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

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

blood-group H type 2 antigen, on the carbohydrate moiety

of mucin

Analysis of the epitope sequence and immunohistochemical study Daigo Tsubokawa1, Yukinobu Goso1, Akira Sawaguchi2, Makoto Kurihara3, Takafumi Ichikawa4, Noriko Sato5, Tatsuo Suganuma2, Kyoko Hotta4and Kazuhiko Ishihara1

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: isiharak@kitasato-u.ac.jp

(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(6SO3H)b1–6(Fuca1–2Galb1–3) GalNAc-ol and Fuca1–2Galb1–4GlcNAc(6SO3H)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(6SO3H)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.

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)

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 to 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.

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,

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 (SO3H)(Hex)(HexNAc)2GalNAc-ol A1-3 675 (Neu5Ac)(Hex)GalNAc-ol

822 (Neu5Ac)(dHex)(Hex)GalNAc-ol

830 (SO3H)(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 (SO3H)(dHex)(Hex)2(HexNAc)GalNAc-ol

1017 (SO 3 H)(dHex)(Hex)(HexNAc) 2 GalNAc-ol A1-5* 976 (SO3H)(dHex)(Hex)2(HexNAc)GalNAc-ol

1121 (SO3H)(dHex)2(Hex)2(HexNAc)GalNAc-ol

1179 (SO 3 H)(dHex)(Hex) 2 (HexNAc) 2 GalNAc-ol A1-6 1179 (SO3H)(dHex)(Hex)2(HexNAc)2GalNAc-ol

1325 (SO3H)(dHex)2(Hex)2(HexNAc)2GalNAc-ol

The putative carbohydrate compositions of the

oligosaccharides that reacted positively with PGM34

were as follows: A1-4c, (SO3H)(dHex)(Hex)(GlcNAc)

(GalNAc)(GalNAc-ol) and⁄ or (SO3H)(dHex)(Hex)2

(GlcNAc)(GalNAc-ol); A1-5a, (SO3H)(dHex)(Hex)2

(GlcNAc)(GalNAc-ol); A1-5b, (SO3H)(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 1H-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 13C 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 (SO3H)(dHex)(Hex)2(HexNAc)GalNAc-ol

A1-5b* 1121 (SO3H)(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).

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 1H of Fuc and 13C at 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 13C chemical shift assess-ment The following remote coupling could also be recognized from the HMBC spectrum: anomeric1H of one b-Gal (4.60 p.p.m.) and position 4 13C of b-Glc-NAc (77.8 p.p.m.), anomeric 1H of another b-Gal (4.53 p.p.m.) and position 3 13C of GalNAc-ol (77.1 p.p.m.), anomeric 1H of b-GlcNAc (4.54 p.p.m.) and position 6 13C of GalNAc-ol (73.9 p.p.m.) A lower field change (+ 8.1 p.p.m.) in the 13C 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 1H 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-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 1H 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 1H 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

Standardsb

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-CH3 2.02 2.01 25.0 21.9

b-Gal 3 a

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-Gal2,4 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-Fuc2,3 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-Fuc2,4 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.bStandards are a and b-methyl derivatives

of each component sugar except GalNAc-ol.

Structure 1

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(6SO3H)-O-CH2-CHO and

Gal-O-CH(CHO)-CH(NHCOCH3)-CH2OH, 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(6SO3

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

CH2-CHO and

Fuc-Gal-O-CH(CHO)-CH(NHC-OCH3)-CH2OH, were obtained (data not shown)

These results strongly indicate that the Fuc residue

attached to the Galb1–4GlcNAc(6SO3H)b- sequence via an a1–2 linkage is an essential component of the epitope of PGM34

Aa

C

D

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.

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(6SO3H)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(6SO3H)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.

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 (SO3H)

(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 sulfarepor-ted 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 migrdifferenti-ate 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