Abstract
Helicobacter pylori infects more than half of the world's population. Although most patients are asymptomatic, persistent infection may cause chronic gastritis and gastric cancer. Adhesion of the bacteria to the gastric mucosa is a necessary prerequisite for the pathogenesis of H. pylorirelated diseases and is mediated by mucin O-glycans. In order to define which glycans may be implicated in the binding of the bacteria to the gastric mucosa in humans, we have characterized the exact pattern of glycosylation of gastric mucins. We have identified that the major component was always a core 2-based glycan carrying two blood group H antigens, whatever was the blood group of individuals. We have also demonstrated that around 80% of Oglycans carried blood group A, B or H antigens, suggesting that the variation of gastric mucin glycosylation between individuals is partly due to the blood group status. This study will help better understanding the role of O-glycans in the physiology and homeostasis of gastric mucosa. Overall, the results reported here give us the necessary background information to begin studies to determine whether individuals who express certain carbohydrate epitopes on specific mucins are predisposed to certain gastric diseases.
Introduction
The mucus layer that covers gastric mucosa is a powerful barrier that protects tissues from mechanical stress as well as erosion by pepsin and HCl (Allen et al. 1993) . Mucus is secreted by epithelial cells and its main components are large glycoproteins called mucins and water. Mucins consist of a long linear protein core (apomucin) carrying hundreds of oligosaccharide chains along the molecule. The extreme length of these molecules, their high degree of glycosylation (more than 50% of their total weight) and their negative charge are major determinants of their ability to form gels, to undertake receptor-ligand interaction and communicate information about the external cell conditions through signal transduction (Hollingsworth and Swanson 2004) .
Introduction
In the adult stomach, two cell types secreting different mucins have been identified. MUC5AC is highly expressed in mucous cells of the surface epithelium of the gastric epithelium, whereas MUC6 is expressed in mucous neck cells and principal cells of the body and in pyloric glands of the antrum (de Bolos et al. 1995; Machado et al. 2000; Corfield et al. 2001; Nordman et al. 2002) . MUC5AC is the common site of Helicobacter pylori localization, whereas MUC6 exerts antibacterial activity (Van den Brink et al. 2000) . Synthesis of mucus begins in fetal stomach by 8 weeks of gestation, long before epithelial cytodifferentiation (Buisine et al. 2000) . Fetal mucosa expresses four different secreted mucins: the two adult gastric mucins MUC5AC and MUC6, but also MUC5B and the intestinal mucin MUC2, which are not recovered in normal adult stomach (Buisine et al. 2000) .
Introduction
The characteristics of the gastric mucous layer reflect its physiological function and changes in its composition can be noted following various insults. Notably, the development of monoclonal antibodies to various mucins has allowed the evaluation of the changes in composition and distribution in diverse pathologies. For example, it has been shown that H. pylori-infected patients exhibit an increase in MUC6 in surface mucous cells with a reduction in MUC5AC. MUC2 mucin expression has been proposed to be a reliable marker of intestinal metaplasia which appears in the context of H. pylori infection (Babu et al. 2006 ). MUC2 and MUC5B were found to be de novo expressed in gastric carcinomas.
Introduction
If alterations in mucin expression have been well documented during gastric pathologies, only few lectin or immunohistochemistry studies have described modification in the pattern of glycosylation of human gastric mucins, concerning mainly the expression of tumor-associated carbohydrate antigens such as the expression of Tn, sialyl-Tn or TF antigens and inflamed associated antigens such as 6-sulfo-sialyl-Le x . However, these studies have not allowed the identification of specific carbohydrate markers reliable for the early diagnosis of gastric cancer, for its prognosis or its differential diagnosis from other nonmalignant gastric diseases.
Introduction
In this way, we have decided to establish the exact structures of mucin O-glycans isolated from healthy adult gastric tissues, using physical techniques such as tandem mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. The results reported here will give us the necessary background information to characterize precisely which oligosaccharides are differently expressed or neo-expressed in the pathological state.
