The epitope recognized by the unique anti-MUC1 monoclonal antibody MY.1E12 involves sialylα2–3galactosylβ1–3N-acetylgalactosaminide linked to a distinct threonine residue in

The epitope recognized by the unique anti-MUC1 monoclonal

antibody MY.1E12 involves sialyla2–3galactosylh1–3N-acetylgalactosaminide linked to a distinct threonine residue

in the MUC1 tandem repeat

Hideyuki Takeuchia, Kentaro Katoa, Kaori Denda-Nagaia, Franz-Georg Hanischb,Henrik Clausenc, Tatsuro Irimuraa,*

aLaboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo,

7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japanb Institute of Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Koln, Germany

cDepartment of Oral Diagnostics, School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Nørre Alle 20, 2200 N, Denmark

Received 16 November 2001; received in revised form 17 July 2002; accepted 17 July 2002

Abstract

The specificity of the MY.1E12 mAb that was generated by immunizing mice with human milk fat globule (HMFG) was

investigated. Fluorescein isothiocyanate (FITC)-conjugated peptides corresponding to a portion of the MUC1 tandem repeat

were enzymatically glycosylated with N-acetylgalactosamine, galactose, and then sialic acid. The MY.1E12 mAb was examined

for its affinity to the resulting glycopeptides by fluorescence polarization. Its affinity for the peptide whose Thr within the VTS

sequence bears a Neu5Aca2–3Galh1–3GalNAc trisaccharide (Kd = 1.4� 10� 7 M) was significantly higher than for the same

peptide whose Thr bears an unsialylated disaccharide (Kd = 3.9� 10� 6 M). The MY.1E12 mAb also bound strongly to a

purified recombinant MUC1 fusion protein with six tandem repeats that was expressed by transfected MCF-7 breast cancer

cells. The removal of sialic acids from the fusion protein significantly decreased MY.1E12 mAb reactivity, much more so than

the MUC1-specific 115D8 antibody, whose epitope is known to be destroyed by desialylation. Thus, the attachment of the

sialyla2–3Galh1–3h1-3GalNAc trisaccharide onto the Thr within the VTS motif significantly increases the binding of the

MY.1E12 antibody to the MUC1 repeat sequence.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Mucin; MUC1; Monoclonal antibody; Glycosylation; Sialic acid

0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0022 -1759 (02 )00298 -3

Abbreviations: ABTS, 2,2V-azino-bis (3-ethlbenzothiazoline-6-sulfonic acid); HPLC, high performance liquid chromatography; pp-GalNAc-

T, UDP-N-acetyl-D-galactosamine:polypeptide UDP-N-acetylgalactosaminyltransferase; HMFG, human milk fat globule; PNA, peanut

agglutinin; VVA, Vicia villosa agglutinin; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

* Corresponding author. Tel.: +81-3-5841-4870; fax: +81-3-5841-4879.

E-mail address: [email protected]. (T. Irimura).

www.elsevier.com/locate/jim

Journal of Immunological Methods 270 (2002) 199–209

1. Introduction

Antibodies generated against O-glycosylated pro-

teins sometimes recognize epitopes consisting of a

combination of peptides and carbohydrates. Classical

examples are some monoclonal antibodies (mAbs)

that are specific for blood group M or N glycophorins,

which are known to recognize epitopes formed by

ionic interactions between the sialic acid residues of

O-glycans and the amino groups at the terminal of

certain peptides (Blumenfeld and Adamany, 1978).

Another example is the anti-human fibronectin mAb

FDC-6 that recognizes an oncofetal structure consist-

ing of a hexapeptide and O-linked carbohydrates

(Matsuura et al., 1988, 1989). A series of mAbs

specific for the human epithelial mucin MUC1 also

often recognize epitopes comprised of peptide and

carbohydrate. Although many of these mAbs are

specific for the polypeptide portions of MUC1, the

binding of other mAbs is known to be influenced by

the glycosylation profile of MUC1 (Price et al., 1998).

One example is the BW835 antibody, which specifi-

cally binds to MUC1 when a Galh1–3GalNAc dis-

accharide is linked to Thr in the GVTSA sequence

present in the MUC1 tandem repeat (Hanisch et al.,

1995). Other MUC1-specific mAbs, such as the SM-3

mAb that also binds to the PDTR sequence in the

tandem repeat, are also influenced by the glycosyla-

tion of MUC1 (Burchell and Taylor-Papadimitriou,

1993). With regard to the SM-3 mAb, core 2 expres-

sion of O-glycans outside the PDTR appears to block

its binding whereas attachment of a GalNAc residue

to the Thr within the PDTR motif enhances this

binding (Lloyd et al., 1996; Karsten et al., 1998).

