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 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 Hotta4 and Kazuhiko Ishihara1 Department of Biochemistry, Kitasato University Graduate School of Medical Sciences, Sagamihara, Japan Department of Anatomy, Ultrastructural Cell Biology, Faculty of Medicine, University of Miyazaki, Japan Isehara Research Laboratory, Kanto Chemical Co Inc., Isehara, Japan Department of Biochemistry, Kitasato University School of Medicine, Sagamihara, Japan Department of Instrumental Analysis, Kitasato University School of Pharmaceutical Sciences, Tokyo, Japan 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, 2288555, Japan Fax ⁄ Tel: +81 42 778 8262 E-mail: isiharak@kitasato-u.ac.jp (Received November 2006, revised 16 January 2007, accepted February 2007) doi:10.1111/j.1742-4658.2007.05731.x Mucin, a major component of mucus, is a highly O-glycosylated, highmolecular-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 reactivity 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, oligosaccharide-alditols obtained from pig gastric mucin were fractionated by successive 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 examination of rat gastrointestinal tract showed that PGM34 stained surface mucous cells close to the generative cell zone in the gastric fundus and goblet 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 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 Abbreviations CCG, cationic colloidal gold; CG, colloidal gold; GalNAc-ol, N-acetylgalactosaminitol; HID, high iron diamine; HMBC, heteronuclear multiplebond 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 mAb against sulfated oligosaccharide of mucin D Tsubokawa et al by a combination of galactose oxidase-cold thionin Schiff staining and paradoxical concanavalin A staining [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 different 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 accumulation 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 produced 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 determined as a peripheral a-linked GlcNAc on the mucin oligosaccharides Interestingly, this epitope is restricted 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 Helicobacter pylori This suggests that mucin bearing a characteristic 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 surface 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 1834 gastric fundus, we were interested in an epitope recognized 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 epitope 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, periodate oxidation and trypsin digestion of the purified mucin were performed to degrade the carbohydrate and peptide moieties, respectively The residual antigenic activity was then tested by ELISA Periodate oxidation 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, and inhibited the reaction of PGM34 with the purified mucin on the ELISA plate, with fraction achieving the strongest inhibition (Fig 1B) Fractions and produced almost the same results as fractions and (data not shown) Although the data indicated that all the oligosaccharide fractions reacted with PGM34, fraction was expected to be the easiest to analyze for the structure of the oligosaccharides because of their relatively small size Therefore, fraction was chosen for epitope analysis, and further purified by anionexchange chromatography on a QAE-Toyopearl-550C column As shown in Fig 2A, one neutral oligosaccharide 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 fractions N and A2 did not (Fig 2B) FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS D Tsubokawa et al mAb against sulfated oligosaccharide of mucin Fig 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 fractions, 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 (s), fraction (d), fraction (n), were examined by competitive ELISA as described in Experimental procedures Data are expressed as mean ± SD from three experiments Fig QAE-Toyopearl-550C anion-exchange chromatography of fraction in Fig and the reactivity of oligosaccharides with PGM34 (A) Fraction 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 acetate (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 described in Experimental procedures Data are expressed as mean ± SD from three experiments Fraction A1 