Results

Purification of gastric mucins
Mucins recovered in the gastric mucus were isolated by a combination of isopycnic density gradient centrifugation and gel chromatography (data not shown). After ultracentrifugation, a carbohydrate-rich peak was observed with a density in the range 1.33-1.45 g/mL, which is typical for the gel forming-gastric mucins (Sheehan et al. 2000) . The mucins were further purified by gel chromatography in GuHCl 4 M on a Sepharose CL-4B column. The mucins eluted in the void volume and were separated from the smaller proteins and glycoproteins that were eluted in the included volume of the column (data not shown). To ascertain the identity of the mucins into the adult stomachs, the density gradient fractions were probed with a number of mucin-specific antibodies. As expected, adult gastric mucins have a strong reactivity with the 45M1 (recognizing MUC5AC) and antibodies. This coincided with the major carbohydrate peak. No reactivity was found with antisera recognizing the MUC5B (LUM5B-3) and MUC2 (LUM2-3) mucins in the adult stomachs. Mucin-containing fractions were pooled, dialyzed into water at 4°C and lyophilized for further study.
Structural features of human gastric mucin O-glycans
Purified mucins were analyzed for their glycan compositions and sequences. Oligosaccharides were released by base/borohydride treatment followed by desalting. The pool of oligosaccharide alditols obtained from each sample was analyzed by nano-ESI quadrupole time-of-flight (Q-TOF) tandem mass spectrometry (Robbe et al. 2004) . Fragment annotations applied in this study were based on the suggested nomenclature (Domon and Costello 1988; Karlsson et al. 1996) . The fragment ions obtained were mainly A i , B i , Y j and Z j ions. Moreover, an α suffix was used to designate cleavages in the 6-linked branch and a β suffix for cleavage in the 3-linked branch from the GalNAcol.
Structural features of human gastric mucin O-glycans
Sequencing of oligosaccharides was acquired in both positive-and negative-ion modes to obtain the complete elucidation of the structure for each mucin oligosaccharide. The positive-ion mode is useful for the determination of the coretype oligosaccharide and the position of fucose, whereas the negative-ion mode produces characteristic cross-ring cleavages necessary for differentiation between isomers and identification of NeuAc (N-acetylneuraminate) substitution. For example, a specific 0,2 A i cleavage with concomitant loss of water from a GlcNAc substituted on C-4 allowed to differentiate between type 1 (Galβ1-3GlcNAc) and type 2 (Galβ1-4GlcNAc) chains. Some characteristic ions also allowed assigning unambiguously the presence of certain terminal structures such as the Lewis determinants. However, in complex oligosaccharides, diagnostic ions are sometimes of lower intensity, rendering the complete elucidation of the structure impossible (Robbe et al. 2006) .
Structural features of human gastric mucin O-glycans
Unfractionated mixtures of O-glycans were also analyzed by one-and two-dimensional COSY, TOCSY and HSQC NMR spectroscopy. All the analyses were performed on the same total amount of oligosaccharide alditols in each fraction to make a qualitative and semi-quantitative comparison of specific sequences on blood group determinants. Despite the complexity, different classes of NMR signals were identified by using previously published spectra of purified reference compounds and by using GLYCOBASE (http://glycobase. univ-lille1.fr/base/) database. Oligosaccharides were further fractionated by high-performance liquid chromatography (HPLC) on primary amino-bounded and reverse-phase columns (Supplementary data, Figures S1 and S2) , and the fractions collected were analyzed by NMR spectroscopy and nano-ESI Q-TOF MS/MS. These studies not only allowed the confirmation of certain glycan structures, but above all were useful for the determination of the relative abundance of major oligosaccharides (Table I) . Figure 1A represents the MS spectrum acquired in the positive-ion mode for the total oligosaccharide fractions from one individual with blood group A. Most of the ions could be related to [M + 2Na] 2+ ions of the theoretical oligosaccharide structures. The spectra illustrate a remarkable structural diversity: the true heterogeneity is likely to be even larger, since a substantial number of the molecular ions may represent oligosaccharide species with isomeric structures. To avoid confusion, the mass values used in the text, tables and illustrated fragmentations are mainly nominal masses for all monocharged ions. Figure 2 corresponds to extended regions of the COSY90 NMR spectrum obtained from the mixture of oligosaccharides from the same blood group A individual. As shown in Figure 2 and Table II, numerous α-(4.8-5.4 ppm) and β-(4.4-4.7 ppm) anomeric protons were observed on the two-dimensional spectra of adult gastric human mucins, indicating a large heterogeneity of O-glycan compositions. β-anomeric protons belonged to Gal or GlcNAc residues as found on lactosaminic chains, whereas α-anomeric protons belonged to Fuc, GlcNAc and GalNAc residues. Table I summarizes all the identified oligosaccharides and their relative abundance in human gastric mucins. Around 70 different structures were characterized by tandem mass spectrometry and NMR spectroscopy, demonstrating a significant heterogeneity. As determined by HPLC and nano-ESI-M/MS ( Figure 1A and B and Table I ), most of the oligosaccharides recovered were neutral, highly fucosylated and carried several lactosaminic units (repetition of Galβ1-3/4GlcNAc). Core 2 was the main core structure detected in these gastric mucins.