Extending this list of glycosylation-affected MUC1-

specific mAbs is the anti-human MUC1 MY.1E12

mAb, which we established by immunizing mice with

human milk fat globules (HMFG) and whose binding

to MUC1 is dependent on sialylation (Yamamoto et

al., 1996). This mAb is particularly interesting

because immunohistochemical analysis showed that

it recognizes MUC1 glycoforms that are expressed by

all invasive ductal carcinomas of the pancreas and

invasive cholangiocarcinomas of the liver but are not

frequently found on noninvasive tumors such as intra-

ductal papillary mucinous tumors of the pancreas and

bile duct cystadenocarcinomas of the liver (Yonezawa

and Sato, 1997; Yonezawa et al., 1998). The MY.1E12

antibody can also recognize histologically high

grades, advanced stages, and metastases of renal cell

carcinomas (Yonezawa and Sato, 1997; Yonezawa et

al., 1998; Higashi et al., 1999; Masaki et al., 1999).

Indeed, we recently showed that the MY.1E12 mAb

could identify MUC1-expressing tumors in pT2 gall-

bladder carcinoma patients that were distributed at the

deepest invading sites and that high expression levels

of the MY.1E12 mAb epitope on the tumor correlate

with an unfavorable postsurgical prognosis (Kawa-

moto et al., 2001). Correlation of mAb MY.1E12 with

poor prognosis was also shown with renal cell carci-

nomas (Fujita et al., 1999). These observations sug-

gest that the elucidation of the epitope recognized by

this mAb could substantially contribute to our under-

standing of carcinoma progression because it could

help reveal the biological function(s) and thus the

clinical significance of the different MUC1 glyco-

forms.

In the present study, we determined the epitope

recognized by the MY.1E12 mAb. We show that the

MY.1E12 antibody binds more profoundly to MUC1

tandem repeats when a carbohydrate chain Neu5-

Aca2–3Galh1–3GalNAc is linked to the Thr residue

in the VTS sequence in the tandem repeat than when

the same residue is linked only with a Galh1–3Gal-NAc. This binding specificity was not affected by the

addition of sialylated antigens at other positions. To

our knowledge, this is the first report that shows that

the epitope recognized by an anti-MUC1 antibody is a

sialylated carbohydrate chain that is attached to a

distinct position of the MUC1 polypeptide. It seems

that sialylation of this carbohydrate chain on VTS in

the tandem repeat of MUC1 has a profound effect on

the biological behavior of carcinoma cells bearing

MUC1.

2. Results

2.1. Preparation of MUC1 glycopeptides with

Sialyla2–3Galb1–3GalNAc trisaccharides at variouspositions

We synthesized fluorescein isothiocyanate (FITC)-

conjugated glycopeptides bearing a2-3-linked sialic

acids to investigate the epitope structure at the level of

O-glycan-peptide conjugates. Two different FITC-

H. Takeuchi et al. / Journal of Immunological Methods 270 (2002) 199–209200

conjugated peptides, FITC–GSTAPPAHGVTSAP-

DTK and FITC–GSTAPPAHGVTSK, were synthe-

sized. The sequences of these peptides correspond to

partial MUC1 tandem repeats. The peptides were

incubated with UDP-GalNAc as the donor substrate

and soluble recombinant UDP-GalNAc:polypeptide

N-acetylgalactosaminyltransferase (pp-GalNAc-T,

EC 2.4.1.41) 1 or pp-GalNAc-T2. The reaction mix-

tures were applied to reversed-phase high perform-

ance liquid chromatography (HPLC) and fractionated.

Molecular weights of the purified (glyco)peptides

were analyzed by matrix-assisted laser desorption

ionization-time of flight mass spectrometry

(MALDI-TOF MS) and the positions of the GalNAc

residues attached to the peptides were determined by

peptide sequencing. We found that the order of

GalNAc incorporation was strictly regulated by the

specificity of these enzymes. The first GalNAc resi-

due transferred by pp-GalNAc-T1 onto the longer

FITC –GSTAPPAHGVTSAPDTK peptide was

attached to its Thr-11. With the shorter FITC–

GSTAPPAHGVTSK peptide, the first GalNAc resi-

due transferred by pp-GalNAc-T2 was attached to

Thr-3, the second to Ser-2, and the third to Thr-11.