was further purified by two-step normal-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, designated 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 significantly 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 FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS 1835 mAb against sulfated oligosaccharide of mucin D Tsubokawa et al Table Oligosaccharide structures separated by the first-step HPLC: identified by MALDI-TOF ⁄ MS Fractions that reacted with PGM34 are indicated with an asterisk Fraction A1-1 A1-2 A1-3 A1-4* Fig First-step HPLC of oligosaccharide fraction A1 in Fig 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 The compositions of all the oligosaccharides tested were assigned to the appropriate acidic oligosaccharide-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 A1-5* A1-6 [M–H]– (m ⁄ z) Expected composition of oligosaccharide-alditols 675 871 675 822 830 871 1017 879 976 1017 976 1121 1179 1179 1325 (Neu5Ac)(Hex)GalNAc-ol (SO3H)(Hex)(HexNAc)2GalNAc-ol (Neu5Ac)(Hex)GalNAc-ol (Neu5Ac)(dHex)(Hex)GalNAc-ol (SO3H)(Hex)2(HexNAc)GalNAc-ol (SO3H)(Hex)(HexNAc)2GalNAc-ol (SO3H)(dHex)(Hex)(HexNAc)2GalNAc-ol (Neu5Ac)(Hex)(HexNAc)GalNAc-ol (SO3H)(dHex)(Hex)2(HexNAc)GalNAc-ol (SO3H)(dHex)(Hex)(HexNAc)2GalNAc-ol (SO3H)(dHex)(Hex)2(HexNAc)GalNAc-ol (SO3H)(dHex)2(Hex)2(HexNAc)GalNAc-ol (SO3H)(dHex)(Hex)2(HexNAc)2GalNAc-ol (SO3H)(dHex)(Hex)2(HexNAc)2GalNAc-ol (SO3H)(dHex)2(Hex)2(HexNAc)2GalNAc-ol 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 Second-step HPLC of oligosaccharide fraction A1-5 in Fig using TSK-Gel Amide80 columns and the reactivity of oligosaccharides with PGM34 (A) Fraction A1-5 was chromatographed on two TSK-Gel Amide-80 columns and eluted under isocratic conditions The absorption was monitored at 210 nm (B) The antigenic activities of various 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 described in Experimental procedures Data are expressed as mean ± SD from three experiments 1836 FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS D Tsubokawa et al mAb against sulfated oligosaccharide of mucin Table 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* A1-5b* A1-5c 976 1121 1179 (SO3H)(dHex)(Hex)2(HexNAc)GalNAc-ol (SO3H)(dHex)2(Hex)2(HexNAc)GalNAc-ol (SO3H)(dHex)(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 subjected to NMR spectroscopy Figure shows the one- dimensional 1H-NMR spectra of these two oligosaccharides In the spectrum of A1-5b, b-anomeric resonances (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-anomeric resonances were also recognized as two residues of the a-linked Fuc by a method similar to that described 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 chemical 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 (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 Fig One-dimensional 1H NMR spectroscopy of oligosaccharides A1-5a (A) and A1-5b (B) FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS 1837 mAb against sulfated oligosaccharide of mucin D Tsubokawa et al Table Chemical shifts of each sugar component Chemical shifts labeled with either an asterisk or a dagger indicate occurrence of glycosylation or sulfation shift, respectively ND, Not determined A1-5b Sugar GalNAc-ol Position Position Position Position Position Position Ac-CH3 b-GlcNAc Position Position Position Position Position Position Ac-CH3 b-Gal3 a Position Position Position Position Position Position b-Gal2,4 a Position Position Position Position Position Position a-Fuc2,3 a Position Position Position Position Position CH3 a-Fuc2,4 a Position Position Position Position Position CH3 A1-5a H H 13 C Standardsb 13 C 3.68 ⁄ 3.74 4.35 4.02* 3.43 4.27 3.62 ⁄ 3.9* 2.03 3.72 ⁄ 3.76 4.35 4.04* 3.47 4.20 3.64 ⁄ 3.9* 2.02 63.0 54.2 77.1* 71.6 70.6 73.9* 25.0 61.5 51.5 68.4 69.4 69.8 63.2 21.7 4.53 3.73 3.64 3.85* 3.62 4.25 ⁄ 4.32  2.02 4.54 3.75 3.65 3.83* 3.63 4.25 ⁄ 4.32  2.01 104.4 57.9 75.0 77.8* 75.7 68.8  25.0 101.9 55.4 73.9 69.9 75.8 60.7 21.9 4.42 3.53 ND ND ND ND 4.53 3.63* 3.80 3.85 ND ND 104.8 81.8* 75.0 71.8 78.0 63.8 103.7 70.7 72.7 68.6 75.1 60.9 4.60 3.59* ND ND ND ND 4.60 3.59* 3.81 3.87 ND ND 102.6 80.1* 76.0 71.2 77.6 63.6 103.7 70.7 72.7 68.6 75.1 60.9 5.21 3.74 3.78 3.77 4.23 1.20 103.8 72.0 74.5 72.2 71.1 18.2 99.4 67.8 71.7 69.5 66.4 15.2 5.19 3.74 3.78 3.77 4.18 1.19 102.6 71.2 74.4 72.1 69.7 