Structural features of human gastric mucin O-glycans
Only few glycans expressed core 1 structures. Numerous fucosylated oligosaccharides carried the blood group O determinant (Fucα1-2Galβ1). They were unambiguously identified in MS/MS fragmentation spectra by two diagnostic ions at m/z 205 and 247 in the negative-ion mode, corresponding, respectively, to the 1,3 A 2 fragment (including fucose and part of the galactose) and a B 2 cleavage accompanied with additional loss of acetate parts (B 2 -C 2 H 4 O 2 ).
Structural features of human gastric mucin O-glycans
As shown in Figure 3 , almost all the gastric O-glycans (more than 80%) were constructed on a unique tetrasaccharide structure: Fucα1-2Galβ1-3[GlcNAcβ1-6]GalNAcol. The lower branch was never elongated, except the presence of blood group A or B antigens. These results suggest that core 2 β6-GlcNAc transferase and FUT2 are key enzymes in the biosynthesis of gastric mucin O-glycans. The extreme heterogeneity of gastric mucin glycosylation is mainly due to the elongation of the upper branch with several lactosaminic units and substitution by peripheral residues.
A weak expression of Lewis determinants
Most of the O-glycans from gastric mucins are fucosylated. However, around 8% of the total oligosaccharides carried Sialyl TF antigen 2.4 ± 1.0* 1.9 ± 0.9*** 1.6 ± 0.1* 675 Sialyl TF antigen 2.2 ± 1.1* 1.6 ± 0.9*** 1.8 ± 0.2*** Structures carrying blood group H antigens are noted in bold. *P < 0.001. **P < 0.01. ***P < 0.05. Variation in gastric mucin glycosylation between individuals is mainly due to expression of blood group antigens Expression of blood group antigens was recovered in gastric mucin O-glycans, in agreement with previous studies (Mollicone et al. 1985; Oberhuber et al. 1997) . For example, the α-fucosyl unit at δ 5.33 which possessed a strong typical Table III) showing H6/H5 correlation from fucosyl residues (A), H3ax/H3eq correlation of sialic acid (B) and main correlations from different sugar units (D). (C) N-acetyl NMR signals extracted from a one-dimensional spectrum indicating the presence of some aminosugars. Signals with asterisk indicate the presence of A blood group sequence. Signals in circle correspond to H3 ( pseudo-doublet) and H4 (singlet) protons of a sialylated galactosyl residue.
Glycosylation of human gastric mucins

Glycosylation of human gastric mucins
A major feature of gastric mucins is the expression of blood group H antigen, recovered in individuals with either blood group O or A and B. For example, the ion at m/z 1065 in the positive-ion mode corresponded to an oligosaccharide carrying two H antigens, one in the upper branch and the other in the lower branch. This ion accounted, respectively, for 19, 18 or 30% of the total quantity of oligosaccharides in blood group A, B or O individuals, respectively. Figure 4 shows the MS/MS spectrum of this ion at m/z 1065. The core 2 was easily identified by the two diagnostic ions Y i ions at m/z 408 (Y 1β/2α corresponding to a Gal linked to the GalNAcol) and m/z 449 (Y 1α/2β corresponding to a GlcNAc linked to the GalNAcol). The Y 1α fragmentation ion at m/z 554 indicated the presence of a group H antigen in the lower branch (Fucα1-2Galβ1-3GalNAcol). In the upper branch, the presence of the B3α ion at m/z 534 (indicating a sequence composed by three monosaccharides: Gal, GlcNAc and Fuc) and the absence of the ion at m/z 591 (corresponding to Fucα1/3-4GlcNAcβ1GalNAcol) confirmed that the Fuc residue is linked to galactose and defined the second group H antigen.
