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Biochemistry and Biophysics |
Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Kyoto 603-8555, Japan [M. I., S. T., I. Y., H. N.]; First Department of Surgery [M. K., Y. K.] and Department of Medical Chemistry [T. O.], Kansai Medical University, Osaka 570-8506, Japan; and Unite de Chimie Organique, Institut Pasteur, Paris 75724, France [S. V-G., D. C.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have prepared some monoclonal antibodies against a human colorectal
cancer cell line, LS 180, and demonstrated that they are strongly
reactive only with clustered O-glycans on peptide. For
instance, the epitopic structures for anti-Tn and anti-sialylTn
antibodies comprise three and four consecutive sequences of
GalNAc-Ser/Thr and SA
2–6GalNAc-Ser/Thr, respectively
(3, 4, 5, 6)
. The clustering of a relatively common structure
could lead to the formation of an uncommon structure exhibiting
antigenicity. Although the roles of these truncated
O-glycans in malignant behavior are not well understood,
they are highly immunogenic and useful as a vaccine (7
, 8)
. Therefore, it is important to elucidate a biosynthetic
mechanism of clustered O-glycans aligned on a core peptide.
In a previous studies (3, 4, 5 , 9) , we reported that an extract of LS 180 cells glycosylated the MUC2 tandem repeat peptide in vitro, leading to the synthesis of Tn antigenic sites recognized by a monoclonal antibody (MLS 128), indicating the synthesis of clustered Thr-GalNAc.
Many mucins contain a large number of Thr and Ser residues within their tandem repeat domains, which include consecutive Thr/Ser residues except for MUC1 (10) . The initial step in the regulation of O-glycosylation is the enzymatic transfer of GalNAc from UDP-GalNAc to Thr and Ser residues (11 , 12) . It is well known that there are multiple GalNAc-Ts3 expressed in various tissues (13) . Thus, GalNAc transferases that regulate the initiation of O-glycosylation of mucins are important for understanding some aspects of tumor-associated aberrant O-glycosylation.
We used a synthetic peptide of MUC2 tandem repeat unit and extracts from a human colonic adenocarcinoma and paired normal mucosa and rGalNAc-Ts as a substrate and enzyme sources, respectively. Because MUC2 is a major secreted mucin of intestinal epithelia, and its tandem repeat unit contains two consecutive parts consisting of three and five Thr residues that potentially serve as a scaffold presenting clustered carbohydrate antigens (14) .
Because the O-glycosylation of one site of a peptide may have some effects on other acceptor sites, we used a whole unit of the MUC2 tandem repeat as a substrate. The peptide was conjugated with DABITC, which has absorbance at 436 nm, to monitor the glycosylated peptides on reverse-phase HPLC. We determined the glycosylated sites of glycopeptides with the maximum number of GalNAc residues incorporated by combining evidence from MALDI-TOF mass spectrometry with sequence data from Edman degradation. First, we found a remarkable difference between glycopeptides synthesized with extracts of colonic adenocarcinoma and with paired normal mucosa. The adenocarcinoma extract glycosylated the peptide to a greater extent than that of the normal mucosa, probably due to enhanced expression of GalNAc-T3. Next, the peptide glycosylated by rGalNAc-T1, -T2, and -T3 was prepared. Maximum numbers of 6, 8, and 11 GalNAc residues were transferred to the peptide by rGalNAc-T1, -T2, and -T3, respectively, and both the consecutive Thr residues were fully glycosylated by rGalNAc-T3 but not by rGalNAc-T1 or -T2.
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MATERIALS AND METHODS |
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Cells.
A human colorectal cancer cell line, LS 180, was obtained from the
American Type Culture Collection and cultured in Eagle’s MEM
supplemented with 10% FCS.
Preparation of Adenocarcinoma and Normal Mucosa Extracts.
A fresh colonic adenocarcinoma and normal mucosa taken at a distance of
more than 10 cm from the adenocarcinoma were frozen immediately after
surgery with liquid nitrogen. Five adenocarcinomas and paired normal
mucosae were always processed in parallel. Extracts of adenocarcinoma
and normal mucosa were obtained as follows. The tissues were
homogenized in 25 mM phosphate buffer (pH 7.5) and 0.15
M NaCl with a Bio-Mixer, and the resultant lysate was
centrifuged at 1,000 x g for 10 min. The
supernatant was sonicated for 1 min and then centrifuged at
10,000 x g for 10 min. The supernatant was
further centrifuged at 105,000 x g for
1 h. After solubilization with 2% octylglucoside, 2
mM phenylmethylsulfonyl fluoride, and 0.1
M Tris-HCl buffer (pH 7.4), the pellet was
centrifuged at 105,000 x g for 1 h, and
the resultant supernatant was used as the enzyme source.