These four GalNAc-glycosylated peptides were

denoted as FITC–GSTAPPAHGVT*SAPDTK,

FITC–GST*APPAHGVTSK, FITC–GS*T*AP-

PAHGVTSK, and FITC–GS*T*APPAHGVT*SK

(T* stands for GalNAca–Thr). Gal was then added

to these four peptides by incubating them with UDP-

Gal as the donor substrate and the detergent-soluble

microsome fraction of human laryngeal carcinoma

H.Ep.2 cells as the source of h1-3Gal-T(s). After

purification of the products with reversed-phase

HPLC, the number of Gal residues incorporated into

the glycopeptides was determined by MALDI-TOF

MS analysis. According to the profiles (data not

shown), incorporation of Gal into all GalNAc residues

occurred and resulted in the following glycopeptides:

FITC – GSTAPPAHGVT*jSAPDTK, FITC –

G S T * jA P P A H G V T S K , F I T C –

G S * jT * jA P PA H G V T S K , a n d F I T C –

GS*jT*jAPPAHGVT*jSK (T*j stands for

Galh1–3GalNAca–Thr). The incorporation of Gal

was confirmed by the binding of the glycopeptides to

peanut agglutinin (PNA) and sensitivity to Bacillus

circulans h-galactosidase specific for h1-3 linkages.

Sialic acid residues were then added to the glycopep-

tides with the Gal–GalNAc glycans by incubating

them with CMP-Neu5Ac and soluble recombinant

ST3Gal II. After purification of the products with

reversed-phase HPLC, the number of Neu5Ac resi-

dues incorporated into each of the glycopeptides was

determined by MALDI-TOF MS. The profile of the

longer FITC-labeled GSTAPPAHGVTSAPDTK gly-

copeptide is shown in Fig. 1A. The molecular mass

Fig. 1. MALDI-TOF MS analysis of the nonbiotinylated (A) or

biotinylated (B) glycopeptides derived from a peptide FITC–

GSTAPPAHGVTSAPDTK. Mass indicates (M+H)+ form. (A) The

profiles of (a) FITC–GSTAPPAHGVT*jsSAPDTK (predicted

mass = 2639.6) and (b) FITC–GSTAPPAHGVT*jSAPDTK (pre-

dicted mass = 2348.4) are shown. (B) The profiles of (a) FITC–

GSTAPPAHGVT*jsSAPDTK–biotin (predicted mass = 2866.9)

and (b) FITC–GSTAPPAHGVT*jSAPDTK–biotin (predicted

mass = 2575.7) are shown.

H. Takeuchi et al. / Journal of Immunological Methods 270 (2002) 199–209 201

of the product corresponds to FITC–GSTAP-

PAHGVT*jsSAPDTK (T*js stands for Neu5-

Aca2–3Galh1–3GalNAca–Thr). The MALDI-

TOF MS analysis on the sialylated derivatives of

the shorter FITC-labeled GSTAPPAHGVTSK glyco-

peptide is shown in Fig. 2. The molecular masses

correspond to FITC –GST*jsAPPAHGVTSK,

FITC –GS*jsT*jsAPPAHGVTSK, and FITC –

GS*jsT*jsAPPAHGVT*jsSK. The anomeric form

and the linkage position of the sialyl residue were

confirmed by its sensitivity to the a2-3 linkage-spe-

cific sialidase from Streptococcus pneumonia.

2.2. Affinity of the MY.1E12 mAb to MUC1 peptides

with sialyla2–3Galb1–3GalNAc residues

The affinity of the purified MY.1E12 mAb to the

sialylated glycopeptides was measured by fluores-

cence polarization. The FP assay in solution is a

unique among various methods used to determine

molecular binding because it gives a direct, nearly

instantaneous measurement of a bound/free ratio of

ligands (Checovich et al., 1995). Rotational relaxa-

tion time is related to viscosity (g), absolute temper-

ature (T), molecular volume (V), and the gas constant

(R). Rotational relaxation time = 3gV/RT. Therefore,

if viscosity and temperature are held constant, polar-

ization is directly related to the molecular volume

(i.e., molecular size). Changes in molecular volume

result from the binding or dissociation of two mol-

Fig. 3. The binding glycopeptides (derived from GSTAPPAHGVT-

SAPDTK) to the MY.1E12 mAb. (A) Fluorescence polarization of

glycopeptides incubated with the MY.1E12 mAb at indicated

concentrations. Open squares, FITC –GSTAPPAHGVT*js

TSAPDTK; closed diamonds, FITC – GSTAPPAHGVT*jTSAPDTK. (B) Absorbance measured at 405 nm in the ELISA

assays to examine the binding of the mAb MY.1E12 to the

biotinylated glycopeptides. Open circles, biotinylated FITC–

GSTAPPAHGVT*jsTSAPDTK; closed circles, biotinylated

FITC–GSTAPPAHGVT*jsTSAPDTK.