Glycosylation of human gastric mucins
Supplementary data, Figure S1 shows the HPLC chromatograms corresponding to O-glycans purified from three individuals with blood group A, B or O, respectively. Important qualitative and quantitative variations in the expression of certain oligosaccharides are observed between individuals. These variations are mainly correlated with the expression of blood group antigens. As shown in Table I , around 80% of structures carried blood group A, B or O determinants. This result demonstrates that glycosylation varies extensively between individuals and that this variation is mainly due to the blood group status. However, it is important to note that The values with hyphen correspond to minimum and maximum chemical shifts observed of corresponding proton. The values with a slash correspond to two protons from distinguishable protons. Identification of one (α1,4GlcNAc)-capped core 2 branched O-glycan Two-dimensional NMR experiments allowed us to confirm the expression of a particular α-N-acetylglucosaminyl residue on gastric mucins. Indeed, starting from the anomeric jason70@example.net ppm with small (S) 3 J 4,5 as being L defining the α-Gluco configuration of this unit (configuration S L L L as described by Koerner et al. 1987) . Moreover, the HSQC spectrum (not shown) indicated that H2 was coupled to a jason70@example.net ppm having attached nitrogen and, therefore, supporting the conclusion that this unit is an N-acetylglucosaminosyl residue. Finally, the absence of the NAc group signal downfield jason70@example.net demonstrated that this unit is linked on the upper branch of oligosaccharide (Kamerling and Vlgenthart 1992) . This unit was not recovered in the lower branch of the oligosaccharide.
Glycosylation of human gastric mucins
After separation of oligosaccharides by HPLC, we have shown that only one glycan carried this particular terminal α1,4GlcNAc-linked residue. It corresponded to the ion at m/z 1122 in the positive-ion mode MS analysis. This glycan is based on a core 2 with a fucose residue linked to a Gal, on the lower branch. The upper branch contains a GlcNAc linked to the GalNAcol, followed by a Gal which is substituted by the α1,4GlcNAc. Moreover, this is not a main component of human gastric mucins, representing less than 3% of the total structures identified.
Glycosylation of human gastric mucins
Gastric mucin O-glycans are mainly neutral with only few sialylated oligosaccharides Most of the NeuAc residues identified were α2-3 linked to Gal residues. Figure 2B shows the correlation between two methylenic protons corresponding to H3eq at δ 2.77 ( Moreover, three jason70@example.net, 4.12 and 3.93 ppm were identified as being the H2, H3 and H4 protons, respectively, of an O-3 sialylated galactosyl residue. These parameters were in agreement with a sialylated galactose on the lower branch of the acidic O-glycans as observed, for example, in the sequences having CCSD numbers 873, 1091 and 11942 from SUGABASE. Considering the low intensity of concerned signals, we must also note that acidic O-glycans were in low amounts compared with neutral glycans. Indeed, only 10% of total oligosaccharides were sialylated and no sulfated glycans were recovered. Only eight structures were characterized carrying NeuAc residues (Table I) . Among them, some oncofetal markers were recovered such as sialyl-Tn at m/z 513 in the negative-ion mode (NeuAcα2-6GalNAcol) and sialyl-TF at m/z 675 in the negative-ion mode (NeuAcα2-3Galβ1-3GalNAcol) ( Table I) .
Binding of H. pylori to human gastric mucins
Because we have found a high level of expression of glycans carrying blood group A, B or H antigens which are known to be ligands for H. pylori, we have decided to analyze the adhesion of the bacteria to purified human gastric mucins. Two strains of H. pylori were used, an ulcer-associated J99 strain and a gastritis-associated 26695 strain. The results (Figure 5 ) Mucins were purified from individuals of different blood group and Lewis status. Two strains of H. pylori, an ulcer-associated J99 strain and a gastritis-associated 26695 strain, were tested. Specific binding to mucin (binding parameter "R") was calculated by subtracting the value of bacteria bound to blocking buffer from the values of bacteria bound to mucin. A value of >1 indicates specific binding.
Glycosylation of human gastric mucins
confirmed that human gastric mucins are recognized by H. pylori and serve as binding sites for the bacteria. The J99 strain presented a stronger adhesion than the 26695 strain, among all the individuals included in the study. Mucins purified from blood group A patients (secretors and non-secretors) interacted very strongly with the J99 strain. Mucins from secretors patients (expressing Lewis b antigens) of blood groups A and B seemed to interact more strongly with the J99 strain of H. pylori than the non-secretors. This is in agreement with previous studies showing that Le b is a preferential ligand for BabA, one of the most studied adhesin of H. pylori (Aspholm-Hurtig et al. 2004 ).