RT-PCR of GalNAc-Ts.
Preparation of total RNA from colonic adenocarcinoma and paired normal
mucosa was performed using Isogen (Nippon Gene, Toyama, Japan)
according to the manufacturer’s instruction. Total RNA (1 µg) was
reverse transcribed using a reaction mixture comprising 5
mM MgCl2, 1 mM
deoxynucleotide triphosphate mixture, 1 unit/µl RNase inhibitor, 1
µM oligodeoxythymidylic acid primer, and 0.25
unit/µl reverse transcriptase in a final volume of 20 µl. The
mixture was incubated at 55°C for 40 min, at 99°C for 5 min, and
then at 5°C for 5 min. The cDNA was then subjected to PCR. To obtain
semiquantitative results, the cycle number and cDNA concentration were
chosen so as to ensure that the amplification was not in the plateau
phase. For GalNAc-T1 and -T2, 20, 26, and 30 cycles were performed, and
for GalNAc-T3, 30, 35, and 40 cycles were performed. The cDNA
amplification of GalNAc-Ts and ß-actin was performed at the same
time. The reaction mixture comprised 2.5 mM
MgCl2, 20 pM forward and reverse
primers, and 2.5 units of TaKaRa Taq (TaKaRa, Tokyo, Japan) in a final
volume of 100 µl. The forward and reverse primers used and the
expected product sizes were as follows: (a) for GalNAc-T1,
5'-CAAAAGCCTCATGAAGGTCC-3' and 5'-ACCCGCCATAGGTCATGT-3', 321 bp;
(b) for GalNAc-T2, 5'-TGGCGGGTGGATCTGCCGGC-3' and
5'-TCTAGGTTTTCTCCTCCCCA-3', 600 bp; (c) for GalNAc-T3,
5'-ACAGCAGCAGAATTGAAGCC-3' and 5'-TTAATCATTTTGGCTAAGTA-3',
1594 bp; and (d) for ß-actin,
5'-ATGGATGATGATATCGCCGC-3' and 5'-ATAGGAATCCTTCTGACCCA-3', 291 bp.
After a polymerase activation step at 94°C for 5 min, samples were
amplified for the indicated numbers of cycles of denaturation at 94°C
for 1 min, annealing at 55°C for 30 s, and extension at 74°C
for 1 min. The amplified cDNAs were run on 1% agarose gels with 0.5
µg/ml ethidium bromide and visualized under UV light.
Preparation of rGalNAc-Ts.
Total RNA was prepared from LS 180 cells using Isogen as described
above. cDNAs encoding the putative ectodomains of rGalNAc-T1, -T2, and
-T3 flanked by artificial sites for BamHI and
EcoRV (rGalNAc-T1) and BamHI and XbaI
(rGalNAc-T2 and -T3) were amplified by PCR. The PCR-generated DNAs were
digested with BamHI and EcoRV for rGalNAc-T1 or
with BamHI and XbaI for rGalNAc-T2 and -T3 and
then inserted into expression vector pSecTag containing an
immunoglobulin 
chain leader sequence, a myc epitope, and
a polyhistidine tag. The constructed plasmid (pSecTag-rGalNAc-T1, -T2,
and -T3) was introduced into TOP10F' cells and amplified according to
the manufacturer’s instructions, and then COS-7 cells were transfected
with pSecTag-rGalNAc-T1, -T2, and -T3 by the lipofection method. The
culture medium was dialyzed against 25 mM
phosphate buffer (pH 7.8) and 15 mM NaCl and then
subjected to DEAE-cellulose column chromatography. This step was
necessary to exclude endogenously synthesized GalNAc transferases. The
excluded fraction was applied to a ProBond column, washed with 20
mM phosphate buffer (pH 7.8) and 0.5
M NaCl, and then eluted with 0.3
M imidazole, 0.5 M NaCl,
and 20 mM phosphate buffer (pH 6.0). Finally,
rGalNAc-Ts were prepared on an immunoaffinity column using
anti-myc/His antibodies according to the manufacturer’s
instructions.
Transfer of GalNAc to a Synthetic Peptide.