Fig. 2. MALDI-TOF MS analysis of the glycopeptides derived from

FITC–GSTAPPAHGVTSK. Mass indicates (M+H)+ form. (a)

FITC–GS*jsT*jsAPPAHGVT*jsSK (predicted mass = 3568.6);

(b) FITC–GS*jsT*jsAPPAHGVTSK (predicted mass = 2911.9);

(c) FITC–GST*jsAPPAHGVTSK (predicted mass = 2255.3); and

(d) FITC–GSTAPPAHGVTSK (predicted mass = 1598.7).


ecules, from degradation, or conformational changes.

The MY.1E12 antibody bound approximately 30-fold

more strongly to FITC –GSTAPPAHGVT*js

SAPDTK (Kd = 1.4� 10� 7 M) than to the unsialy-

lated form (FITC–GSTAPPAHGVT*jSAPDTK,

Kd = 3.9� 10� 6 M) (Fig. 3A). Fluorescence polar-

ization did not increase upon incubation of the anti-

body with the FITC-conjugated MUC2 peptide

FITC–PTTTPITTTTK (data not shown). In order

to eliminate a possibility that these results were

obtained by fluorescence polarization (FP) assays,

the interaction between MY.1E12 mAb and the

glycopeptides was also determined by ELISA. For

this purpose, the glycopeptides, GSTAPPAH-

GVT*jsTSAPDTK and GSTAPPAHGVT*jTSAPDTK, were biotinylated at q-amino groups of

Lys residues. Successful biotinylation of the glyco-

peptides was confirmed by MALDI-TOF MS (Fig.

1B). The MY.1E12 mAb bound more strongly to

biotinylated FITC–GSTAPPAHGVT*jsTSAPDTKthan to biotinylated FITC–GSTAPPAHGVT*jTSAPDTK. The binding of the MY.1E12 mAb to

the latter peptide was not detected under the con-

dition used in these experiments. Importance of

sialylation of the Galh1–3GalNAc residue attached

to VTS sequence was confirmed by the ELISA

assays with FITC-labeled biotinylated glycopeptides

(Fig. 3B). With regard to comparisons of the second

FITC-labeled repeat peptide shorter in size, attach-

ment of a Neu5Aca2–3Galh1–3GalNAc trisacchar-

ide onto the Thr-3 residue alone or both the Thr-3

and Ser-2 residues did not increase the binding

activity of mAb MY.1E12. On the contrary, substan-

tial mAb binding occurred after the attachment of the

Neu5Aca2–3Galh1–3GalNAc trisaccharide to the

Thr-11 residue (Fig. 4). Thus, the attachment of the

Neu5Aca2–3Galh1–3GalNAc moiety onto Thr-11

within a VTS sequence significantly increases the

binding affinity of mAb MY.1E12.

Fig. 4. Fluorescence polarization of the shorter glycopeptides

incubated with the MY.1E12 mAb of the indicated concentrations.

Closed circles, FITC –GS*jsT*jsAPPAHGVT*jsSK; open

squares, FITC –GS*jsT*jsAPPAHGVTSK; open triangles,

FITC–GST*jsAPPAHGVTSK; open circles, FITC–GSTAP-

PAHGVTSK. The symbol @ in this panel indicates Neu5Aca2–

3Ga1h1–3GalNAc.

Fig. 5. Binding of MUCl-specific antibodies to native and sialidase-

treated MFP6 fusion protein derived from transfected breast cancer

MCF7 cells as measured by ELISA. (A) Profiles with the MY.1E12

mAb: closed diamonds, native MFP6 not treated with sialidase;

closed squares, sialidase-treated MFP6. (B) Profiles with the 115D8

mAb: closed diamonds, native MFP6 not treated with sialidase;

closed squares, sialidase-treated MFP6.