Discussion
In the present paper, we provide the first detailed structural analysis of O-glycans originating from healthy adult gastric mucins. Until today, only few studies have dealt with the glycosylation of normal adult gastric mucins. Hanisch et al. (1993) have demonstrated the predominant expression of core 1 and 2 structures. Combining different mass spectrometry techniques, they also identified around 30 neutral oligosaccharides. Many more structures have been identified in the present work. This can be readily explained by the use of nano-ESI mass spectrometry methodology, which is known to be more sensitive than NMR or negative-ion liquid secondary electron impact mass spectrometry. However, our results are in agreement with this previous work in that all the structures described before have been recovered in this study and we also found the major expression of core 2 glycans in adult gastric samples. Jiang et al. (2004) have performed a lectin histochemical study to determine the glycoprofile of the pit, the neck and the gland epithelia of the normal gastric epithelium. In contrast to our study where only mucin O-glycans were analyzed, this previous work gave information about oligosaccharide sequences recovered on all the "glycocomponents" of gastric mucosa. Most of the peripheral and internal substitutions stained by lectins were also structurally characterized and are recovered in gastric mucin O-glycans in our study. For example, we demonstrated, by NMR, the expression of blood group antigens in gastric tissues and Jiang et al. showed staining with DBA lectin, which specifically recognizes the blood group A epitope, and with UEA-I lectin, which stains L-fucosyl terminal residues and especially Fucα1-2Galβ1-4GlcNAcβ1.
Discussion
Among the 70 oligosaccharides identified in this paper, most of them were neutral and highly fucosylated. Only few structures were acidic carrying sialic acid residues. No sulfated O-glycans were recovered. This is in agreement with the previous histochemical studies showing the absence of dark brown staining with the high-iron diamine-Alcian blue staining, which is specific for sulfated mucins, in the normal gastric mucosa. Sulfomucins gradually make their appearance as the metaplastic process advances (Correa 1988) . However, this is in contrast to rat gastric mucins where sulfation has been found to cause the heterogeneity of such mucins (Van Beurden-Lamers et al. 1989) . Several hypotheses may explain the weak sialylation and the absence of sulfation of human gastric mucins. First, sialic acids and sulfate residues are mainly implicated in protection against bacterial attack and confer resistance to protease degradation (Corfield et al. 1992 ). However, due to the acidity of the pH in men, the level of the microflora in the stomach is low: around 10 3 -10 4 bacteria/mL in human stomach, with a flora composed of Gram-positive facultative anaerobic bacteria such as streptococci. In contrast, because the pH is relatively high in rat stomach, more bacteria are found: around 10 5 -10 6 bacteria/ mL (Dethlefsen et al. 2007 ). Secondly, the extremely acidic pH ( pH 1-2) in men, which is due to HCl secretion from parietal cells of the gastric mucosa, may directly affect the peripheral substitution of glycans. Indeed, low pH causes a protonation of sulfate and carboxyl groups resulting in a low stability of these residues, due to the formation of electrostatic repulsion of charged groups. ABH histo-blood group status are α1,2-fucosyloligosaccharides found on glycoproteins and glycolipids. They are synthesized through the stepwise action of several glycosyltransferases, from a disaccharide precursor. In mucin O-glycans, three precursors exist: type 1, Galβ1-3GlcNAcβ1-R; type 2, Galβ1-4GlcNAcβ1-R; type 3, Galβ1-3GalNAcα1-R. The first biosynthesis step is a transfer of a fucose residue α1,2 linked to the Gal residue. This step may be catalyzed by two fucosyltransferases encoded by FUT1 and FUT2 genes. The FUT2 "secretor" gene encodes the fucosyltransferase that synthesizes the H-type 1 antigen. The lack of functional FUT2 allele is responsible for the nonsecretor phenotype, which is characterized, as opposed to the secretor status, by the absence of ABH antigens in saliva and on various epithelial cell types. Further addition of GalNAc or Galα1-3 linked to the H antigen leads to the synthesis of blood group A or B antigens and are catalyzed by α1,3-N-acetylgalactosaminyltransferases or α1,3-galactosyl transferases, respectively. Individuals included in this study were either secretor or non-secretor. As expected, we found differences in the expression of structures carrying histo-blood group Le b antigens. For example, the ion at m/z 1211 corresponding to a core 2 glycan with an Le b antigen and the ion at m/z 1227 corresponding to a core 2 glycan with a BLe b antigen were exclusively recovered in secretor individuals.
Discussion
However, most of the O-glycans identified on gastric mucins were similar between individuals with the same blood group status whatever was the Lewis status. This was confirmed by the statistical analysis of variation of glycosylation between individuals. Indeed, almost all the O-glycans were constructed on a tetrasaccharide structure: Fucα1,2Galβ1,3 [GlcNAcβ1,6]GalNAcol. The lower branch was never elongated, except the presence of blood group A or B antigens. The extreme heterogeneity of gastric mucin glycosylation is mainly due to the elongation of the upper branch with several lactosaminic units, mainly of type 2, and substitution by peripheral residues such as blood group A, B or H antigens.
