Báo cáo khoa học: A monoclonal antibody, PGM34, against 6-sulfated blood-group H type 2 antigen, on the carbohydrate moiety of mucin doc

A monoclonal antibody, PGM34, against 6-sulfated
blood-group H type 2 antigen, on the carbohydrate moiety
of mucin
Analysis of the epitope sequence and immunohistochemical study
Daigo Tsubokawa
1
, Yukinobu Goso
1
, Akira Sawaguchi
2
, Makoto Kurihara
3
, Takafumi Ichikawa
4
,
Noriko Sato
5
, Tatsuo Suganuma
2
, Kyoko Hotta
4
and Kazuhiko Ishihara
1
1 Department of Biochemistry, Kitasato University Graduate School of Medical Sciences, Sagamihara, Japan
2 Department of Anatomy, Ultrastructural Cell Biology, Faculty of Medicine, University of Miyazaki, Japan
3 Isehara Research Laboratory, Kanto Chemical Co. Inc., Isehara, Japan
4 Department of Biochemistry, Kitasato University School of Medicine, Sagamihara, Japan
5 Department of Instrumental Analysis, Kitasato University School of Pharmaceutical Sciences, Tokyo, Japan
The gastric mucus which covers the mucosal surface is
considered to be a major factor in the gastric defense
mechanism against various aggressive factors, such as

gastric acid and pepsin [1]. Mucus-secreting cells of the
mammalian gastric mucosa have been mainly classified
into surface mucous and gland mucous cells [2,3]. The
types of mucus accumulated in and⁄ or secreted from
these two types of cell are individually characterized
Keywords
monoclonal antibody; mucin; mucous cells;
sulfated oligosaccharide
Correspondence
K. Ishihara, Department of Biochemistry,
Kitasato University School of Allied Health
Sciences, 1-15-1 Kitasato, Sagamihara, 228-
8555, Japan
Fax ⁄ Tel: +81 42 778 8262
E-mail:
(Received 9 November 2006, revised 16
January 2007, accepted 7 February 2007)
doi:10.1111/j.1742-4658.2007.05731.x
Mucin, a major component of mucus, is a highly O-glycosylated, high-
molecular-mass glycoprotein extensively involved in the physiology of
gastrointestinal mucosa. To detect and characterize mucins derived from
site-specific mucous cells, we developed a monoclonal antibody, designated
PGM34, by immunizing a mouse with purified pig gastric mucin. The reac-
tivity of PGM34 with mucin was inhibited by periodate treatment of the
mucin, but not by trypsin digestion. This suggests that PGM34 recognizes
the carbohydrate portion of mucin. To determine the epitope, oligosaccha-
ride-alditols obtained from pig gastric mucin were fractionated by succes-
sive gel-filtration, ion-exchange, and normal-phase HPLC, and tested for
reactivity with PGM34. Two purified oligosaccharide-alditols that reacted
with PGM34 were obtained. Their structures were determined by NMR

spectroscopy as Fuca1–2Galb1–4GlcNAc(6SO
3
H)b1–6(Fuca1–2Galb1–3)
GalNAc-ol and Fuca1–2Galb1–4GlcNAc(6SO
3
H)b1–6(Galb1–3)GalNAc-ol.
None of the defucosylated or desulfated forms of these oligosaccharides
reacted with PGM34. Thus, the epitope of PGM34 was determined as the
Fuca1–2Galb1–4GlcNAc(6SO
3
H)b- sequence. Immunohistochemical exam-
ination of rat gastrointestinal tract showed that PGM34 stained surface
mucous cells close to the generative cell zone in the gastric fundus and gob-
let cells in the small intestine, but only slightly stained antral mucous cells
in the stomach. These data, taken together, show that PGM34 is a very
useful tool for elucidating the role of mucins with characteristic sulfated
oligosaccharides.
Abbreviations
CCG, cationic colloidal gold; CG, colloidal gold; GalNAc-ol, N-acetylgalactosaminitol; HID, high iron diamine; HMBC, heteronuclear multiple-
bond correlation; HMQC, heteronuclear multiple-quantum coherence; NHS, normal horse serum.
FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS 1833
by a combination of galactose oxidase-cold thionin
Schiff staining and paradoxical concanavalin A stain-
ing [4]. This method showed that these two types of
mucus cooperatively construct a stable mucus gel
layer, and therefore, it is postulated that these two
types of mucus have distinct physiological roles in the
gastric mucosal defense mechanism [5].
Mucin, a highly O-glycosylated, high-molecular-mass
glycoprotein, is a major component of gastrointestinal

mucus and plays important roles. In the stomach, the
protein parts of the surface and gland mucins are dif-
ferent from each other: MUC5AC is the dominant
mucin in surface mucus, and MUC6 is the dominant
mucin in gland mucus [6,7]. The carbohydrate parts of
the two mucins are also different from each other as
already described. Furthermore, a distinct control
mechanism underlies the biosynthesis and accumula-
tion of a mucin in a specific region and layer of the
gastric mucosa [8,9]. For the precise characterization
of individual mucins on biochemical and physiological
bases, mAbs that recognize the mucin in one cell type
and not in the other are needed. Many mAbs that
react with specific mucin molecules obtained from
mammalian gastric mucosa have been developed in
our laboratory, and their properties histochemically
and biochemically characterized. The histochemical
study showed that the different types of mucin pro-
duced by the surface and gland mucous cells of the
gastric mucosa are stained differently by mAbs
[10–12]. For instance, mucin derived from surface
mucous cells of the rat stomach was stained with the
mAb, RGM11 [12], whereas mucins derived from neck
cell and pyloric gland cell mucus were stained with the
mAb, HIK1083 [13]. Although the epitope of RGM11
is not yet resolved, that of HIK1083 has been deter-
mined as a peripheral a-linked GlcNAc on the mucin
oligosaccharides. Interestingly, this epitope is restric-
ted to gastrointestinal mucus [14]. Furthermore,
Kawakubo et al. [15] reported that glycoproteins with

this epitope on their oligosaccharides function as a
natural antibiotic, protecting the host from Helico-
bacter pylori. This suggests that mucin bearing a char-
acteristic oligosaccharide chain has a specific biological
function. Thus, epitope analysis of a mAb that reacts
with a specific oligosaccharide chain bound to the
mucin molecules is needed to clarify the biological
function of the particular oligosaccharide.
In this study, mAb PGM34 was established as an
antigen with purified pig gastric mucin. Because PGM34
selectively reacts with mucin obtained from gastric sur-
face mucous cells and small intestinal goblet cells of the
rat, and immunohistochemically stains the generative
cell zone specifically in the surface mucosa of the rat
gastric fundus, we were interested in an epitope recog-
nized by PGM34 which may have a specific biological
role. This paper presents the unique epitope sequence of
PGM34 containing a sulfate residue and histochemical
observations showing the unique distribution of this epi-
tope sequence in the rat gastrointestinal tract.
Results
Study of the antigenic determinant of PGM34
by modification of mucin
PGM34 was developed using pig gastric mucin as an
antigen. To characterize the epitope of PGM34, perio-
date oxidation and trypsin digestion of the purified
mucin were performed to degrade the carbohydrate
and peptide moieties, respectively. The residual anti-
genic activity was then tested by ELISA. Periodate oxi-
dation reduced the antigenic activity with PGM34,

whereas trypsin digestion did not affect the reactivity
with this mAb (data not shown). These results indicate
that the carbohydrate moieties of the mucin are
involved in the epitope of PGM34.
Reactivity of PGM34 with oligosaccharides
obtained from pig gastric mucin
For characterization of the epitope of PGM34,
reduced oligosaccharides were prepared from partially
purified pig gastric mucin by alkaline borohydride
reduction, fractionated on a Bio-Gel P-6 column, and
tested for reactivity with this mAb. Five fractions,
monitored by hexose measurement, were obtained
(Fig. 1A), and their antigenic activity with PGM34
was examined by competitive ELISA. Fractions 1, 3
and 5 inhibited the reaction of PGM34 with the puri-
fied mucin on the ELISA plate, with fraction 1 achiev-
ing the strongest inhibition (Fig. 1B). Fractions 2 and
4 produced almost the same results as fractions 3 and
5 (data not shown). Although the data indicated that
all the oligosaccharide fractions reacted with PGM34,
fraction 5 was expected to be the easiest to analyze for
the structure of the oligosaccharides because of their
relatively small size. Therefore, fraction 5 was chosen
for epitope analysis, and further purified by anion-
exchange chromatography on a QAE-Toyopearl-550C
column. As shown in Fig. 2A, one neutral oligosaccha-
ride fraction, N, eluted with distilled water, and two
acidic oligosaccharide fractions, A1 and A2, eluted
from the column with 0.2–0.3 m sodium acetate, were
obtained. The inhibition assay indicated that fraction