2.3. Binding of the MY.1E12 mAb to the MUC1 fusion

protein expressed by a tumor cell line

The breast cancer cell line MCF-7 was transfected

by a plasmid encoding an MUC1 fusion protein

(MFP6) containing six tandem repeats. The recombi-

nant protein was purified and the ability of the

MY.1E12 mAb to recognize it was examined by

ELISA. As a control, the 115D8 antibody, which

recognizes the sialylated repeat region of MUC1

(Price et al., 1998), was used. Both mAbs bound

strongly to MFP6 which indicates that, like the

115D8 mAb, the MY.1E12 mAb recognizes the

tandem repeat domain of the mucin. The removal

of sialic acid from MFP6 with the sialidase from

Clostridium perfringens significantly reduced the

binding of the mAb MY.1E12 (Fig. 5A), revealing

that sialylation is an important component of its

epitope. The removal of sialic acid from MFP6 also

reduced the binding of the 115D8 mAb, albeit to a

much smaller extent (Fig. 5B).

3. Discussion

The glycosylation status of MUC1 expressed by

tumor cells is an important issue in tumor immunol-

ogy as MUC1 glycosylation appears to determine its

immunogenicity as well as its biological function

(Denda-Nagai and Irimura, 2000; Hanisch and Muller,

2000). MUC1-specific mAbs that recognize particular

glycosylation patterns would be of great value in

research and clinical applications. Here we have

characterized the epitope recognized by one such

mAb, which is denoted as MY.1E12.

Characterization of the precise epitope structure

of the MY.1E12 mAb is required to clarify exactly

which amino acids within these peptides are

involved in the interaction with the antibody. For

example, it is not clear whether the Neu5Aca2–

3Galh1–3GalNAc is directly involved in the bind-

ing or whether the sialoglycan serves to create

conformational alterations in the repeat peptide that

generate the epitope recognized by this mAb. A

previous work by Live et al. (1999) suggested that

a-GalNAc-transfers induced conformational changes

in the peptides. Kirnarsky et al. (2000) also showed

that the attachment of an a-GalNAc residue to Thr

caused mild but distinct effects on the conformation

of the GVTSA sequence in MUC1 tandem repeats.

However, other peptide residues remote from VTS

contributing to the antibody binding due to conforma-

tional changes that occur when Galh1–3GalNAcattached to this Thr is sialylated remains to be eluci-

dated.

It is important to elucidate how the MY.1E12 mAb

epitope is biosynthesized because of its association

with highly malignant phenotypes of carcinomas. O-

Glycans are synthesized by the stepwise transfer of

monosaccharides (Clausen and Bennett, 1996), with

the first step being the addition of a GalNAc residue

onto Ser or Thr residues in mucin polypeptides. This

step is catalyzed by a family of pp-GalNAc-Ts (EC

2.4.1.41). The addition of GalNAc residues to mucin

polypeptides was found to be strictly regulated by the

coordinated action of the various pp-GalNAc-Ts

based on their substrate specificities and to be affected

by carbohydrate extensions on neighboring Ser and

Thr residues (Iida et al., 1999; Irimura et al., 1999). It

is known that pp-GalNAc-T1 and T3 specifically

transfer a GalNAc residue onto the Thr residue in a

VTS sequence (Wandall et al., 1997; Bennett et al.,

1998; Hassan et al., 2000), which corresponds to Thr-

11 in the repeat peptides used in our study. While the

expression of pp-GalNAc-T3 is known to be tissue-

specific, various carcinoma cell lines have been

shown to have an elevated expression of pp-Gal-

NAc-T3, as shown by Western blot analysis (Bennett

et al., 1996; Sutherlin et al., 1997; Shibao et al.,

2002). It will be interesting to examine whether the

ability of the MY.1E12 mAb to bind to tumor cells

correlates with their enhanced expression of pp-Gal-

NAc-T3. It is also unclear whether the sialic acid a3

linked to the Galh1–3GalNAc moiety is involved in

the direct binding of the MY.1E12 mAb to the

sialylated glycopeptides. Modification of the 6-

hydroxyl groups of GalNAc residues, such as a2-6

sialylation or h1-6 N-acetylglucosaminylation, may

also change the affinity of the MY.1E12 mAb for

MUC1.

The biological significance of the MY.1E12

epitope is not yet clear. MUC1 on breast cancer

cells was found to be a potential counter-receptor

for sialoadhesin, which is expressed on primary

human breast tumor-infiltrating macrophages and

binds specifically to Neu5Aca2–3Gal moieties


(Nath et al., 1999). The expression of the MY.1E12

epitope by carcinoma cells may also linked to other

biological properties. Recently, Dalziel and co-

workers showed that, regardless of expression of

Core 2 N-acetylglucosaminyltransferase-I, the O-

glycans of MUC1 became core 1-dominated struc-

tures when ST3Gal I expression was increased, as

occurs in breast cancer (Burchell et al., 1999;

Dalziel et al., 2001). Transcriptional regulation of

the ST3Gal I gene was found to be at least in part

regulated by the transcription factor Sp1 (Taniguchi

et al., 2001). Transcription of MUC1 gene is also

regulated by Sp1 (Kovarik et al., 1993, 1996;

Morris and Taylor-Papadimitriou, 2001). Thus, the

ST3Gal I and MUC1 genes, which both contribute

to the expression of the MY.1E12 mAb epitope,

may be regulated in concert at the level of tran-

scription.