Discussion
Helicobacter pylori is the major pathogen of the gastric mucosa. It is one of the most common infections in human, affecting more than half of the world's population. If most infected individuals develop only gastritis and remain asymptomatic, around 10% of infected individuals develop three important upper gastrointestinal diseases: gastroduodenal ulcer disease, gastric carcinoma and mucosal associated lymphoid tissue lymphoma (Marshall and Warren 1984; Wotherspoon et al. 1993; Blaser et al. 1995) . The ability of the organism to cause disease depends on a variety of host, environmental and bacterial factors. Among them, the attachment of the bacteria to the mucous epithelial cells and the mucous layer lining the gastric epithelium is a crucial step for the pathogenesis of H. pylori-related diseases (Linden et al. 2008; McGuckin et al. 2011) . Adhesion of H. pylori is mediated by bacterial outer membrane proteins which recognize carbohydrate ligands on the host surface of gastric mucosa. In particular, Boren et al. (1993) ). Based on these observations, it has been proposed a higher adhesion of the bacteria to the gastric mucosa of blood group O individuals, contributing to their higher risk of peptic ulcer disease (Clarke et al. 1955 ). Our major results in this study concerns the quantitative demonstration that (i) more than 80% of human mucin O-glycans carry blood group antigens on their periphery, (ii) the variation of glycosylation between individuals is mainly due to blood group determinants expression and (iii) the binding of H. pylori to human gastric mucins depends on H. pylori strains and the level of expression of glycans carrying blood group and Lewis antigens. Thus, the present work reinforce previous observations about a closely link between H. pylori pathogenicity and the expression of ABH antigens in the gastric mucosa.
Discussion
Using NMR spectroscopy, we have confirmed the expression of gastric mucin O-glycans carrying a particular GlcNAc residue α1-4 linked to β-Gal residues. In normal human tissues, immunohistochemical studies have shown that glycoproteins having GlcNAcα1-4Gal β1-R structures at non-reducing ends are exclusively limited to the mucins secreted from gland mucous cells of gastric mucosa and Brunner's gland of duodenum (Nakamura et al. 1998) . Several studies have shown that gastric mucins containing α1-4GlcNAc capped O-glycans inhibit H. pylori growth (Kawakubo et al. 2004; Lee et al. 2008 ) by inhibiting cholesterol α-glucosyltransferase which synthesizes α-glucosyl cholesterol. α-Glucosyl cholesterol and its derivatives are major components of the cell wall of Helicobacter species and α-glucosylation of cholesterol in H. pylori facilitates both infectivity and pathogenicity of the bacteria. Here, we found a weak relative level of expression of α1-4GlcNAc (around 2 ± 0.6%) in all healthy individuals studied. This result may be partly explained by the fact that the synthesis of this structure belongs to an innate immune defense mechanism. One hypothesis may be that an increased expression of the synthesis of this residue is observed in response to an infection by H. pylori.
Discussion
In conclusion, this study provides for the first time the exact structural characterization of human gastric mucin O-glycans isolated from healthy adults, showing a high heterogeneity in the pattern of glycosylation of these mucins and an important variation of glycosylation between individuals. In future works, it will help to identify which oligosaccharides are differently expressed or neo-expressed in the pathological state and will help to identify, for example, carbohydrate markers reliable for the early diagnosis of gastric cancer or its prognosis.
Materials and methods
Human samples and mucin preparation Gastric tissues were collected by Dr Bara according to the protocols approved by the National Ethical Committee. Thirty-two human adult stomachs were obtained from France Transplant Association from kidney donors. A summary of age, sex and blood group status of the donors is given in Table III . Blood group activity was detected by hemagglutination inhibition tests and confirmed by immunofluorescence on gastric sections. Briefly, crude stomach extracts at 10 mg/mL in saline were used for hemagglutination inhibition Samples of mucosa were scraped and snapfrozen in liquid nitrogen and stored in liquid nitrogen until used. Samples collected both included the inner firmly adherent and the outer loosely adherent mucus gel layers.
Materials and methods
After thawing, the tissue was kept at 4°C, homogenized in distilled water (Ultra-turrax, Jankee and Kunkel, Stauffer, Germany) and centrifuged (1 h at 48,000 × g). The resultant supernatant was dialyzed against distilled water for 2 days at 4°C and lyophilized (Lesuffleur et al. 1995) .
