GalNAc-T1 (485 bp) 5'-CAA TGA GGC TTG GAG CAC ACT T-3' and 5'- TGA CAG GAA GAG TCC GAT CAC C-3'
pp-GalNAc-T2 (655 bp) 5'-TAC ATG ACG CCT GAG CAG AGA AG-3' and 5'- GGT CCA CCA CAG TAA GGC ACA A-3'
pp-GalNAc-T3 (650 bp) 5'-TGG TTG GCT AGA ACC TCT GTT GG-3' and 5'-GGT CTG GCA CAT ACA CCT CTG G-3'
pp-GalNAc-T4 (464 bp) 5'-ACT CCA GCT CAA CGA GGA TGA A-3' and 5'- GCC AAC CGG AAT TAC ACT CAC A-3'
pp-GalNAc-T6 (575 bp) 5'-ATG CCA GCA CAG AGG AGC AC-3' and 5'-CAC ATG GCC TAC GAC AGA GCA-3'
pp-GalNAc-T7 (670 bp) 5'-TTG CTC ACT TCG AGC GTT GTC-3' and 5'-TGC CAC CAC ACT GCC ATA TCT T-3'
pp-GalNAc-T8 (499 bp) 5'-GTG GAG CTT AGC CTG AGG GTG T-3' and 5'-CTG GCC TCT GCA ATC AGT TGT C-3'
Forty-five PCR cycles were performed under the following condtitons: denaturation at 94°C for 1 min, annealing at 58°C (hT1, 2, 3, 4, 6, and 7) or at 60°C (T8) for 1 min, and elongation at 72°C for 1 min. The PCR reaction products were analyzed by electrophoresis on 1.2% agarose gels.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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