The desialylation of MUC1 was previously shown

to decrease the binding of some anti-MUC1 mAbs.

According to Dai et al. (1998), the most prominent

mAb affected was the 115D8 mAb. As a control for the

effect of desialylation on the ability of the MY.1E12

mAb to recognize recombinant MUC1 generated by

transfected breast cancer cells, we also tested the

115D8 mAb. We found that the decrease in 115D8

mAb binding to the sialidase-treated protein was much

smaller than the decrease seen with the MY.1E12

mAb. These observations indicate that the MY.1E12

mAb is very useful in detecting the expression of

MUC1 bearing a2-3-linked sialic acid moieties.

Although the binding of many anti-MUC1 mAbs is

dependent on MUC1 glycosylation, there are few

mAbs that have been shown to recognize carbohy-

drates attached to a distinct Thr or Ser residue within

the MUC1 tandem repeats. An exception is the

BW835 mAb, which recognizes an epitope generated

by Galh1–3GalNAc moiety that is also attached to the

VTS motif (Hanisch et al., 1995). Thus, the specificity

of the MY.1E12 mAb is highly unique because this

antibody recognizes the Neu5Aca2–3Galh1–3Gal-NAc trisaccharide linked to the Thr residue within

the VTS sequence.

In conclusion, the present study has revealed that

the MY.1E12 mAb binds to MUC1 containing the

Neu5Aca2–3Galh1–3GalNAc trisaccharide at a dis-

tinct Thr residue within the VTS sequence present in

its tandem repeat.

4. Materials and methods

4.1. Antibodies

The anti-MUC1 mucin mAb MY.1E12 was estab-

lished by immunizing mice with HMFG (Yamamoto

et al., 1996). Purified MUC1-specific mAb 115D8

was purchased from Centocore or supplied in the

context of the ISOBM TD-5 workshop on monoclonal

antibodies against MUC1.

4.2. Synthesis and labeling of peptides

The synthetic peptides GSTAPPAHGVTSAPDTK

and GSTAPPAHGVTSK, which correspond to partial

tandem repeat sequences in MUC1, were synthesized

by the standard Fmoc-solid phase method on a Model

9020 peptide synthesizer (Milligen, Burlington, MA)

and purified by reversed-phase HPLC. Molecular

weights and sequences of the peptides were confirmed

by MALDI-TOF MS analysis (see below) and amino

acid sequencing (see below), respectively. The pep-

tides were labeled exclusively at their N-terminal

amino acids with FITC at pH 7.5 adjusted with 100

mM HEPES buffer.

4.3. Enzymatic synthesis of glycopeptides

Synthesis of GalNAc-attached peptides was per-

formed by incubating the peptides with soluble

recombinant pp-GalNAc-T1 or T2, which were pre-

pared as described previously (Wandall et al., 1997).

The standard reaction mixture consisted of 50 mM

HEPES buffer (pH 7.5), 5 mM MnCl2, 5 mM 2-

mercaptoethanol, 0.1% Triton X-100, 1 mM UDP-

GalNAc (Sigma, St. Louis, MO), 5–50 AM FITC-

conjugated peptides, and recombinant pp-GalNAc-T1

or T2. All reactions were performed at 37 jC and the

products were separated by reversed-phase HPLC (see

below). After the molecular weights of the GalNAc-

glycosylated peptides had been measured by MALDI-

TOF MS analysis (see below), the sites where Gal-

NAc had been incorporated into the peptides were

determined by amino acid sequencing (see below).

Gal residues were transferred onto GalNAc resi-

dues on the glycosylated peptides by using the deter-

gent-soluble microsome fraction of human laryngeal

carcinoma H.Ep.2 cells as the enzymatic source. The


reaction conditions of Gal incorporation into peptides

with GalNAc residues were the same as for the

GalNAc incorporation except for the enzyme source

(final concentration 0.7 mg/ml) and the fact that UDP-

Gal (final concentration 1 mM) was used. All reac-

tions were performed at 37 jC, and the products were

separated by reversed-phase HPLC. Gal incorporation

in a h1-3 linkage were confirmed by the molecular

weight of the products, the binding of PNA, and the

sensitivity to the h1-3 linkage-specific h-galactosi-dase derived from B. circulans (Fujimoto et al., 1998).