A synthetic peptide including 1 unit of the MUC2 tandem repeat was
conjugated with DABITC as described by Chang (16)
. The
incubation mixture comprised 5 mM
MgCl2, 5 mM 2-mercaptoethanol, 5
mM CDP-choline, 43 nmol of UDP-GalNAc, 4 µg of
DABITC-MUC2 peptide, and an appropriate amount of a tissue extract or
rGalNAc-Ts in a final volume of 100 µl. The mixture was incubated at
37°C for the times indicated in Figs. 1
, 3
, and 4
.
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Mass Spectrometry of the Glycopeptides.
Mass spectra of the glycopeptides were obtained by MALDI-TOF mass
spectrometry (Kratos Analytical, Manchester). Samples (10 pmol)
dissolved in 1 µl of distilled water were placed on a stainless steel
probe tip along with 1 µl of the matrix (2,5-dihydroxybenzoin acid in
0.1% trifluoroacetic acid/acetonitrile, 7:3). Measurements were
performed in the linear mode using an appropriate delay time and a
potential to focus the ions of interest.
Identification of O-Glycosylation Sites of the
Glycopeptides.
The glycopeptides were degraded with cyanogen bromide (17)
and then purified by the same reverse phase HPLC. Pulsed liquid phase
Edman degradation amino acid sequencing was performed with a Procise
492 protein sequencer (PE Biosystems, Foster City, CA).
O-Glycosylation sites were determined by elution profile of
PTH-Thr-GalNAc, which are eluted as two diastereotopic peaks at unique
positions on the chromatogram as reported by Gerken et al.
(18)
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RESULTS |
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To detect the products directly, the synthetic peptide was conjugated
with DABITC, which exhibits absorbance at 436 nm. Furthermore, as
described in "Materials and Methods," four amino acids including
Met were inserted between DABITC and the
NH2-terminal of the MUC2 tandem repeat to
minimize the influence of DABITC and to exclude DABITC released through
cyanogen bromide degradation after fractionation of the glycopeptides.
The effects of various assay parameters (cell extract, MUC2 peptide,
and UDP-GalNAc) were evaluated, and appropriate conditions were
selected. Five colonic adenocarcinomas and paired normal mucosae were
examined. Fig. 1
shows a representative elution pattern on HPLC of the
glycopeptides synthesized by the adenocarcinoma or normal mucosa
extract. The nonglycosylated DABITC-MUC2 peptide was eluted at 30.5 min
(fraction 6). Other fractions (fractions 1–5) contained glycopeptides
with various numbers of GalNAc residues transferred. The peaks
that eluted faster (fractions 1 and 2) contained glycopeptides with
higher contents of GalNAc residues and showed the clustered Tn
antigenicity, as reported previously (9)
. It is
notable that the adenocarcinoma extract could transfer GalNAc to the
MUC2 peptide to a much higher extent compared with that of the normal
paired mucosa, as shown in Fig. 1, A and B
.
Expression of GalNAc-T1, -T2, and -T3 mRNA was analyzed
semiquantitatively by RT-PCR. Representative results for the same
specimen seen in Fig. 1
are shown in Fig. 2
. The mRNA levels for all GalNAc-T probes were quantified with a
densitometer and normalized relative to the level of ß-actin mRNA. It
was found that the level of GalNAc-T3 mRNA in an adenocarcinoma was
significantly higher than that in the paired normal mucosa.
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. Because fractions 1 and 2 in Fig. 1
contained the clustered Tn
antigenicity as reported previously (9)
, the relative
activity producing the clustered GalNAc-Thr is shown by comparing the
area of fractions 1 and 2.
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Characterization of the MUC2 Peptide Glycosylated by
rGalNAc-Ts.
It is generally agreed that there are multiple polypeptide GalNAc-Ts
expressed in various tissues (13)
. It was interesting to
determine which GalNAc-T is responsible for the synthesis of clustered
carbohydrate antigens. GalNAc-T1, -T2, and -T3 have been cloned and
extensively characterized (19, 20, 21, 22)
. rGalNAc-Ts were
prepared as described in "Materials and Methods." Each enzyme was
subjected to SDS-PAGE followed by Western blotting. After staining
through successive incubation with anti-myc/His antibodies
and protein G-peroxidase, the relative amounts of the enzymes were
determined with a densitometer. Bands corresponding to molecular
weights of 79,000 73,000, and 85,000 were detected in the
rGalNAc-T1, -T2, and -T3 lanes, respectively (data not shown). These
molecular weights were larger than the sizes expected from the cDNAs
inserted in the vector, and this difference was probably due to
glycosylation during the synthesis in COS-7 cells.