A1 significantly reacted with PGM34, whereas frac-
tions N and A2 did not (Fig. 2B).
mAb against sulfated oligosaccharide of mucin D. Tsubokawa et al.
1834 FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS
Fraction A1 was further purified by two-step nor-
mal-phase HPLC using a TSK-Gel Amide-80 column.
From the first step, six major fractions, designated
A1-1 to A1-6, and several minor fractions were
obtained (Fig. 3). The inhibition assay showed that
fractions A1-4 and A1-5 reacted significantly with
PGM34. Therefore, these two fractions were further
purified individually by the second-step HPLC. As
shown in Fig. 4A, fraction A1-5 separated into three
fractions, designated A1-5a, A1-5b and A1-5c. Fraction
A1-4 also separated into three fractions, designa-
ted A1-4a, A1-4b and A1-4c (data not shown). The
inhibition assay indicated that fractions A1-5a, A1-5b
(Fig. 4B) and A1-4c (data not shown) reacted signifi-
cantly with PGM34, but the other fractions did not
react with PGM34.
Determination of carbohydrate composition
of the oligosaccharides
The oligosaccharides fractionated by the first-step
HPLC were analyzed by MALDI-TOF ⁄ MS (Table 1).
The masses of the oligosaccharides ranged from 675 to
1325, corresponding to trisaccharides t o heptasaccharides.
Fig. 1. Bio-Gel P-6 column chromatography of oligosaccharides prepared from partially purified pig gastric mucin by alkaline borohydride
reduction and the reactivity of oligosaccharides with PGM34. (A) Reduced oligosaccharide sample was loaded on to a Bio-Gel P-6 column
and eluted with water. The hexose content (s) of each fraction was assessed by the phenol ⁄ sulfuric acid method. Five oligosaccharide frac-
tions, 1–5, were pooled and used for further analysis. Elution positions of (A) Dextran T-500 (500 kDa) (B) maltohexaose and (C) glucose are

indicated. (B) The antigenic activities of various amounts of the oligosaccharides from the pooled fractions, fraction 1 (s), fraction 3 (d), frac-
tion 5 (n), were examined by competitive ELISA as described in Experimental procedures. Data are expressed as mean ± SD from three
experiments.
Fig. 2. QAE-Toyopearl-550C anion-exchange chromatography of fraction 5 in Fig. 1 and the reactivity of oligosaccharides with PGM34. (A)
Fraction 5 was loaded on to a column of QAE-Toyopearl-550C and eluted with water followed by a linear gradient of 0.0–0.6
M sodium acet-
ate (dashed line). The hexose content (s ) of each fraction was assessed by the phenol ⁄ sulfuric acid method. The neutral oligosaccharide
fractions (N) and two acidic oligosaccharide fractions (A1 and A2) were pooled and used for further analysis. (B) The antigenic activities of
various amounts of the oligosaccharides from the pooled fractions, N (s), A1 (n) and A2 (d), were examined by competitive ELISA as des-
cribed in Experimental procedures. Data are expressed as mean ± SD from three experiments.
D. Tsubokawa et al. mAb against sulfated oligosaccharide of mucin
FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS 1835
The compositions of all the oligosaccharides tested
were assigned to the appropriate acidic oligosaccha-
ride-alditols, bearing either a sulfate or a sialic acid
residue, as well as having N-acetylgalactosaminitol
(GalNAc-ol) at the reducing terminus based on their
masses. Fraction A1-5 contained three oligosaccharides
with m ⁄ z 976, 1121 and 1179. These were separated
into the three fractions A1-5a, A1-5b and A1-5c with
m ⁄ z 976, 1121 and 1179, respectively, indicating that
highly purified oligosaccharides were obtained after the
second-step HPLC in this case (Table 2).
Amino sugar analyses of oligosaccharides A1-5a
and A1-5b showed that the molar ratio of GalNAc-ol,
GalNAc and GlcNAc was 1.0 : 0.0 : 1.1 and
1.0 : 0.0 : 0.9, respectively. These results agree with the
carbohydrate compositions of the oligosaccharides
expected from the molecular mass data.
Fig. 3. First-step HPLC of oligosaccharide fraction A1 in Fig. 2 using

TSK-Gel Amide-80 columns. Fraction A1 was chromatographed on
two TSK-Gel Amide-80 columns and eluted by a linear gradient of
acetonitrile. Absorption was monitored at 210 nm. Oligosaccharide
fractions A1-4 and A1-5 were further characterized.
Fig. 4. Second-step HPLC of oligosaccharide
fraction A1-5 in Fig. 3 using TSK-Gel Amide-
80 columns and the reactivity of oligosac-
charides with PGM34. (A) Fraction A1-5 was
chromatographed on two TSK-Gel Amide-80
columns and eluted under isocratic condi-
tions. The absorption was monitored at
210 nm. (B) The antigenic activities of var-
ious amounts of the oligosaccharides from
three purified oligosaccharide fractions,
A1-5a (n), A1-5b (m) and A1-5c (s), were
examined by competitive ELISA as des-
cribed in Experimental procedures. Data are
expressed as mean ± SD from three
experiments.
Table 1. Oligosaccharide structures separated by the first-step
HPLC: identified by MALDI-TOF ⁄ MS. Fractions that reacted with
PGM34 are indicated with an asterisk.
Fraction
[M–H]
–
(m ⁄ z)
Expected composition of
oligosaccharide-alditols
A1-1 675 (Neu5Ac)(Hex)GalNAc-ol
A1-2 871 (SO

3
H)(Hex)(HexNAc)
2
GalNAc-ol
A1-3 675 (Neu5Ac)(Hex)GalNAc-ol
822 (Neu5Ac)(dHex)(Hex)GalNAc-ol
830 (SO
3
H)(Hex)
2
(HexNAc)GalNAc-ol
871 (SO
3
H)(Hex)(HexNAc)
2
GalNAc-ol
1017 (SO
3
H)(dHex)(Hex)(HexNAc)
2
GalNAc-ol
A1-4* 879 (Neu5Ac)(Hex)(HexNAc)GalNAc-ol
976 (SO
3
H)(dHex)(Hex)
2
(HexNAc)GalNAc-ol
1017 (SO
3
H)(dHex)(Hex)(HexNAc)

2
GalNAc-ol
A1-5* 976 (SO
3
H)(dHex)(Hex)
2
(HexNAc)GalNAc-ol
1121 (SO
3
H)(dHex)
2
(Hex)
2
(HexNAc)GalNAc-ol
1179 (SO
3
H)(dHex)(Hex)
2
(HexNAc)
2
GalNAc-ol
A1-6 1179 (SO
3
H)(dHex)(Hex)
2
(HexNAc)
2
GalNAc-ol
1325 (SO
3

H)(dHex)
2
(Hex)
2
(HexNAc)
2
GalNAc-ol
mAb against sulfated oligosaccharide of mucin D. Tsubokawa et al.
1836 FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS
The putative carbohydrate compositions of the
oligosaccharides that reacted positively with PGM34
were as follows: A1-4c, (SO
3
H)(dHex)(Hex)(GlcNAc)
(GalNAc)(GalNAc-ol) and ⁄ or (SO
3
H)(dHex)(Hex)
2
(GlcNAc)(GalNAc-ol); A1-5a, (SO
3
H)(dHex)(Hex)
2
(GlcNAc)(GalNAc-ol); A1-5b, (SO
3
H)(dHex)
2
(Hex)
2
(GlcNAc)(GalNAc-ol).
NMR spectroscopy

Purified oligosaccharides with reactivity with PGM34,
A1-5a ( 0.3 mg) and A1-5b ( 1.8 mg), were subjec-
ted to NMR spectroscopy. Figure 5 shows the one-
dimensional
1
H-NMR spectra of these two oligosac-
charides. In the spectrum of A1-5b, b-anomeric reso-
nances (4.60 p.p.m., 4.53 p.p.m. and 4.54 p.p.m.) were
recognized as two residues of the b-linked Gal, and
that of the b-linked GlcNAc, respectively, by their
coupling to a high-field H-2 resonance and pattern of
the cross-peaks in the TOCSY spectrum (data not
shown). As shown in Fig. 5B, two lower-field a-ano-
meric resonances were also recognized as two residues
of the a-linked Fuc by a method similar to that des-
cribed above. The carbohydrate composition of A1-5b
obtained from the NMR spectrum agreed with that
expected from the data obtained from the molecular
mass and amino sugar analyses. From the
13
C chem-
ical shifts of the heteronuclear multiple-quantum
coherence (HMQC) spectra of A1-5b (Table 3), there
was no substitution on the two a-linked Fuc residues,
indicating that these Fuc residues are present at the
nonreducing terminus in this structure. These two Fuc
residues attached to the two b-linked Gal residues at
position 2 (3.59 p.p.m., 3.63 p.p.m.), which could be
confirmed by the lower field changes in the HMQC
spectrum (+ 11.1 p.p.m., +9.4 p.p.m.) of Gal as