Sialic acids (Neu5Ac) residues were transferred

onto Gal residues on the glycopeptides by using

recombinant ST3Gal II (Calbiochem, La Jolla, CA)

as the enzyme and CMP-Neu5Ac as the donor sub-

strate. The sialylation reaction mixture consisted of 100

mM cacodylate–HCl (pH 6.0), 20 mM MnCl2, 5 AMFITC-conjugated glycopeptides, 1 mM CMP-Neu5Ac,

1 mM PMSF, 1 Ag/ml aprotinin, 1 Ag/ml leupeptin, 0.5

Ag/ml pepstatin A, 5 mM 2-mercaptoethanol, 3.4 mU

recombinant ST3Gal II, and 0.1% Triton CF-54. All

reactions were performed at 37 jC and the products

were separated by reversed-phase HPLC. Sialylation in

an a2-3 linkage was confirmed by molecular weights

of the products and the sensitivity of the products to the

a2-3 linkage-specific sialidase derived from S. pneu-

monia (Prozyme, San Leandro, CA).

4.4. Biotinylation of glycopeptides

The glycopeptides were incubated with large

excess amounts of N-hydroxysuccinimido-biotin

(Sigma) at room temperature for 4 h in 0.05 M

Na2CO3. The biotinylated glycopeptides were purified

by reversed-phase HPLC (see below). Successful

bitotinylation corresponding to the theoretical increase

in the molecular mass (227.3) was confirmed by

MALDI-TOF MS (see below).

4.5. Reversed-phase HPLC

The (glyco)peptides were separated by reversed-

phase HPLC (JASCO, Tokyo, Japan) with a Cosmosil

column (C18, 10� 250 mm, Nacalai tesque, Japan).

The column was eluted with a linear gradient ranging

from 0% to 50% solvent B (0.05% trifluoroacetic

acid, 70% 2-propanol in acetonitrile) in solvent A

(0.05% trifluoroacetic acid in water) at a flow rate of 2

ml/min for 30 min. Eluates were monitored by fluo-

rescence intensity at 520 nm.

4.6. MALDI-TOF MS analysis of peptides

The peptides were applied on a tip and mixed with

a 10 mg/ml solution of a-cyano-4-hydroxycinnamic

acid dissolved in 0.1% trifluoroacetic acid–50% etha-

nol in water. All mass spectra were obtained on a

Voyager Elite instrument (Nippon PerSeptive Biosys-

tems, 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 processed by

using GRAMS/386 software. Mass calibration was

performed using ACTH and angiotensin as standards.

4.7. Amino acid sequencing

Pulsed liquid Edman degradation amino acid

sequencing of the peptides was performed with the

Applied Biosystems 490 Procise protein sequencing

system (Perkin Elmer, Norwalk, CT). This system

identified a phenylthiohydantoin (PTH)-derivative of

the GalNAc-attached Thr residue as a pair of peaks

eluting near the positions of PTH–Ser and PTH–Thr

(Gerken et al., 1997). These eluting positions were

confirmed by amino acid sequencing of the fully

GalNAc-glycosylated peptide, PT*T*T*PLK (T*

stands for a GalNAc-attached Thr residue).

4.8. Fluorescence polarization

Glycopeptides (final concentration 10 nM) were

incubated at room temperature for 2 h with the

purified MY.1E12 antibody at various indicated

concentrations in 50 mM HEPES (pH 7.4), 150

mM NaCl, 2 mM CaCl2. Subsequently, the fluores-

cence intensity and fluorescence polarization of the

reaction mixture were measured by BEACON 2000

(TAKARA, Japan).

4.9. Construction of a plasmid encoding the MUCI

fusion protein (MFP6)

An MUC1 tandem repeat sequence containing

terminal restriction sites was constructed by annealing

and ligating four 5Vphosphorylated 60-mer oligonu-


cleotides. The procedure resulted in several clones

that contained 2–16 tandem repeats flanked by the 3Vand 5V sequences of the original pBS TRI. A clone

containing six tandem repeats was chosen and subcl-

oned into the pCEP-PU expression vector using the

5VNheI and the 3VNotI sites (Kohfeldt et al., 1997).

pCEP-PU already contained the signal peptide of the

BM40 extracellular matrix protein, followed by a

hexa-histidine sequence, a myc tag, and the NheI

and NotI restriction sites that were used for the in-

frame insertion of the MUC1 tandem repeat construct.