18.0 99.4 67.8 71.7 69.5 66.4 15.2 5.19 3.74 3.78 3.77 4.17 1.19 compared with the standard b-methylated Gal [13] A heteronuclear multiple-bond correlation (HMBC) spectrum supported this by the presence of remote coupling between the anomeric 1H of Fuc and 13C at position 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 and 6, respectively, based on the 13C chemical shift assessment The following remote coupling could also be recognized from the HMBC spectrum: anomeric 1H of one b-Gal (4.60 p.p.m.) and position 13C of b-GlcNAc (77.8 p.p.m.), anomeric 1H of another b-Gal (4.53 p.p.m.) and position 13C of GalNAc-ol (77.1 p.p.m.), anomeric 1H of b-GlcNAc (4.54 p.p.m.) and position 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 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 sulfation of position of the GlcNAc residue [16] These NMR spectral data support the hypothesis that oligosaccharide 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 blinked 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 1H chemical shift assessment Higher field changes at anomeric ()0.11 p.p.m.) and position ()0.10 p.p.m.) protons 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: 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 1838 FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS D Tsubokawa et al mAb against sulfated oligosaccharide of mucin This structure is supported by the glycosylation shifts observed at position 2, positions and 6, and positions and 6, of the b-linked Gal-bearing Fuc, b-linked GlcNAc and GalNAc-ol, respectively, identified by the cross-peak patterns in the HOHAHA and TOCSY spectra Despite these data, another structure may be possible: Structure To obtain conclusive evidence for the oligosaccharide 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-OCH(CHO)-CH(NHCOCH3)-CH2OH, were obtained from A1-5a (data not shown) The results clearly show that A1-5a was structure and not structure 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 MALDITOF ⁄ 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 determined 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 residue, 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 This was confirmed by the mild periodate oxidation: two fragments, corresponding to Gal-GlcNAc(6SO3H)-O- Fig Effects of defucosylation on antigenic activity with PGM34 (A) After being defucosylated, oligosaccharide A1-5b was chromatographed on two TSK-Gel Amide-80 columns and eluted under isocratic conditions 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 oligosaccharides from these fractions, I (s), II (d), III (n) and IV (m), were examined by the competitive ELISA as described in Experimental procedures Data are expressed as mean ± SD from three experiments FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS 1839 mAb against sulfated oligosaccharide of mucin A D Tsubokawa et al B Ab Aa Bb Ba C Ca Cb Cc D Da Db CH2-CHO and Fuc-Gal-O-CH(CHO)-CH(NHCOCH3)-CH2OH, were obtained (data not shown) These results strongly indicate that the Fuc residue 1840 Fig Immunostaining of the rat gastrointestinal mucosae with PGM34 Immunostaining of the fundic region (Aa), the pyloric region (Ba), the small intestinal region, duodenum (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 performed to compare the immnostaining of these regions, Aa and Bb, respectively Bars ¼ 50 lm attached to the Galb1–4GlcNAc(6SO3H)b- sequence via an a1–2 linkage is an essential component of the epitope of PGM34 FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS D Tsubokawa et al mAb against sulfated oligosaccharide of mucin Table Reactivity of PGM34 with gastrointestinal tissues obtained from rat –, Negative; +, presence of positive cells + ⁄ –, rare presence of positive cells Tissue Stomach Cardia Fundus Pylorus Small intestine Duodenum Jejunum lleum Large intestine Site Reactivity Surface mucous cell Cardiac gland cell Surface mucous cell Mucous neck cell Surface mucous cell Pyloric gland cell – – + – – +⁄– Goblet cell Goblet cell Goblet cell + + + +⁄– extensively stains mucin molecules bearing sulfate residues (Fig 7Ab) This result was supported by the electron 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-secreting 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 Immunohistochemical study of rat gastrointestinal tract with PGM34 