Table 2. Oligosaccharide structures separated by the second-step
HPLC: identified by MALDI-TOF ⁄ MS. Fractions that reacted with
PGM34 are indicated by an asterisk.
Fraction
[M–H]
–
(m ⁄ z)
Expected composition of
oligosaccharide-alditols
A1-5a* 976 (SO
3
H)(dHex)(Hex)
2
(HexNAc)GalNAc-ol
A1-5b* 1121 (SO
3
H)(dHex)
2
(Hex)
2
(HexNAc)GalNAc-ol
A1-5c 1179 (SO
3
H)(dHex)(Hex)
2
(HexNAc)
2
GalNAc-ol
Fig. 5. One-dimensional
1

H NMR spectroscopy of oligosaccharides A1-5a (A) and A1-5b (B).
D. Tsubokawa et al. mAb against sulfated oligosaccharide of mucin
FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS 1837
compared with the standard b-methylated Gal [13]. A
heteronuclear multiple-bond correlation (HMBC) spec-
trum supported this by the presence of remote coup-
ling between the anomeric
1
H of Fuc and
13
Cat
position 2 of Gal. The residue of the b-linked GlcNAc
and GalNAc-ol at the reducing terminus showed the
glycosylation shift at position 4, and positions 3 and 6,
respectively, based on the
13
C chemical shift assess-
ment. The following remote coupling could also be
recognized from the HMBC spectrum: anomeric
1
Hof
one b-Gal (4.60 p.p.m.) and position 4
13
Cofb-Glc-
NAc (77.8 p.p.m.), anomeric
1
H of another b-Gal
(4.53 p.p.m.) and position 3
13
C of GalNAc-ol

(77.1 p.p.m.), anomeric
1
Hofb-GlcNAc (4.54 p.p.m.)
and position 6
13
C of GalNAc-ol (73.9 p.p.m.). A
lower field change (+ 8.1 p.p.m.) in the
13
C chemical
shifts at position 6 indicated the substitution by the
sulfate residue as compared with that of the standard
b-methylated GlcNAc. Furthermore, the lower field
shift of the anomeric proton of a b-linked Gal
(4.60 p.p.m.) attached to b-GlcNAc supported the sulf-
ation of position 6 of the GlcNAc residue [16]. These
NMR spectral data support the hypothesis that oligo-
saccharide A1-5b has the following structure:
Owing to the lower amount applied, only the
1
H
NMR spectra could be obtained for the A1-5a analysis.
Three b-anomeric proton signals around 4.5 p.p.m. and
one lower-field a-anomeric signal were observed in the
A1-5a spectrum (Fig. 5A). The chemical shifts of the b-
linked GlcNAc (4.53 p.p.m.), one of the two b-linked
Gal (4.60 p.p.m.) and a-linked Fuc (5.19 p.p.m.) are
almost identical with those of A1-5b based on a
1
H
chemical shift assessment. Higher field changes at ano-

meric ()0.11 p.p.m.) and position 2 ()0.10 p.p.m.) pro-
tons of another b-linked Gal (4.42 p.p.m.) were
interpreted as no substitution with the a-linked Fuc as
compared with A1-5b in the
1
H chemical shifts. The
structure of A1-5a is estimated to be as follows from
the common chemical shifts with A1-5b:
Table 3. Chemical shifts of each sugar component. Chemical
shifts labeled with either an asterisk or a dagger indicate occur-
rence of glycosylation or sulfation shift, respectively. ND, Not
determined.
Sugar
A1-5a
1
H
A1-5b
Standards
b
13
C
1
H
13
C
GalNAc-ol
Position 1 3.68 ⁄ 3.74 3.72 ⁄ 3.76 63.0 61.5
Position 2 4.35 4.35 54.2 51.5
Position 3 4.02* 4.04* 77.1* 68.4
Position 4 3.43 3.47 71.6 69.4

Position 5 4.27 4.20 70.6 69.8
Position 6 3.62 ⁄ 3.9* 3.64 ⁄ 3.9* 73.9* 63.2
Ac-CH
3
2.03 2.02 25.0 21.7
b-GlcNAc
Position 1 4.53 4.54 104.4 101.9
Position 2 3.73 3.75 57.9 55.4
Position 3 3.64 3.65 75.0 73.9
Position 4 3.85* 3.83* 77.8* 69.9
Position 5 3.62 3.63 75.7 75.8
Position 6 4.25 ⁄ 4.32 4.25 ⁄ 4.32 68.8 60.7
Ac-CH
3
2.02 2.01 25.0 21.9
b-Gal
3a
Position 1 4.42 4.53 104.8 103.7
Position 2 3.53 3.63* 81.8* 70.7
Position 3 ND 3.80 75.0 72.7
Position 4 ND 3.85 71.8 68.6
Position 5 ND ND 78.0 75.1
Position 6 ND ND 63.8 60.9
b-Gal
2,4 a
Position 1 4.60 4.60 102.6 103.7
Position 2 3.59* 3.59* 80.1* 70.7
Position 3 ND 3.81 76.0 72.7
Position 4 ND 3.87 71.2 68.6
Position 5 ND ND 77.6 75.1

Position 6 ND ND 63.6 60.9
a-Fuc
2,3 a
Position 1 5.21 103.8 99.4
Position 2 3.74 72.0 67.8
Position 3 3.78 74.5 71.7
Position 4 3.77 72.2 69.5
Position 5 4.23 71.1 66.4
CH
3
1.20 18.2 15.2
a-Fuc
2,4 a
Position 1 5.19 5.19 102.6 99.4
Position 2 3.74 3.74 71.2 67.8
Position 3 3.78 3.78 74.4 71.7
Position 4 3.77 3.77 72.1 69.5
Position 5 4.17 4.18 69.7 66.4
CH
3
1.19 1.19 18.0 15.2
a
A superscript at a monosaccharide residue indicates to which
position of the adjacent monosaccharide it is glycosidically linked.
Two superscripts map out the pathway from the residue toward
the GalNAc-ol residue.
b
Standards are a and b-methyl derivatives
of each component sugar except GalNAc-ol.
Structure 1

mAb against sulfated oligosaccharide of mucin D. Tsubokawa et al.
1838 FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS
This structure is supported by the glycosylation
shifts observed at position 2, positions 4 and 6, and
positions 3 and 6, of the b-linked Gal-bearing Fuc,
b-linked GlcNAc and GalNAc-ol, respectively, identi-
fied by the cross-peak patterns in the HOHAHA and
TOCSY spectra. Despite these data, another structure
may be possible:
To obtain conclusive evidence for the oligosaccha-
ride structure of A1-5a, mild periodate oxidation, by
which the GalNAc-ol at the reducing terminus was
cleaved between C4 and C5 [17], was performed. The
molecular masses of the fragments were estimated
by MALDI-TOF ⁄ MS. Two fragments, corresponding
to Fuc-Gal-GlcNAc(6SO
3
H)-O-CH
2
-CHO and Gal-O-
CH(CHO)-CH(NHCOCH
3
)-CH
2
OH, were obtained
from A1-5a (data not shown). The results clearly show
that A1-5a was structure 1 and not structure 2.
Effect of defucosylation on the reactivity
with PGM34
The Fuc residue attached via the a1–2 linkage was

removed in order to determine the involvement of this
residue in the epitope of PGM34. Oligosaccharides
generated from A1-5b by mild acid hydrolysis were
separated into four fractions, I–IV, by HPLC using
an Amide-80 column (Fig. 6A). The molecular masses
of these fractions were estimated by MALDI-
TOF ⁄ MS, and these fractions were tested for their
reactivity with PGM34 (Fig. 6B). Fraction IV, which
reacted with PGM34, was the original A1-5b as deter-
mined from the mass and retention time on HPLC.
Fraction I had no Fuc residue and did not react with
PGM34. Both fractions II and III had one Fuc resi-
due, but only fraction III reacted with PGM34. As
fraction III had the same retention time as A1-5a,
fraction III appeared to be A1-5a. The mild periodate
oxidation of fraction III supports this, because the
same fragments as for A1-5a were obtained (data not
shown). Fraction II had the same mass as fraction
III, but their retention times were different. Therefore,
it was expected that fraction II had structure 2. This
was confirmed by the mild periodate oxidation: two
fragments, corresponding to Gal-GlcNAc(6SO
3
H)-O-
Fig. 6. Effects of defucosylation on anti-
genic activity with PGM34. (A) After being
defucosylated, oligosaccharide A1-5b was
chromatographed on two TSK-Gel Amide-80
columns and eluted under isocratic condi-
tions. The absorption was monitored at