Restriction enzymes and all other DNA modifying

enzymes were obtained from New England Biolabs,

Frankfurt am Main, Germany.

4.10. Generation of MFP6 by a transfected tumor cell

line

The breast cancer cell line MCF-7 was obtained

from the American Type Culture Collection and was

cultured in 80- or 160-cm2 flasks at the same con-

ditions. Minimal essential medium supplemented with

Glutamax I, 10% fetal calf serum, 0.1 mM sodium

pyruvate, 100 IU penicillin, and 100 Ag/ml strepto-

mycin was used. The cell line was transfected with 0.1

Ag/ml of the plasmid encoding MFP6 using the Super-

fect (Quiagen, Hilden, Germany) lipofection reagent,

after which puromycin was added to all media.

4.11. Purification of MFP6

Conditioned supernatants from confluent cell layers

were collected, centrifuged at 1000� g at 4 jC for 10

min and dialyzed against several changes of deminer-

alized water. Dialyzed supernatants were adjusted to 50

mM sodium phosphate pH 8.0, 200 mM sodium

chloride, 1 mM imidazol, 5 mM 2-mercaptoethanol,

and 10% ethanol, and centrifuged at 20,000� g for 45

min at 4 jC. Up to 1 l of the adjusted supernatant was

loaded onto a column of 2 ml Ni–NTA Superflow

(Quiagen) and chromatographed according to standard

protocols. Fusion proteins in fractions 2–5 were sub-

jected to HPLC purification on a reversed phase

column (Vydac 208TPI015, MZ Analysentechnik,

Mainz, Germany). The quality of the preparations

was analyzed by sodium dodecylsulfate polyacryla-

mide gel electrophoresis andWestern blot analysis with

anti-myc antibody.

4.12. ELISA assays

Enzyme immunoassays with biotinylated glyco-

peptides were performed in polystyrene 96-well plates

(greiner bio-one, Germany) that had been precoated

with 200 Al/well of ascitic fluids of mAb MY.1E12

diluted at 1:1000 with 0.05 N NaHCO3, pH 9.6. After

blocking with 3% BSA in PBS, pH 7.4, for 3 h at

room temperature, a 50-Al aliquot of the biotinylated

glycopeptides were serially diluted twofold in 50 mM

HEPES, pH 7.4, 150 mM NaCl, 2 mM CaCl2 and

incubated for 1.5 h at room temperature. One hundred

microliters of ZyMAXk streptavidin–HRP Conju-

gate (Zymed, San Francisco, CA, USA) diluted at

1:1000 with 1% BSA in PBS, pH 7.5, was added into

each well and incubated for 1 h at room temperature.

One hundred microliters of 1 mM 2,2V-azinobis(3-ethylbenzthiazoline-sulfonic acid (ABTS) in 100 mM

citric acid, pH 4.2, containing 1/1000 volume of H2O2

was added into each well. Absorbance at 405 nm was

measured on a microplate reader.

Enzyme immunoassays with MFP6 fusion proteins

were performed in polystyrene microtitration plates

(Nunc, Wiesbaden, Germany) that had been precoated

with MFP6 (2 Ag/ml) by drying 50-Al solutions in 0.1

M carbonate buffer, pH 9.6. After blocking with 5%

BSA in PBS, pH 7.2, for 1 h at 37 jC, a 50-Al aliquotof the primary antibody (MY.1E12 prediluted at

1:1000 or 115D8 prediluted at 10 Ag/ml) was serially

diluted twofold in washing buffer (0.5% BSA in PBS)

and incubated overnight at 4 jC. Bound antibody was

quantitated by incubation with anti-mouse Ig–biotin

(E413, Dako, Hamburg, Germany) diluted 1:5000 (1

h, 37 jC) followed by streptavidin–alkaline phospha-

tase (Roche) diluted 1:5000 (30 min, 30 jC). Theduplicate assays were developed with p-nitrophenyl-

phosphate (1 mg/ml) in 50 mM diethanolamine buffer,

pH 9.8, containing 0.5 mM MgC12, and read at 405

nm. To liberate sialic acid from MFP6, the coated

antigen was treated with 50 Al of C. perfringens

neuraminidase (4 mU/ml) in 50 mM sodium citrate

buffer, pH 4.5, at 37 jC.

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Documents

The epitope recognized by the unique anti-MUC1 monoclonal antibody MY.1E12 involves sialylα2–3galactosylβ1–3N-acetylgalactosaminide linked to a distinct threonine residue in