Materials and methods
Isolation and purification of mucins from gastric tissues Mucins were solubilized in 4 M guanidine chloride solution containing 5 mM ethylenediaminetetraacetic acid, 10 mM benzamidine, 5 mM N-ethylmaleimide, 0.1 mg/mL soy bean trypsin inhibitor and 1 mM phenylmethanesulfonyl fluoride. CsCl was added to an initial density of 1.4 g/mL and mucins were purified by isopycnic density-gradient centrifugation (Beckman Coulter LE80K ultracentrifuge; 70.1 Ti rotor, 417 600 g at 15°C for 72 h). Fractions of 1 mL were collected from the bottom of the tube and analyzed for periodic acid-Schiff (PAS) reactivity, density and reactivity against MUC5AC and MUC6 antibodies.
Materials and methods
The mucin-containing fractions were pooled, dialyzed into 4 M guanidinium chloride buffer and subjected to gel chromatography on Sepharose CL-4B. The column (85 × 2 cm, Pharmacia, GE Healthcare, France) was eluted at a flow rate of 8 mL/h with 4 M guanidinium chloride.
Materials and methods
Analytical methods Slot-blotting of mucin proteins. Polyclonal antibodies (LUM2-3, LUM5B-3 and LUM6-1) used in this study were kind gifts from Dr Ingemar Carlstedt, University of Lund, Sweden. One monoclonal antibody recognizing MUC5AC, kindly provided by Dr Jacques Bara (45M1), was also used. Aliquots of purified mucins were slot-blotted onto polyvinylidene fluoride membranes. The membranes were then blocked with 0.5% (w/v) skimmed milk in tris-buffered saline containing 0.1% (v/v) Tween 20 (blocking solution) for 1 h and incubated with mono-or polyclonal antibodies at a dilution of 1:1000 in blocking solution. Mono-or polyclonal antibodies used in this study were LUM2-3, LUM5B-3, 45-M1 and LUM6-1 antisera recognizing MUC2, MUC5B, MUC5AC and MUC6, respectively. Bound antibody was detected by incubation with horseradish peroxidaseconjugated anti-rabbit (1:10,000) antibody in blocking solution for 1 h followed by the ECL Western detection kit. All incubations were carried out at room temperature. Total carbohydrate was determined using a PAS assay after slot blotting of mucins onto nitrocellulose (Thornton et al. 1989 ).
Materials and methods
Release of oligosaccharides from mucin by alkaline borohydride treatment The gastric mucins were submitted to β-elimination under reductive conditions (0.1 M KOH, 1 M KBH 4 for 24 h at 45°C; Carlson 1968) . The mixture of oligosaccharide alditols was purified by size exclusion chromatography on a column of Bio-Gel P2 (85 × 2 cm ID, 400 mesh, Bio-Rad, Richmond, CA) equilibrated and eluted with water (10 mL/h) at room temperature. The oligosaccharide fractions, detected by UV absorption at 206 nm, were pooled for structural analysis.
Materials and methods
Electrospray mass spectrometry (nano-ESI-MS/MS). All analyses were performed on a Q-STAR Pulsar Q-TOF mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, Canada) fitted with a nano-electrospray ion source (Protana, Odense, Denmark). Oligosaccharides dissolved in water (60 pmol/µL) were acidified by addition of an equal volume of methanol/0.1% formic acid and sprayed from gold-coated "medium length" borosilicate capillaries (Protana). A potential of −800 V was applied to the capillary tip and the focusing potential was set at −100 V, the declustering potential varying between −60 and −110 V. For the recording of conventional mass spectra, TOF data were acquired by accumulation of 10 multiple channel acquisition scans over mass ranges of m/z 400-2000. In the collision-induced dissociation (CID) tandem MS analyses, multiple charged ions were fragmented using nitrogen as collision gas (5.3 × 10 −5 Torr), the collision energy varying between −40 and −90 eV to obtain optimal fragmentation. The CID spectra were recorded on the orthogonal TOF analyzer over a range of m/z 80-2000. Data acquisition was optimized to supply the highest possible resolution and the best signal-to-noise ratio even in the case of low-abundance signals. Typically, the full width at half maximum was 7000 in the measured mass ranges. External calibration was performed prior to each measure using a 4-pmol/µL solution of taurocholic acid in acetonitrile/water (50:50, v/v) Fractionation of the oligosaccharide alditols by HPLC. The mixture of oligosaccharide alditols released from each sample was subjected to fractionation by HPLC (Dionex Chromeleon System, Sunnyvale, CA) on a primary amino-bonded silica column (Supelcosyl, LC-NH 2 , 4.6 × 250 mm, Supelco, Bellefonte, CA). The column was equilibrated with the initial solvent using a mixture of acetonitrile/H 2 O (80:20, by vol) with a flow rate of 1 mL/min. After the injection, a linear gradient from 80:20 to 40:60 (acetonitrile/H 2 O, by vol) for 80 min was applied followed by isocratic conditions [40:60 (acetonitrile/ H 2 O, by vol)] for 20 min. A second step of HPLC on a C18 reverse-phase column was conducted for the fraction containing several oligosaccharides in one peak. The column was equilibrated with H 2 O at a flow rate of 1 mL/min and oligosaccharides were eluted with H 2 O. Oligosaccharides were detected by UV spectroscopy at 200 nm using an UVD 170U detector (Dionex Chromeleon System).