210 nm. The arrows indicate the elution
positions of A1-5a and 5b. (B) The antigenic
activities of various amounts of the oligosac-
charides from these fractions, I (s), II (d),
III (n) and IV (m), were examined by the
competitive ELISA as described in Experi-
mental procedures. Data are expressed as
mean ± SD from three experiments.
Structure 2
D. Tsubokawa et al. mAb against sulfated oligosaccharide of mucin
FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS 1839
CH
2
-CHO and Fuc-Gal-O-CH(CHO)-CH(NHC-
OCH
3
)-CH
2
OH, were obtained (data not shown).
These results strongly indicate that the Fuc residue
attached to the Galb1–4GlcNAc(6SO
3
H)b- sequence
via an a1–2 linkage is an essential component of the
epitope of PGM34.
Aa
AB
C
D
Ca Cb Cc

Da Db
Ab Ba
Bb
Fig. 7. Immunostaining of the rat gastroin-
testinal mucosae with PGM34. Immuno-
staining of the fundic region (Aa), the pyloric
region (Ba), the small intestinal region, duo-
denum (Ca), jejunum (Cb), ileum (Cc), and
the colonic region, proximal (Da) and distal
(Db). HID staining of the fundic region (Ab)
and the pyloric region (Bb) was also per-
formed to compare the immnostaining of
these regions, Aa and Bb, respectively.
Bars ¼ 50 lm.
mAb against sulfated oligosaccharide of mucin D. Tsubokawa et al.
1840 FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS
Immunohistochemical study of rat
gastrointestinal tract with PGM34
Figure 7 shows the immunohistochemical reactivity of
PGM34 with different sections of rat gastrointestinal
mucosa. In the lower part of the pit region of the gas-
tric fundus, the surface mucous cells were specifically
stained with PGM34 (Fig. 7Aa). On the other hand,
some mucous cells in the deep region of the pyloric
gland were stained with this mAb in the antral mucosa
(Fig. 7Ba). The goblet cells in the small intestinal mu-
cosae, duodenum (Fig. 7Ca), jejunum (Fig. 7Cb), and
ileum (Fig. 7Cc) were extensively stained, whereas the
gland cells in the colon were only partly stained with
PGM34 (Figs 7Da and 7Db). Table 4 summarizes the

immunohistochemical reactivity of this mAb with rat
gastrointestinal tissues.
In the gastric fundic region, the surface mucous cells
stained with PGM34 were almost identical with those
stained by the high iron diamine (HID) method, which
extensively stains mucin molecules bearing sulfate resi-
dues (Fig. 7Ab). This result was supported by the elec-
tron microscopic observation, which showed that all
the mucous cells that reacted positively with PGM34
(14 nm) were co-labeled with cationic colloidal gold
(CCG) (8 nm) for the nonspecific sulfated mucin-secre-
ting cells in the fundic mucosa (Fig. 8B). On the other
hand, antral mucous cells were more extensively
stained by HID than by PGM34 (Fig. 7Bb). In the top
region above the lower part of the pit region of the
gastric fundus, surface mucous cells were rarely stained
with PGM34, indicating that mature surface mucous
cells could not generate the sulfated mucin stained
with this mAb (Figs 7Aa and 8A).
Discussion
This study indicates that the epitope of PGM34 is a
trisaccharide sequence with a sulfate residue, Fuca1–
2Galb1–4GlcNAc(6SO
3
H)b-, 6-sulfated blood-group H
type 2 sequence (6-sulfo H), based on the following.
(a) The two PGM34-reactive oligosaccharides, A1-5a
and A1-5b, contain this common trisaccharide
sequence with a sulfate residue. (b) The oligosaccha-
rides with the 6-sulfo N-acetyl-lactosamine sequence,

the defucosylated form of 6-sulfo H, generated from
A1-5b by mild acid hydrolysis, did not show any reac-
tivity with PGM34 (Fig. 6). Thus, the Fuc residue
linked to the Galb1–4GlcNAc(6SO
3
H)b- sequence via
an a1–2 linkage is required for the reaction with
PGM34. (c) Fuca1–2Galb1–4GlcNAcb1–6(Fuca1–
2Galb1–3)GalNAc-ol, the desulfated form of A1-5b,
did not inhibit binding of PGM34 to mucin (data not
shown). This indicates that the sulfate residue linked
to the 6 position of GlcNAc is essential for the reac-
tion with PGM34. (d) The reduced oligosaccharides
showed inhibitory activity toward the binding of
Table 4. Reactivity of PGM34 with gastrointestinal tissues obtained
from rat. –, Negative; +, presence of positive cells. + ⁄ –, rare pres-
ence of positive cells.
Tissue Site Reactivity
Stomach
Cardia Surface mucous cell –
Cardiac gland cell –
Fundus Surface mucous cell +
Mucous neck cell –
Pylorus Surface mucous cell –
Pyloric gland cell + ⁄ –
Small intestine
Duodenum Goblet cell +
Jejunum Goblet cell +
lleum Goblet cell +
Large intestine + ⁄ –

A
B
Fig. 8. Electron micrographs of surface
mucosa in rat gastric fundus. Dual labeling
of PGM34 (14 nm CG) and CCG (8 nm CG)
at pH 1.0 of the top pit region (A) and the
mid pit region (B). Bars ¼ 0.5 lm.
D. Tsubokawa et al. mAb against sulfated oligosaccharide of mucin
FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS 1841
PGM34 to mucin; therefore, the reducing-end GalNAc
seems not to participate in the reactivity.
A1-3 and A1-6 did not react with PGM34, whereas
they possibly contained oligosaccharides with 6-sulfo H,
because oligosaccharides containing the (SO
3
H)
(dHex)(Hex)(HexNAc) composition appeared in A1-3
and A1-6. Although we did not analyze these oligosac-
charides further, because of their low amounts, they
may be additionally modified. For instance, A1-6 may
contain the 6-sulfated blood group A sequence. This is
possible because oligosaccharides with the blood group
A sequence are present in pig gastric mucin [18],
although they do not have the sulfate residue. If this is
the case, the addition of GalNAc to Gal may cause loss
of reactivity with PGM34. Another possibility is that
A1-3 and A1-6 contain 6-sulfo H in the core 3 or core 4
branch. The core structure may influence the reactivity
of PGM34–6-sulfo H. Furthermore, A1-6 may con-
tain the 6-sulfo Lewis y structure. The addition of

the Fuc residue to the 6-sulfated GlcNAc may cause
loss of reactivity with PGM34. Further study needs to
clarify the factors that modify the reactivity with
PGM34.
Although PGM34 recognizes the acidic oligosaccha-
ride with the 6-sulfo H sequence, the acidic oligosac-
charide fraction A2 did not inhibit binding of PGM34
to pig gastric mucin (Fig. 2B). As fraction A2 was
more acidic than fraction A1, fraction A2 may consist
of disialylated or sialylated and sulfated oligosaccha-
rides. In the latter case, reactivity with PGM34 may be
blocked by the addition of sialic acid to the 6-sulfo H
sequence. Although fraction A2 has not been analyzed
because of its small amount, further analysis may clar-
ify this point.
PGM34 extensively stained surface mucous cells in
the fundic region, but only slightly stained pyloric
gland cells (Figs 7Aa and 7Bb). In our previous study,
we demonstrated that the 6-sulfo H sequence is pre-
dominantly found on oligosaccharides of mucin present
in the fundus region, whereas this sequence was only
rarely found in the pyloric region [19]. Thus, this bio-
chemical result is compatible with the immunohisto-
chemical reactivity of PGM34 in the rat gastric section.
The surface mucous cells in the fundic region stained
by PGM34 were almost identical with those stained by
the HID and CCG labeling method (Figs 7Ab and 8).
The specificities of HID staining and labeling of CCG
for sulfated mucin in the rat gastric gland have been
reported by Spicer et al. [20] and Yang et al. [21],