The MUC2 tandem repeat unit conjugated with DABITC was incubated with
each rGalNAc-T for up to 5 days to determine the maximal number of
GalNAc residues transferred and identify the sites of
O-glycosylation. The glycopeptides synthesized with the
recombinant enzymes were directly subjected to reverse phase HPLC, as
shown in Fig. 3
. Although the glycopeptides synthesized with rGalNAc-T1 and -T2
gave symmetrical peaks with apparent retention times of 28.07 and 27.32
min, respectively (Fig. 3, A and B)
, they
contained glycopeptides with a few different numbers of incorporated
GalNAc residues, as described later. The elution pattern of the
peptides glycosylated by rGalNAc-T3 showed a broad peak even on
incubation for 5 days (Fig. 3C)
. The peak was tentatively
divided into three fractions. The first fraction eluted at about 27.20
min was purified by reverse phase HPLC.
The DABITC-glycopeptides and -peptide were analyzed by MALDI-TOF mass
spectrometry, as shown in Fig. 4
. Peaks a–c and d showed DABITC-glycopeptides
synthesized by GalNAc-Ts and DABITC-peptides, respectively. Peak
a, which corresponds to the highest molecular weight
synthesized by rGalNAc-T1, showed a Mr
of 4177.8, corresponding to the addition of six GalNAc residues to the
peptide (Fig. 4A)
. Peptides with five or four GalNAc
residues attached were also observed in the lower molecular weight
region. The maximum numbers of GalNAc residues incorporated by
rGalNAc-T2 and -T3 were calculated to be 8 and 11 from the molecular
weights of 4582.3 (peak b) and 5187.0 (peak c),
respectively, as shown in Fig. 4, B and C
. To
exclude DABITC, the glycopeptides were degraded by cyanogen bromide
treatment and purified again by the same reverse phase HPLC. To analyze
the glycopeptides with a maximum number of incorporated GalNAc
residues, the first peak with a symmetrical elution profile was
subjected to analysis by an amino acid sequencer. It has been reported
that PTH-Thr/Ser-GalNAc derivatives are eluted as two diastereotopic
peaks at unique positions on the chromatogram (18)
. To
determine the retention time of PTH-Thr-GalNAc in our system, we used a
reference glycopeptide containing
SerGalNAc-Thr-ThrGalNAc
(Fig. 5)
. Chromatography of PTH-Thr-GalNAc gave two peaks eluted at 5.24 min
(peak T*) and 5.94 min (peak T**). The second
peak was slightly behind the peak of PTH-Thr (retention time, 5.91 min)
on the chromatogram. Based on these data, the sites of
O-glycosylation were determined. Fig. 6
shows the relative areas of the peaks (T*) derived from the
PTH-Thr-GalNAc at each cycle. Based on the amount of the first peak
(T*) and the retention time of the second peak (T**), we determined the
O-glycosylation sites to be the Thr-2, -4, -10, -11, -13,
and –15; the Thr-2, -4, -8, -10, -11, -15, -17, and –19; and the
Thr-2, -3, -4, -7, -8, -9, -10, -11, -15, -17, and -19 residues of the
MUC2 peptides glycosylated by GalNAc-T1, -T2, and -T3,
respectively. In addition to these O-glycosylation
sites, two Thr residues, i.e., Thr-13 (Fig. 6B)
and Thr-21 (Fig. 6C)
, appeared to be poorly glycosylated in
some cases.
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DISCUSSION |
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One is an unmasked core peptide produced by decreased glycosylation. MUC1 on breast cancer cells has been characterized by this unmasked core peptide and is assumed to permit preferential interaction with cytotoxic lymphocytes (23 , 24) . In contrast to these hypoglycosylation, Muller et al. (25) demonstrated that breast cancer cell line T47D glycosylates the MUC1 tandem repeat peptide at a higher density than lactating breast epithelia. This discrepancy may be explained by the finding (26) that a specific GalNAc-T named GalNAc-T4 is responsible for the glycosylation of the PDTR motif within the MUC1 tandem repeat peptide. Thus, the expression of these epitope structures seems to be dependent on the expression of particular GalNAc-Ts but not on the decrease or increase of generally expressed GalNAc-Ts.