Materials and methods
Statistical analysis. To determine whether differences in the level of expression of each glycan from individuals with the same blood group and Lewis status were statistically significant, a Student's t-test was applied.
Materials and methods
Bacterial strains and growth media. Helicobacter pylori strains 26695 (Tomb et al. 1997 ) and J99 (Alm et al. 1999) were cultivated on blood agar plates or BHI with 10% of decomplemented fetal calf serum, both supplemented or not, with an antibiotic-antifungal mix (0.31 μg/mL of polymyxin B, 2.5 μg/mL of amphotericin B, 12.5 μg/mL of vancomycin and 6.25 μg/mL of trimethoprim) and appropriated antibiotics (20 μg/mL of kanamycin, 5 μg/mL of apramycin or 5 μg/mL of chloramphenicol), at 37°C in a microaerophilic atmosphere.
Materials and methods
Enzyme-linked immunosorbent assay for H. pylori binding to human gastric mucins This enzyme-linked immunosorbent assay (ELISA) was modified from Tzouvelekis et al. (1991) . Briefly, microtitre plates (Greiner Bio-One, Wemmel, Belgium) were coated with purified gastric mucins (50 µg of mucin per well) in 0.05 M carbonate buffer ( pH 9.6) and incubated overnight at 4°C. The unbound mucins were removed by three washes with 300 µL of phosphate-buffered saline (PBS). Residual binding sites were blocked with protein-free blocking buffer (PFBB, Thermo Fisher Scientific Inc.) and incubated at room temperature for 2 h. After removing unbound blocking buffer by two washes with PBS followed by one wash with PBS-T (0.1% Tween-20), 100 µL of a solution containing 10 6 bacteria/mL was added to each well and incubated at 37°C for 2 h. The unbound bacteria were removed by two washes with PBS followed by one wash with PBS-T. A total of 100 µL of rabbit monoclonal antibody against H. pylori (Abcam, Cambridge, UK; 1:1000 in PFBB) was added to each well and incubated at room temperature for 2 h. The unbound antibodies were removed by two washes with PBS followed by one wash with PBS-T. About 100 µL of horseradish peroxidase-conjugated anti-rabbit (1:1000 in PFBB) was added to each well and incubated at 37°C for 1 h. The unbound antibodies were removed by two washes with PBS followed by one wash with PBS-T. The peroxidase staining reaction was developed in the presence of 2,2′-azino-bis(3-ethylbenz-tiazoline-6-sulfonic acid, diammonium salt) (Sigma-Aldrich; 22 mg in 100 mL of 50 mM sodium citrate, pH 4.0) completed with 0.05% H 2 O 2 . After 30-60 min of incubation at room temperature, the OD 405 values were measured on a BioTek microplate reader. All experiments were done in triplicate and repeated thrice. Specific binding to mucin (binding parameter "R") was calculated by subtracting the value of bacteria bound to blocking buffer from the values of bacteria bound to mucin. A value of >1 indicates specific binding.
Supplementary data
Supplementary data for this article is available online at http:// glycob.oxfordjournals.org/.
Funding
This investigation was supported in part by the CNRS (Unité Mixte de Recherche CNRS/USTL 8576; Director Dr Jean-Claude Michalski) and by the Ministère de la Recherche et de l'Enseignement Supérieur. The mass spectrometry and NMR facility used in this study was funded by the European Community (FEDER), the Région Nord-Pas de Calais (France) and the CNRS and the Université des Sciences et Technologies de Lille.

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