Figure shows the immunohistochemical reactivity of PGM34 with different sections of rat gastrointestinal mucosa In the lower part of the pit region of the gastric 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 mucosae, 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 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 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 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 oligosaccharides 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 reactivity 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 position of GlcNAc is essential for the reaction with PGM34 (d) The reduced oligosaccharides showed inhibitory activity toward the binding of B A Fig 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 FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS 1841 mAb against sulfated oligosaccharide of mucin D Tsubokawa et al 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 oligosaccharides 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 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 or core branch The core structure may influence the reactivity of PGM34–6-sulfo H Furthermore, A1-6 may contain 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 oligosaccharide with the 6-sulfo H sequence, the acidic oligosaccharide 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 oligosaccharides 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 clarify 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 predominantly found on oligosaccharides of mucin present in the fundus region, whereas this sequence was only rarely found in the pyloric region [19] Thus, this biochemical result is compatible with the immunohistochemical 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] repor1842 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-glycans 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 glycoproteins 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 highpressure freezing ⁄ freeze substitution method, the excretory 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 surface of the mucosa [24] The lower part of the pit cells of the rat gastric fundus stained by PGM34 is close to the generative cell zone stained by antiproliferating cell nuclear antigen [25] The possibility that mucous cells that secrete sulfomucin are localized in the generative cell zone is supported by a previous study [26] Undifferentiated, granule-free stem cells predominate in the rat isthmus region of the gastric mucosa; these stem cells differentiate and migrate upwards and downwards, replacing the surface mucous cells and glandular cells, respectively [27] In this study, PGM34 did not stain the glandular 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 FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS D Tsubokawa et al 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 clarify the physiological function of sulfomucin in generating cells The mAb, HCM31, recognizes a particular carbohydrate structure of mucin with sialic acid residues [28] Some goblet cells that are stained by HCM31 are distributed 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 extensively 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 biological functions in the human body The mAb, HIK1083, which stains the glandular mucosa 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 mAb against sulfated oligosaccharide of mucin In summary, PGM34 is a very useful tool for recognizing the specific sulfomucin molecule bearing the 6-sulfo H sequence in immunochemical and immunohistochemical 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 precipitating crude pig gastric mucin (Type I; Sigma, St Louis, MO, USA) with ethanol as previously described [35] The oligosaccharide, Fuca1–2Galb1–4GlcNAcb1–6(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) Dowex-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 Kohler & Milstein [36] with the modication of Groth & ă Scheidegger [37] as previously described [10,13] The antibody 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 NaBH4 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 BioGel P-6 column (3.4 cm · 100 cm) The column was eluted with distilled water, and the eluate was monitored by hexose 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 measurement The