respectively. These correspond to the histochemical
data in this paper. On the other hand, pyloric mucous
cells were more extensively stained by the HID method
than by PGM34 (Fig. 7Bb). Goso & Hotta [22] repor-
ted that the sulfated oligosaccharide structure differs
according to the region in the rat gastrointestinal
mucin. These facts indicate that mucous cells that
secrete the specific sulfomucin with the 6-sulfo H
sequence are localized in the fundus in rat gastric mucosa.
Although the 6-sulfo H sequence is located in O-gly-
cans of gastric mucin molecules, this sequence may also
be found in N-glycans of glycoproteins present in
mucous cells. However, this is not likely because glyco-
proteins other than the high-molecular-mass mucins
extracted from rat stomach did not react with PGM34
(unpublished data). It is not clear whether the 6-sulfo H
sequence is contained in glycolipids. Further study may
clarify this point.
Sawaguchi et al. [23] demonstrated, by the high-
pressure freezing ⁄ freeze substitution method, the excre-
tory flow of zymogenic and mucin contents in the
lumen of the rat fundic gland. At the base and neck
regions, where mucous neck, parietal and chief cells
are dominant, the exocytosed zymogenic contents have
a droplet-like appearance in mucin derived from
mucous neck cells. In the pit region above the neck
and isthmus regions, where surface mucous cells are
dominant, not only mucin derived from mucous neck
cells, but also sulfated mucin form the intraluminal
mucous channels. Upon reaching the pit region, the

zymogenic contents merge into the mucous neck cell
mucous channel. The mucous-neck-cell-derived mucin
is confined to the central portion of the glandular
lumen, surrounded by sulfomucin secreted from the
lower part of the pit cells. It should be noted that a
distinct interface is formed between these two types of
mucin. PGM34 recognized the surface mucous cells in
the lower part of the pit region (Fig. 7Aa). Therefore,
the sulfomucin containing the 6-sulfo H sequence,
which has an antipepsin action, may have the function
of protecting the mucosa from zymogenic contents
merged into the mucous neck cells by covering the sur-
face of the mucosa [24].
The lower part of the pit cells of the rat gastric fun-
dus stained by PGM34 is close to the generative cell
zone stained by antiproliferating cell nuclear antigen
[25]. The possibility that mucous cells that secrete sul-
fomucin are localized in the generative cell zone is sup-
ported by a previous study [26]. Undifferentiated,
granule-free stem cells predominate in the rat isthmus
region of the gastric mucosa; these stem cells differenti-
ate and migrate upwards and downwards, replacing
the surface mucous cells and glandular cells, respect-
ively [27]. In this study, PGM34 did not stain the glan-
dular cell zone below the isthmus region. Thus, as the
mucous cells that secrete sulfomucin with the 6-sulfo H
sequence have a site-specific localization as described
mAb against sulfated oligosaccharide of mucin D. Tsubokawa et al.
1842 FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS
above, they may be the characteristic precursor cells

that differentiate into the mature surface mucous cells
in rat gastric mucosa. Further study is needed to clar-
ify the physiological function of sulfomucin in gener-
ating cells.
The mAb, HCM31, recognizes a particular carbohy-
drate structure of mucin with sialic acid residues [28].
Some goblet cells that are stained by HCM31 are distri-
buted throughout the intestine in aged rats, but goblet
cells in the distal colon and rectum of young rats are
not stained with this mAb [29]. Thus, HCM31 staining
revealed physiological changes in sialomucin expression
in the rat intestinal mucosa during aging. In our study,
goblet cells in rat small intestinal mucosa were exten-
sively stained with PGM34 (Fig. 7Ca,7Cb and 7Cc),
but gland cells in the colon were only partly stained
with this mAb (Fig. 7Da and 7Db). PGM34 staining
reveals the distribution of a specific sulfomucin in rat
intestinal mucosa. Therefore, the combined use of
PGM34 and HCM31 should reveal distributional
changes in specific sulfomucins and sialomucins during
various pathophysiological alterations of rat intestinal
mucosa. This may clarify the biological significance of
sulfation and ⁄ or sialylation of mucin oligosaccharides
in the gastrointestinal tract.
Sulfated oligosaccharides with the sialyl 6-sulfo
Lewis x sequence is expressed on the high endothelial
venules in human lymph nodes as a major ligand for
L-selectin in order to allow lymphocyte homing
[30,31]. The sequence of the sialyl 6-sulfo Lewis x is
also expressed in nonmalignant colonic epithelia [32]

and changes along with malignant alteration of the
colon. Thus, sugar chains with 6-sulfate linked to the
GlcNAc residue have been implicated in the mutual
recognition of and pathological change in cells in the
human body. The 6-sulfo H sequence may have biolo-
gical functions in the human body.
The mAb, HIK1083, which stains the glandular mu-
cosa of the stomach, has been a useful clinical marker
for adenoma malignum of the uterine cervix [33]. In
this case, a peripheral a-linked GlcNAc on mucin
oligosaccharides recognized by HIK1083 appear with
malignancy, whereas this a-linked GlcNAc is restricted
to a few sections of normal mucosa [14]. This indicates
that other mAbs developed by our laboratory may be
possible clinical markers. As PGM34 recognized the
characteristic sequence, 6-sulfo H, and oligosaccharides
with this sequence have been obtained from respiratory
mucins of a secretor patient suffering from human
chronic bronchitis [34], PGM34 could potentially be
developed into an important clinical marker for the
early diagnosis of various human diseases, such as
chronic bronchitis.
In summary, PGM34 is a very useful tool for recog-
nizing the specific sulfomucin molecule bearing the
6-sulfo H sequence in immunochemical and immu-
nohistochemical methods. Further research using this
mAb should elucidate the biological significance of
sulfomucins containing the 6-sulfo H sequence.
Experimental procedures
Materials

Partially purified pig gastric mucin was obtained by precipi-
tating crude pig gastric mucin (Type I; Sigma, St Louis,
MO, USA) with ethanol as previously described [35].
The oligosaccharide, Fuca1–2Galb1–4GlcNAc b 1–6(Fuca1–
2Galb1–3)GalNAc-ol, was a product of Kanto Chemical,
(Tokyo, Japan). Bio-Gel P-2 and P-6 resins were purchased
from Bio-Rad Laboratories (Richmond, CA, USA). Do-
wex-50 resin was purchased from Dow Chemical Company
(Midland, MI, USA). QAE-Toyopearl-550C resin and
TSK-Gel Amide-80 column were purchased from Tosoh
(Tokyo, Japan). Sephadex G-10 resin was a product of GE
Healthcare Bio-Sciences (Uppsala, Sweden).
PGM34 was produced in our laboratory using highly
purified pig gastric mucin as an antigen, by the method of
Ko
¨
hler & Milstein [36] with the modification of Groth &
Scheidegger [37] as previously described [10,13]. The anti-
body subclass was determined as IgM j by ELISA using
an isotyping kit (PharMingen, San Diego, CA, USA).
Preparation and purification of oligosaccharides
from pig gastric mucin
Alkaline borohydride treatment of the partially purified pig
gastric mucin was carried out by the method of Carlson
[38] with 0.05 m NaOH in 1.0 m NaBH
4
at 50 °C for 24 h.
The reaction mixture, after being acidified by the dropwise
addition of acetic acid (final pH ¼ 4), was applied to a Bio-
Gel P-6 column (3.4 cm · 100 cm). The column was eluted

with distilled water, and the eluate was monitored by hex-
ose measurement [39]. Oligosaccharide fractions were then
applied to a column of Dowex-50 H
+
-form to remove the
peptides. The eluted fractions were subsequently applied to
an anion-exchange column, QAE-Toyopearl-550C, and this
column was washed with distilled water, and then eluted
with a linear gradient of 0–0.6 m sodium acetate. Elution of
the oligosaccharides was monitored by hexose measure-
ment. The fractions were collected and desalted on a col-
umn of Bio-Gel P-2.
Normal-phase HPLC
The two-step normal-phase HPLC using TSK-Gel Amide-
80 (7.8 mm · 300 mm · 2 columns) was used. Two buffer
D. Tsubokawa et al. mAb against sulfated oligosaccharide of mucin
FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS 1843
systems were used: buffer A [80% (v ⁄ v) acetonitrile in
2.5 mm NaH
2
PO
4
] and buffer B (30% acetonitrile in
2.5 mm NaH
2
PO
4
). In the first step, the column was equili-
brated with 75% A, and the gradient was initiated after
injection and increased to 50% B over 30 min at a flow rate

of 2.0 mLÆmin
)1
. In the second step, the fractions obtained
from the first-step HPLC were rechromatographed under
isocratic conditions of 88% A for 1 h at a flow rate of
1.0 mLÆmin
)1
. UV absorption of the eluate was monitored
at 210 nm. For the removal of NaH
2
PO
4
, the fractions
were chromatographed on Sephadex G-10 using distilled
water as the eluent.
Amino sugar analysis
The oligosaccharides were hydrolyzed with 6 m HCl at
98 °C for 4 h using the Waters’ Workstation. The amino
sugars obtained were derivatized with phenylisothiocyanate
according to the instructions of the Pico-Tag amino
acid analysis [40], then analyzed by HPLC using a Pico-Tag
column (3.9 mm · 150 mm) and the buffer system as previ-
ously described [41]. UV absorption of the eluate was mon-
itored at 254 nm. The monosaccharide mixture containing
GlcNAc, GalNAc and GalNAc-ol (molar ratios, 1 : 1 : 1)
was used as the standard sample.
MALDI-TOF ⁄ MS analysis
The molecular masses of the oligosaccharides were meas-
ured by MALDI-TOF ⁄ MS using the Voyager DE-PRO
(Applied Biosystems, Foster City, CA, USA) instrument.