Another is a truncated O-glycan, in which Tn and T antigens
and their sialylated counterparts, sialylTn and sialylT antigens, are
involved. Many mucins (except for MUC1) have unique tandem repeats
containing consecutive Ser/Thr residues. MUC2 potentially serves as a
scaffold, presenting a variety of carbohydrate epitopes on its abundant
Thr residues. Byrd et al. (27)
reported that
78% of the Thr residues are glycosylated in the LS 174T colon
carcinoma cell line. However, whether or not the degree of
glycosylation changes due to malignancy and how clustered
O-glycosylation occurs have not been studied in detail. We
have shown that the antigenic sites of the Tn and sialylTn antigens are
built up clusters of GalNAc-Ser/Thr and SA2–6GalNAc-Ser/Thr,
respectively (3, 4, 5, 6)
. Although the function of these
antigens is not understood, their increased expression has some
correlation to advanced malignancy. It has been reported that about
73% and 96% of human colon cancers express the Tn and sialylTn
antigens, respectively (28)
.
The present study was designed to compare the glycosylation of the MUC2
peptide by extracts of colonic adenocarcinoma and paired normal mucosa
and to determine which GalNAc-Ts are relevant to the synthesis of
consecutive Thr-GalNAcs. Our previous work showed that microsomal
membranes of LS 180 cells glycosylated the MUC2 tandem repeat peptide,
leading to the synthesis of clustered Tn epitopes, and the peptide
glycosylated most extensively was determined to be a glycopeptide with
11 attached GalNAc residues. It is notable that the colonic
adenocarcinoma extract exhibited a higher level of enzyme activity than
did the normal mucosa (Fig. 1)
. In addition, as judged from the elution
profile of the glycopeptides on reverse phase HPLC, the colonic
adenocarcinoma extract could glycosylate the peptide at the same level
as the microsomal membranes of LS 180 cells, as reported previously
(9)
.
First, we estimated the expression of GalNAc-T mRNA semiquantitatively
by RT-PCR. Only GalNAc-T3 mRNA was elevated remarkably in the colonic
adenocarcinoma, whereas the level of GalNAc-T1 and -T2 mRNA increased
slightly (Fig. 2
and Table 1
), and these findings were consistent with
previous reports that in all human organs examined, GalNAc-T1 and -T2
are expressed universally at low to moderate levels, but that the
expression of GalNAc-T3 mRNA is highly tissue specific
(19
, 21
, 22
, 29) . GalNAc-T4 was expressed at a
very low level in both adenocarcinoma and normal mucosa (data not
shown). These results prompted us to examine whether or not GalNAc-T3
could glycosylate the MUC2 peptide more extensively than the other
enzymes could. Although O-glycosylation on the MUC1 tandem
repeat unit has been studied extensively (25
, 30
, 31)
,
little is known about the O-glycosylation sites on the MUC2
peptide. Iida et al. (32)
demonstrated the
acceptor specificity of rGalNAc-T1, -T2, and -T3 toward a short
peptide, PTTTLK, mimicking a part of the MUC2 tandem repeat unit, in
which rGalNAc-T3 was shown to have a unique acceptor specificity
differing from those of rGalNAc-T1 and -T2. Because substrate activity
is significantly influenced by certain aspects of the primary amino
acid sequence in the region adjoining the glycosylated position, it
seems to be preferable to use a long peptide as a substrate within the
range to be analyzed. In fact, Nishimori et al.
(31)
reported that the length for an enzyme-substrate
interaction may extend to at least nine residues, and minimum activity
is seen with substrates with one residue on the
NH2-terminal side and four residues to the
COOH-terminal side. In addition, Hanisch et al.
(33)
also demonstrated that the initial glycosylation of a
peptide substrate influences its subsequent glycosylation, including
both vicinal and distal glycosylation sites. Therefore, we used the
whole MUC2 tandem repeat composed of 23 amino acid residues as a
substrate.
The MUC2 peptide was glycosylated extensively on incubation with
rGalNAc-T1, -T2, and -T3 for a long period with daily supplementation
of the recombinant enzymes. Although the potential glycosylation sites
were glycosylated completely or almost completely, the products
comprised several glycopeptides with different numbers of incorporated
GalNAc residues, as shown in Fig. 4
.
Muller et al. (25)
reported that glycosylation
of the tandem repeat peptides within individual MUC1 molecules is not
uniform. However, the multiple Thr residues seem to be glycosylated in
an ordered manner, at least under the experimental conditions used in
which the peptides are glycosylated by a single enzyme in
vitro. The fact that a preferential order for the incorporation of
GalNAc to the Thr residues of the MUC2 peptide exists (32)
supports this view. Although the order remains to be elucidated,
it was clearly demonstrated that rGalNAc-T3 could glycosylate the MUC2
peptide most extensively, including both consecutive parts composed of
three and five Thr residues (Fig. 6)
. This distinct substrate
specificity of rGalNAc-T3 is very significant in light of the fact
that in contrast to the universal expression of GalNAc-T1 and -T2 mRNA
in all human organs and malignant cells, GalNAc-T3 shows a quite unique
expression pattern. These results are also consistent with previous
reports (30
, 32)
, which found that although there is
overlapping of acceptor-substrate specificities due to some redundant
functions of these enzymes, each enzyme gives a specific pattern of
glycosylated Thr residues on the MUC2 peptide.