fractions were collected and desalted on a column of Bio-Gel P-2 Normal-phase HPLC The two-step normal-phase HPLC using TSK-Gel Amide80 (7.8 mm · 300 mm · columns) was used Two buffer FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS 1843 mAb against sulfated oligosaccharide of mucin D Tsubokawa et al systems were used: buffer A [80% (v ⁄ v) acetonitrile in 2.5 mm NaH2PO4] and buffer B (30% acetonitrile in 2.5 mm NaH2PO4) In the first step, the column was equilibrated with 75% A, and the gradient was initiated after injection and increased to 50% B over 30 at a flow rate of 2.0 mLỈmin)1 In the second step, the fractions obtained from the first-step HPLC were rechromatographed under isocratic conditions of 88% A for h at a flow rate of 1.0 mLỈmin)1 UV absorption of the eluate was monitored at 210 nm For the removal of NaH2PO4, the fractions were chromatographed on Sephadex G-10 using distilled water as the eluent residues were derived from an HMBC spectrum The lyophilized powder of the purified oligosaccharides was dissolved in deuterium oxide (2H2O) and evaporated to exchange the unstable 1H with 2H The evaporation and dissolution were repeated five times, and the sample was finally dissolved in 0.75 mL 2H2O 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 Amino sugar analysis Defucosylation of oligosaccharides by mild acid hydrolysis The oligosaccharides were hydrolyzed with m HCl at 98 °C for 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 previously described [41] UV absorption of the eluate was monitored at 254 nm The monosaccharide mixture containing GlcNAc, GalNAc and GalNAc-ol (molar ratios, : : 1) was used as the standard sample MALDI-TOF ⁄ MS analysis The molecular masses of the oligosaccharides were measured 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-dihydroxybenzoic acid dissolved in distilled water ⁄ acetonitrile (1 : 1, v ⁄ v) at 10 mgỈmL)1 as the matrix solutions [42] A 2-lL sample of this mixture was then applied to a stainlesssteel 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 accumulating 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 1H[15N-31P] pulse field gradient indirect-detecting probe Standard pulse sequences were used throughout The 1H NMR spectrum was assigned through pulse field gradient multiple-quantum-correlation spectroscopy and one-dimensional HOHAHA spectroscopy The 13C assignments were made from an HMQC spectrum obtained with carbon decoupling Additional assignments and information on the sequence and linkage of the sugar 1844 The oligosaccharides were hydrolyzed with 0.1 m HCl at 80 °C for h The defucosylated oligosaccharides were separated by normal-phase HPLC using a TSK-Gel Amide-80 column Mild periodate oxidation of oligosaccharides The mild periodate oxidation of oligosaccharides was performed as described by Chai et al [17] The oligosaccharides ( 10 lg) were oxidized with sodium periodate in imidazole buffer, pH 6.5, at °C for After the excess periodate had been destroyed by incubation with butane-2,3-diol at °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 °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 ambient temperature for h The wells were successively incubated with horseradish peroxidase-conjugated goat anti-mouse immunogloblins (Dako, Kyoto, Japan) and 2,2¢-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) ⁄ H2O2 solution (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA), and the color was allowed to develop [44] The wells were washed three times with NaCl ⁄ Pi containing 0.05% Tween-20 between each process The absorption was measured at 405 nm (reference at 492 nm) at 15 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 previously described At the same time, NaCl ⁄ Pi solutions of the oligosaccharide fraction, each containing 20–400 lg per FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS D Tsubokawa et al well as the hexose base, were preincubated with a specified amount of PGM34 for h at ambient temperature As a negative control, instead of the sugar-containing solution, NaCl ⁄ Pi was preincubated with PGM34 The preincubated mixtures were then added to the antigen-coated wells and incubated for 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 NaIO4 in 50 mm sodium acetate, pH 4.5, for h at room temperature Trypsin digestion was performed by exposing the mucin