Each sample was mixed with an equal volume of 2,5-dihyd-
roxybenzoic acid dissolved in distilled water ⁄ acetonitrile
(1 : 1, v ⁄ v) at 10 mgÆmL
)1
as the matrix solutions [42]. A
2-lL sample of this mixture was then applied to a stainless-
steel target plate and air-dried at room temperature before
the target was introduced into the spectrometer. The mass
spectra were obtained in the reflection mode by accumula-
ting 150 laser shots using the following conditions: polarity,
negative; accelerating voltage, 20 000 V; grid voltage, 76%;
extraction delay time, 100 ns.
NMR spectroscopy
The NMR spectra were obtained using a Varian Unity 400
NMR spectrometer (Varian Associates, Palo Alto, CA,
USA) equipped with an
1
H[
15
N-
31
P] pulse field gradient
indirect-detecting probe. Standard pulse sequences were
used throughout. The
1
H NMR spectrum was assigned
through pulse field gradient multiple-quantum-correlation
spectroscopy and one-dimensional HOHAHA spectroscopy.
The
13

C assignments were made from an HMQC spectrum
obtained with carbon decoupling. Additional assignments
and information on the sequence and linkage of the sugar
residues were derived from an HMBC spectrum. The lyoph-
ilized powder of the purified oligosaccharides was dissolved
in deuterium oxide (
2
H
2
O) and evaporated to exchange the
unstable
1
H with
2
H. The evaporation and dissolution were
repeated five times, and the sample was finally dissolved in
0.75 mL
2
H
2
O and then subjected to NMR spectroscopy.
Chemical shifts of the reduced oligosaccharide structures
were referred to those described by Kamerling &
Vliegenthart [16]. The NMR spectral data of the standard
a and b methylated monosaccharides as well as those of
GalNAc-ol reported by Ishihara et al. [13] were also used
as references for the chemical shift assignment.
Defucosylation of oligosaccharides by mild acid
hydrolysis
The oligosaccharides were hydrolyzed with 0.1 m HCl at

80 °C for 1 h. The defucosylated oligosaccharides were sep-
arated by normal-phase HPLC using a TSK-Gel Amide-80
column.
Mild periodate oxidation of oligosaccharides
The mild periodate oxidation of oligosaccharides was per-
formed as described by Chai et al. [17]. The oligosaccha-
rides ( 10 lg) were oxidized with sodium periodate in
imidazole buffer, pH 6.5, at 0 °C for 5 min. After the
excess periodate had been destroyed by incubation with
butane-2,3-diol at 0 °C for 40 min, the oligosaccharides
were purified using a column of graphitized carbon [43].
ELISA and competitive ELISA
The ELISA well of a microtiter plate was coated with
100 ng of the purified mucin and kept overnight at 4 °C; this
was followed by blocking with 2% skimmed milk [10]. After
the wells had been washed, a specific amount of PGM34 was
added to each well; this was followed by incubation at ambi-
ent temperature for 1 h. The wells were successively incuba-
ted with horseradish peroxidase-conjugated goat anti-mouse
immunogloblins (Dako, Kyoto, Japan) and 2,2¢-azino-bis-
(3-ethylbenzthiazoline-6-sulfonate) (ABTS) ⁄ H
2
O
2
solution
(Kirkegaard & Perry Laboratories, Gaithersburg, MD,
USA), and the color was allowed to develop [44]. The wells
were washed three times with NaCl ⁄ P
i
containing 0.05%

Tween-20 between each process. The absorption was meas-
ured at 405 nm (reference at 492 nm) at 15 min thereafter
using a Bio-Rad model 550 microplate reader.
A competitive ELISA was applied to detect the reactivity
of PGM34 with the purified oligosaccharide fractions. The
microtiter plate coated with the purified mucin followed by
blocking with 2% skimmed milk was prepared as previ-
ously described. At the same time, NaCl ⁄ P
i
solutions of the
oligosaccharide fraction, each containing 20–400 lg per
mAb against sulfated oligosaccharide of mucin D. Tsubokawa et al.
1844 FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS
well as the hexose base, were preincubated with a specified
amount of PGM34 for 2 h at ambient temperature. As a
negative control, instead of the sugar-containing solution,
NaCl ⁄ P
i
was preincubated with PGM34. The preincubated
mixtures were then added to the antigen-coated wells and
incubated for 1 h. The remaining ELISA steps were the
same as already described.
Treatment with periodate or trypsin
Periodate treatment was performed by exposing the
mucin antigen coated on the microtiter wells to 0.1–2.5 mm
NaIO
4
in 50 mm sodium acetate, pH 4.5, for 1 h at room
temperature.
Trypsin digestion was performed by exposing the mucin

antigen coated on the microtiter wells to trypsin for 1 h at
37 °C. A 2.5 mgÆmL
)1
trypsin sample in 10 mm Tris ⁄ HCl,
pH 8.0, containing 2 mm CaCl
2
was used with the twofold
serial dilution.
Each of the remaining ELISA steps was as already
described.
Light microscopic immunohistochemistry
Male Wistar rats, 8–10 weeks old, were deeply anesthetized
with diethyl ether and sodium pentobarbital, and small
pieces of gastrointestinal tissues were carefully excised. The
specimens were attached to the tips of small bamboo skew-
ers and plunged into a liquid isopentane ⁄ propane mixture
cooled by liquid nitrogen. After immersion for at least 20 s,
the specimens were quickly transferred to liquid nitrogen
and held there until further processing of the freeze substitu-
tion. Freeze substitution was carried out in 0.1% glutaralde-
hyde in acetone at )80 °C for 16 h. Specimens were then
gradually warmed ()50 °C for 2 h, )20 °C for 1 h, and 4 °C
for 1 h) to room temperature. After several washes with
pure ethanol, the specimens were embedded in paraffin.
The paraffin sections, 4 lm thick, were deparaffinized, re-
hydrated, and incubated in methanol containing 0.3% H
2
O
2
for 30 min to block endogenous peroxidase activity. After

several rinses in NaCl ⁄ P
i
, the sections were incubated in 5%
normal horse serum (NHS) ⁄ 1% BSA in NaCl ⁄ P
i
for 10 min
to block nonspecific binding and then incubated with
PGM34 (diluted 1 : 100 with 5% NHS ⁄ 1% BSA in
NaCl ⁄ P
i
)at4°C overnight. After being washed with
NaCl ⁄ P
i
, the sections were incubated with biotinylated
horse anti-mouse IgM (Vector Laboratories, Burlingame,
CA, USA; diluted 1 : 200 with 1% BSA in NaCl ⁄ P
i
)at
room temperature for 40 min, followed by washing with
NaCl ⁄ P
i
. The sections were then incubated in a freshly pre-
pared solution of the avidin-biotinylated horseradish peroxi-
dase complex (ABC) kit (Vector Laboratories) for 30 min.
After a wash with NaCl ⁄ P
i
, the peroxidase reaction was
developed by incubating in 0.05% 3,3 ¢-diaminobenzidine
tetrahydrochloride in 50 mm Tris ⁄ HCl buffer, pH 7.6, con-
taining 0.001% H