It has been reported that GalNAc-T3 is differentially expressed by adenocarcinoma cell lines, with a tendency for more differentiated cell lines to express it more extensively (29) . More detailed analysis to determine the glycosylation pattern on the MUC2 peptide by using various adenocarcinomas differentiated to various degrees is necessary.
Recently, Bennett et al. (34) demonstrated that a new isozyme, designated GalNAc-T6, exhibits a similar substrate specificity to GalNAc-T3. GalNAc-T3 and probably GalNAc-T6 are essential for glycosylating consecutive Thr residues, which might play significant roles in the expression of not only clustered tumor-associated carbohydrate antigens but also of carbohydrate ligands for cellular lectins.
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FOOTNOTES |
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1 Supported in part by Grant-in-Aid for Scientific
Research on Priority Areas 10178102 from the Ministry of Education,
Science and Culture of Japan; by the Foundation for Bio-venture
Research Center from the Ministry of Education, Science and Culture of
Japan; and by the Fugaku Trust for Medicinal Research. 
2 To whom requests for reprints should be
addressed, at Department of Biotechnology, Faculty of Engineering,
Kyoto Sangyo University, Kita-ku, Kyoto 603-8555, Japan. Phone:
81-75-705-1897; Fax: 81-75-705-1888; E-mail: hnakada{at}cc.kyoto-su.ac.jp
3 The abbreviations used are: GalNAc-T,
UDP-GalNAc:polypeptide
N-acetylgalactosaminyltransferase; rGalNAc-T,
recombinant GalNAc-T; HPLC, high-performance liquid chromatography;
MALDI-TOF, matrix-assisted laser desorption/ionization time of flight
mass spectrometry; DABITC,
4-N,N-dimethylaminoazobenzene-4-isothiocyanate;
RT-PCR, reverse transcription-PCR; PTH, phenylthiohydantoin.
Received 4/24/00. Accepted 11/29/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Ser(Thr) monoclonal antibody (MLS 128).. J. Biol. Chem., 266: 12402-12406, 1991.2–6GalNAc-Ser/Thr (sialyl Tn) monoclonal antibody (MLS 132).. Eur. J. Biochem., 263: 27-32, 1999.[Medline]
-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase.. J. Biol. Chem., 270: 24156-24165, 1995.-D-galactosamine.. J. Biol. Chem., 271: 17006-17012, 1996.-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase that complements other GalNAc-transferases in complete O-glycosylation of the MUC1 tandem repeat.. J. Biol. Chem., 273: 30472-30481, 1998.-D-galactosamine: polypeptide N-acetylgalactosaminyltransferases in adenocarcinoma cell lines.. Cancer Res., 57: 4744-4748, 1997. D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-T3 designated GalNAc-T6.. J. Biol. Chem., 274: 25362-25370, 1999.This article has been cited by other articles:
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  T. Freire, S. Bay, S. von Mensdorff-Pouilly, and E. Osinaga Molecular Basis of Incomplete O-Glycan Synthesis in MCF-7 Breast Cancer Cells: Putative Role of MUC6 in Tn Antigen Expression Cancer Res., September 1, 2005; 65(17): 7880 - 7887. [Abstract] [Full Text] [PDF]
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R. Lo-Man, S. Vichier-Guerre, R. Perraut, E. Deriaud, V. Huteau, L. BenMohamed, O. M. Diop, P. O. Livingston, S. Bay, and C. Leclerc A Fully Synthetic Therapeutic Vaccine Candidate Targeting Carcinoma-Associated Tn Carbohydrate Antigen Induces Tumor-Specific Antibodies in Nonhuman Primates Cancer Res., July 15, 2004; 64(14): 4987 - 4994. [Abstract] [Full Text] [PDF]
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 K. G. Ten Hagen, T. A. Fritz, and L. A. Tabak All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases Glycobiology, January 1, 2003; 13(1): 1R - 16R. [Abstract] [Full Text] [PDF]
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, adenocarcinoma; 
, normal tissue).