antigen coated on the microtiter wells to trypsin for h at 37 °C A 2.5 mgỈmL)1 trypsin sample in 10 mm Tris ⁄ HCl, pH 8.0, containing mm CaCl2 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 skewers 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 substitution Freeze substitution was carried out in 0.1% glutaraldehyde in acetone at )80 °C for 16 h Specimens were then gradually warmed ()50 °C for h, )20 °C for h, and °C for h) to room temperature After several washes with pure ethanol, the specimens were embedded in paraffin The paraffin sections, lm thick, were deparaffinized, rehydrated, and incubated in methanol containing 0.3% H2O2 for 30 to block endogenous peroxidase activity After several rinses in NaCl ⁄ Pi, the sections were incubated in 5% normal horse serum (NHS) ⁄ 1% BSA in NaCl ⁄ Pi for 10 to block nonspecific binding and then incubated with PGM34 (diluted : 100 with 5% NHS ⁄ 1% BSA in NaCl ⁄ Pi) at °C overnight After being washed with NaCl ⁄ Pi, the sections were incubated with biotinylated horse anti-mouse IgM (Vector Laboratories, Burlingame, CA, USA; diluted : 200 with 1% BSA in NaCl ⁄ Pi) at room temperature for 40 min, followed by washing with NaCl ⁄ Pi The sections were then incubated in a freshly prepared solution of the avidin-biotinylated horseradish peroxidase complex (ABC) kit (Vector Laboratories) for 30 After a wash with NaCl ⁄ Pi, the peroxidase reaction was developed by incubating in 0.05% 3,3¢-diaminobenzidine tetrahydrochloride in 50 mm Tris ⁄ HCl buffer, pH 7.6, con- mAb against sulfated oligosaccharide of mucin taining 0.001% H2O2 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 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] Conjugation 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 transferred 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 °C at 10 °C ⁄ h, the substitution medium was replaced with pure ethanol (three changes each of 10 duration) and then gradually raised to +18 °C and left for h to remove the remaining hydration shell of protein [50] After complete substitution, the temperature was gradually lowered to )35 °C, and infiltration with Lowicryl K4M was performed in mixtures of : and : (v ⁄ v) 100% ethanol ⁄ Lowicryl K4M (60 each) and in pure Lowicryl K4M overnight at )35 °C The polymerization was performed using a UV lamp from the AFS machine for 24 h at )35 °C and for a further h at 18 °C Ultrathin sections, 70–80 nm thick, were treated with 1% BSA in NaCl ⁄ Pi for 10 to block nonspecific binding, and the sections were passed through 50 mm Tris ⁄ HCl buffer 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 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 ⁄ Pi for 10 to block nonspecific binding The sections were incubated with PGM34 (diluted : 50 with 5% NHS ⁄ 1% BSA in NaCl ⁄ Pi) at °C overnight After being washed with NaCl ⁄ Pi, the sections were incubated with biotinylated horse anti-mouse IgM (diluted : 200 with 1% BSA in NaCl ⁄ Pi) at room temperature for 40 After being washed with NaCl ⁄ Pi, the sections were incubated with goat anti-biotin IgG–CG (14 nm) conjugate (diluted with 1% BSA in NaCl ⁄ Pi) at room temperature for 30 FEBS Journal 274 (2007) 1833–1848 ª 2007 The Authors Journal compilation ª 2007 FEBS 1845 mAb against sulfated oligosaccharide of mucin D Tsubokawa et al After being washed with distilled water and dried, the sections were contrasted by KMnO4 ⁄ 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 (containing N,N-dimethyl-m-phenylenediamine, N,N-dimethylp-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 Nakamura 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 Graduate School of Medical Sciences, Kitasato University References 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... (SO 3H) (dHex)(Hex )2( HexNAc)GalNAc-ol (SO 3H) (dHex)(Hex)(HexNAc)2GalNAc-ol (SO 3H) (dHex)(Hex )2( HexNAc)GalNAc-ol (SO 3H) (dHex )2( Hex )2( HexNAc)GalNAc-ol (SO 3H) (dHex)(Hex )2( HexNAc)2GalNAc-ol (SO 3H) (dHex)(Hex )2( HexNAc)2GalNAc-ol... composition of oligosaccharide-alditols A1 - 5a* A1 -5b* A1 -5c 976 1 121 1179 (SO 3H) (dHex)(Hex )2( HexNAc)GalNAc-ol (SO 3H) (dHex )2( Hex )2( HexNAc)GalNAc-ol (SO 3H) (dHex)(Hex )2( HexNAc)2GalNAc-ol The putative carbohydrate. .. newly characterized monoclonal 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 (20 01) Cell lineage