2
O
2
. After being washed, the sections were
briefly counterstained with hematoxylin. For the controls,
the primary antibodies were omitted from the procedure.
Preparation of colloidal gold (CG) and
immunoglobulin–gold complex
Monodisperse CG 8 nm in diameter was prepared by the
method of Slot & Geuze [45], and CG 14 nm in diameter
was prepared by the modified method of Frens [46]. Conju-
gation of goat anti-biotin IgG (Vector Laboratories) was
performed by the modified method of De Mey et al. [47].
CCG was prepared by the modified method of Goode et al.
[48] and Kashio et al. [49].
Electron microscopic immunohistochemistry
Small fragments of the stomach were excised from deeply
anesthetized male Wistar rats, 8–10 weeks old, and
promptly cut into 0.2–0.3 mm slices to be sandwiched in
the cavity of the specimen carrier. The specimen was
immediately frozen at a pressure of 210 MPa (2100 bar)
using a high-pressure freezing machine (HPM 010;
BAL-TEC, Balzers, Liechtenstein), and then rapidly trans-
ferred to liquid nitrogen for storage until required for
further processing. Freeze substitution was carried out
using a Reichert AFS system (Leica, Wien, Austria). After
programmed warming to )30 °Cat10°C ⁄ h, the substi-
tution medium was replaced with pure ethanol (three chan-
ges each of 10 min duration) and then gradually raised to
+18 °C and left for 2 h to remove the remaining hydration

shell of protein [50]. After complete substitution, the tem-
perature was gradually lowered to )35 °C, and infiltration
with Lowicryl K4M was performed in mixtures of 1 : 1 and
1:2(v⁄ v) 100% ethanol ⁄ Lowicryl K4M (60 min each) and
in pure Lowicryl K4M overnight at )35 °C. The polymer-
ization was performed using a UV lamp from the AFS
machine for 24 h at )35 °C and for a further 8 h at 18 °C.
Ultrathin sections, 70–80 nm thick, were treated with 1%
BSA in NaCl ⁄ P
i
for 10 min to block nonspecific binding,
and the sections were passed through 50 mm Tris ⁄ HCl buf-
fer containing 0.2% BSA (Tris ⁄ HCl ⁄ BSA) at pH 1.0. The
sections were then incubated with CCG (8 nm) at pH 1.0
for 40 min at room temperature. After a brief incubation
with Tris ⁄ HCl ⁄ BSA at pH 1.0, the sections were washed
with distilled water and incubated in 5% NHS ⁄ 1% BSA in
NaCl ⁄ P
i
for 10 min to block nonspecific binding. The sec-
tions were incubated with PGM34 (diluted 1 : 50 with 5%
NHS ⁄ 1% BSA in NaCl⁄ P
i
)at4°C overnight. After being
washed with NaCl ⁄ P
i
, the sections were incubated with bio-
tinylated horse anti-mouse IgM (diluted 1 : 200 with 1%
BSA in NaCl ⁄ P
i

) at room temperature for 40 min. After
being washed with NaCl ⁄ P
i
, the sections were incubated
with goat anti-biotin IgG–CG (14 nm) conjugate (diluted
with 1% BSA in NaCl ⁄ P
i
) at room temperature for 30 min.
D. Tsubokawa et al. mAb against sulfated oligosaccharide of mucin
FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS 1845
After being washed with distilled water and dried, the
sections were contrasted by KMnO
4
⁄ UA ⁄ Pb staining as
previously described [50].
HID staining
Histochemical detection of whole sulfated mucins was
performed by HID staining [51]. The previously described
paraffin sections were treated with diamine solution (con-
taining N,N-dimethyl-m-phenylenediamine, N,N-dimethyl-
p-phenylenediamine and iron chloride) for 20 h at 20 °C.
The sections were briefly washed with distilled water and
then dehydrated, passed through xylene, and mounted.
Acknowledgements
We express our sincere appreciation to Drs T. Nakam-
ura and T. Ikezawa, for valuable discussion, and
Ms. S. Sugawara and Y. Ito for technical assistance.
This work was supported in part by Grants-in-Aid for
Scientific Research from the Ministry of Education,
Science, Sports and Culture of Japan and by a Grant

from the Integrative Research Program of the Gradu-
ate School of Medical Sciences, Kitasato University.
References
1 Corfield AP, Myerscough N, Longman R, Sylvester P,
Arul S & Pignatelli M (2000) Mucins and mucosal pro-
tection in the gastrointestinal tract: new prospects for
mucins in the pathology of gastrointestinal disease. Gut
47, 589–594.
2 Suganuma T, Katsuyama T, Tsukahara M, Tatematsu
M, Sakakura Y & Murata F (1981) Comparative histo-
chemical study of alimentary tracts with special refer-
ence to the mucous neck cells of stomach. Am J Anat
161, 219–238.
3 Katsuyama T, Ota H, Ishii K, Nakayama J, Kanai M,
Akamatsu T & Sugiyama A (1991) In Gastrointestinal
Function. Regulation and Disturbances (Kasuya, Y, Tsu-
chiya, M, Nagao, F & Matsuo, Y, eds), Vol. 9, pp. 145–
165. Excerpta Medica, Amsterdam.
4 Ota H, Katsuyama T, Ishii K, Nakayama J, Shiozawa
T & Tsukahara Y (1991) A dual staining method for
identifying mucins of different gastric epithelial mucous
cells. Histochem J 23, 22–28.
5 Ota H & Katsuyama T (1992) Alternating laminated
array of two types of mucin in the human gastric sur-
face mucous layer. Histochem J 24,
86–92.
6 de Bolos C, Garrido M & Real FX (1995) MUC6 apo-
mucin shows a distinct normal tissue distribution that
correlates with Lewis antigen expression in the human
stomach. Gastroenterology 109, 723–734.

7 Ho SB, Roberton AM, Shekels LL, Lyftogt CT,
Niehans GA & Toribara NW (1995) Expression cloning
of gastric mucin complementary DNA and localization of
mucin gene expression. Gastroenterology 109, 735–747.
8 Ichikawa T, Ishihara K, Saigenji K & Hotta K (1993) Sti-
mulation of mucus glycoprotein biosynthesis in rat gastric
mucosa by gastrin. Biochem Pharmacol 46, 1551–1557.
9 Ichikawa T, Endoh H, Hotta K & Ishihara K (2000)
The mucin biosynthesis stimulated by epidermal growth
factor occurs in surface mucus cells, but not in gland
mucus cells, of rat stomach. Life Sci 67, 1095–1101.
10 Ishihara K, Kurihara M, Goso Y, Ota H, Katsuyama T
& Hotta K (1996) Establishment of monoclonal antibo-
dies against carbohydrate moiety of gastric mucins dis-
tributed in the different sites and layers of rat gastric
mucosa. Glycoconj J 13, 857–864.
11 Katsuyama T, Ono K, Nakayama J, Akamatsu T &
Honda T (1985) Mucosubstance histochemistry of the
normal mucosa and carcinoma of the large intestine.
Galactose oxidase-Schiff reaction and lectin stainings.
Acta Pathol Jpn 35, 1409–1425.
12 Ishihara K, Kurihara M, Eto H, Kasai K, Shimauchi S
& Hotta K (1993) A monoclonal antibody against car-
bohydrate moiety of rat gastric surface epithelial cell-
derived mucin. Hybridoma 12, 609–620.
13 Ishihara K, Kurihara M, Goso Y, Urata T, Ota H,
Katsuyama T & Hotta K (1996) Peripheral a-linked
N-acetylglucosamine on the carbohydrate moiety of
mucin derived from mammalian gastric gland mucous
cells: epitope recognized by newly characterized mono-

clonal antibody. Biochem J 318, 409–416.
14 Ota H, Hayama M, Nakayama J, Hidaka H, Honda T,
Ishii K, Fukushima M, Uehara T, Kurihara M, Ishihara
K et al. (2001) Cell lineage specifically of newly raised
monoclonal antibodies against gastric mucins in normal,
metaplastic and neoplastic human tissues and their
application to pathology diagnosis. Am J Clin Pathol
115, 69–79.
15 Kawakubo Y, Ito Y, Okimura Y, Kobayashi M, Sakura
K, Kasama S, Fukuda MN, Fukuda M, Katsuyama T
& Nakayama J (2004) Natural antibiotic function of
human gastric mucin against Helicobacter pylori infec-
tion. Science 305, 1003–1006.
16 Kamerling JP & Vliegenthart JFG (1992) High-resolu-
tion
1
H-nuclear magnetic resonance spectroscopy of
oligosaccharides released from mucin-type O-glyco-
proteins. Biol Magnetic Resonance 10, 1–287.
17 Chai W, Stoll MS, Galustion C, Lawson AM & Feizi T
(2003) Neoglycolipid technology: deciphering informa-
tion content of glycome. Methods Enzymol 362, 160–
195.
18 Monferran CG, Roth GA & Cumar FA (1990) Inhibi-
tion of cholera toxin binding to membrane receptors by
pig gastric mucin-derived glycopeptides: differential
mAb against sulfated oligosaccharide of mucin D. Tsubokawa et al.
1846 FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS
effect depending on the ABO blood group antigenic
determinants. Infect Immun 58, 3966–3972.

19 Goso Y & Hotta K (1989) Types of oligosaccharide
sulphation, depending on mucus glycoprotein source,
corpus or antral, in rat stomach. Biochem J 264, 805–
812.
20 Spicer SS, Katsuyama T & Sannes PL (1978) Ultra-
structural carbohydrate cytochemistry of gastric epithe-
lium. Histochem J 10, 309–331.
21 Yang DH, Kasamo H, Miyauchi M, Tsuyama S &
Murata F (1996) Ontogeny of sulfated glycoconjugate-
producing cells in the rat fundic gland. Histochem J 28,
33–43.
22 Goso Y & Hotta K (1993) Regional differences in
sulfated oligosaccharides of rat gastrointestinal mucin
as detected by two-dimensional chromatography. Arch
Biochem Biophys 302, 212–217.
23 Sawaguchi A, Ishihara K, Kawano J, Oinuma T, Hotta
K & Suganuma T (2002) Fluid dynamics of the excre-
tory flow of zymogenic and mucin contents in rat gas-
tric gland processed by high-pressure freezing ⁄ freeze
substitution. J Histochem Cytochem 50, 223–234.
24 Takagaki YM & Hotta K (1979) Characterization of
peptic inhibitory activity associated with sulfated glyco-
protein isolated from gastric mucosa. Biochim Biophys
Acta 584, 288–297.
25 Jones HB, Clarke NA & Barrass NC (1993) Phenobarbi-
tal-induced hepatocellular proliferation: anti-bromode-
oxyuridine and anti-proliferating cell nuclear antigen
immunocytochemistry. J Histochem Cytochem 41, 21–17.
26 Wattel W, Geuze JJ & de Rooij DG (1977) Ultrastruc-
tural and carbohydrate histochemical studies on the

differentiation and renewal of mucous cells in the rat
gastric fundus. Cell Tissue Res 176, 445–462.
27 Yang DH, Tsuyama S, Ge YB, Wakamatsu D, Ohmori
J & Murata F (1997) Proliferation and migration
kinetics of stem cells in the rat fundic gland. Histol
Histopathol 12, 719–727.
28 Hayashida H, Ishihara K, Ichikawa T, Okayasu I,
Saigenji K & Hotta K (2001) Expression of a specific
mucin type recognized by monoclonal antibodies in the
gastric mucosa regenerating from acetic acid-induced
ulcer. Scand J Gastroenterol 5, 467–473.
29 Ikezawa T, Goso Y, Ichikawa T, Hayashida H,
Nakamura T, Kurihara M, Okayasu I, Saigenji K &
Ishihara K (2002) Immunohistochemical localization in
rat gastrointestinal tract of a sialomucin species
recognized by HCM31, a new anti-mucin monoclonal
antibody. Biomed Res 23, 63–68.
30 Mitsuoka C, Sawada-Kasugai M, Ando-Furui K, Izawa
M, Nakanishi H, Nakamura S, Ishida H, Kiso M &
Kannagi R (1998) Identification of a major carbohy-
drate capping group of the 1-selectin ligand on high
endothelial venules in human lymph nodes as 6-sulfo
sialyl Lewis X. J Biol Chem 273, 11225–11233.
31 Kimura N, Mitsuoka C, Kanamori A, Hiraiwa N,
Uchimura K, Muramatsu T, Tamatani T, Kansas GS &
Kannagi R (1999) Reconstitution of functional 1-selec-
tin ligands on a cultured human endothelial cell line by
cotransfection of a1–3 fucosyltransferase VII and newly
cloned GlcNAcb:6-sulfotransferase cDNA. Proc Natl
Acad Sci USA 96, 4530–4535.

32 Izawa M, Kumamoto K, Mitsuoka C, Kanamori C,
Kanamori A, Ohmori K, Ishida H, Nakamura S,
Kurata-Miura K, Sasaki K, et al. (2000) Expression of
sialyl 6-sulfo Lewis X is inversely correlated with con-
ventional sialyl Lewis X expression in human colorectal
cancer. Cancer Res 60, 1410–1416.
33 Ishii K, Kumagai T, Tozuka M, Ota H, Katsuyama T,
Kurihara M, Shiozawa T & Noguchi H (2001) A new
diagnostic method for adenoma malignum and related
lesions: latex agglutination test with a new monoclonal
antibody, HIK1083. Clin Chim Acta
312, 231–233.
34 Degroote S, Maes E, Humbert P, Delmotte P, Lamblin
G & Roussel P (2003) Sulfated oligosaccharides isolated
from the respiratory mucins of a secretor patient suffer-
ing from chronic bronchitis. Biochimie 85, 369–379.
35 Azuumi Y, Ichikawa T, Ishihara K & Hotta K (1993)
The validity of the ethanol precipitation method for the
measurement of mucin content in human gastric juices
and its possible relationship to gastroduodenal diseases.
Clin Chim Acta 221, 219–225.
36 Ko
¨
hler G & Milstein C (1975) Continuous cultures of
fused cells secreting antibody of predefined specificity.
Nature 256, 616–626.
37 Groth S & Scheidegger D (1980) Production of mono-
clonal antibodies: strategy and tactics. J Immunol
Methods 35, 1–21.
38 Carlson DM (1968) Structures and immunochemical

properties of oligosaccharides isolated from pig submax-
illary mucins. J Biol Chem 243, 616–626.
39 Dubois M, Gilles KA, Hamilton JK, Rebers PA &
Smith F (1956) Colorimetric method for determination
of sugars and related substances. Anal Chem 28, 350–
356.
40 Bidlingmeyer BA, Cohen SA & Travin TL (1984) Rapid
analysis of amino acids using pre-column derivatization.
J Chromatogr 336, 93–104.
41 Ishihara K, Kameyama J & Hotta K (1993) Develop-
ment of an HPLC method to estimate hexosamine and
its application to determine mucin content in rat and
human gastric mucosa. Comp Biochem Physiol 104B,
781–786.
42 Harvey DJ (1993) Quantitative aspects of the matrix-
assisted laser desorption mass spectrometry of complex
oligosaccharides. Rapid Commun Mass Spectrom 7,
614–619.
43 Packer NH, Lawson MA, Jardine DR & Redmond JW
(1998) A general approach to desalting oligosaccharides
released from glycoproteins. Glycoconj J 15, 737–747.
D. Tsubokawa et al. mAb against sulfated oligosaccharide of mucin
FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS 1847
44 Komuro Y, Ishihara K, Ishii K, Ota H, Katsuyama T,
Saigenji K & Hotta K (1992) A separating method for
quantifying mucus glycoprotein localized in the different
layer of rat gastric mucosa. Gastroenterol Jpn 27, 466–
472.
45 Slot JW & Geuze HJ (1985) A new method of preparing
gold probes for multiple-labeling cytochemistry. Eur

J Cell Biol 38, 87–93.
46 Frens G (1973) Controlled nucleation for the regulation
of the particle size in monodisperse gold suspensions.
Nature 241, 20–22.
47 De Mey J, Moeremans M, Geuens R, Nuydens R &
Brabander MD (1981) High resolution light and elec-
tron microscopic localization of tubulin with the IGS
(immuno gold staining) method. Cell Biol Int Rep 5,
889–899.
48 Goode NP, Shires M & Davison AM (1992) Prepa-
ration and use of the poly-L-lysine-gold probe:
a differential marker of glomerular anionic sites. Histo-
chemistry 98, 67–72.
49 Kashio N, Tsuyama S, Ihida K & Murata F (1992)
Cationic colloidal gold: a probe for light- and electron-
microscopic characterization of acidic glycoconjugates
using poly-L-lysine gold complex. Histochem J 24, 419–
430.
50 Sawaguchi A, Ide S, Kawano J, Nagaike R, Oinuma T,
Tojo H, Okamoto M & Suganuma T (1999) Reappraisal
of potassium permanganate oxidation applied to
Lowicryl K4M embedded tissues processed by high
pressure freezing ⁄ freeze substitution, with special
reference to differential staining of the zymogen gran-
ules of rat gastric chief cells. Arch Histol Cytol 62,
447–458.
51 Spicer SS & Henson JG (1967) Methods for localizing
mucosubstances in epithelial and connective tissues.
Methods Achiev Exp Pathol 2, 78–112.
mAb against sulfated oligosaccharide of mucin D. Tsubokawa et al.

1848 FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS