Glycobiology Advance Access originally published online on April 9, 2008
Glycobiology 2008 18(7):494-501; doi:10.1093/glycob/cwn030
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Studies of Lewis antigens and H. pylori adhesion in CHO cell lines engineered to express Lewis b determinants
2 Division of Clinical Immunology and Transfusion Medicine, Karolinska Institute, SE 14186 Stockholm
3 Department of Surgery, Sahlgrenska University Hospital, SE 41345 Göteborg
4 Department of Medical Chemistry and Biophysics, Umeå University, SE 90187 Umeå, Sweden
1 To whom correspondence should be addressed: Division of Clinical Immunology and Transfusion Medicine, F79 Karolinska University Hospital, Huddinge S-141 86 Stockholm, Sweden. Tel: +46-8-585-81384; Fax: +46-8-585-81390; e-mail: jan.holgersson{at}ki.se
Received on February 15, 2008; revised on April 4, 2008; accepted on April 5, 2008
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Abstract |
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Many microbes bind and adhere via adhesins to host cell carbohydrates as an initial step for infection. Therefore, cell lines expressing Lewis b (Leb) determinants were generated as a potential model system for Helicobacter pylori colonization and infection, and their expression of blood group Lewis determinants was characterized. CHO-K1 cells were stably transfected with selected glycosyltransferase cDNAs, and two Leb positive clones, 1C5 and 2C2, were identified. Expression of Lewis (Lea, Leb, Lex, and Ley) determinants was analyzed by flow cytometry of intact cells, SDS–PAGE/Western blot of solubilized glycoproteins, and thin layer chromatography immunostaining of isolated glycolipids (GL). Binding of H. pylori to cells was examined by microscopy and quantified. Flow cytometry showed that 1C5 and 2C2 were Lea and Leb positive. 1C5 expressed Leb on O-linked, but not N-linked, glycans and only weakly on GLs. In contrast, 2C2 expressed Leb on N-, O-glycans, and GLs. Furthermore, both clones expressed Lea on N- and O-glycans but not on GLs. 2C2, but not 1C5, stained positively for Ley on N-linked glycans and GLs. Both clones, as well as the parental CHO-K1 cells, expressed Lex on GLs. A Leb-binding H. pylori strain bound to the 1C5 and 2C2 cells. In summary, two glycosyltransferase transfected CHO-K1 cell clones differed regarding Lewis antigen expression on N- and O-linked glycans as well as on GLs. Both clones examined supported adhesion of a Leb-binding H. pylori strain and may thus be a useful in vitro model system for H. pylori colonization/infection studies.
Key words: Bacterial adhesion / fucosyltransferase / Helicobacter pylori / Lewis antigens
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Introduction |
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With the cloning of the majority of known glycosyltransferase genes and the ability to express those genes in different cells, specific glycan structures can be engineered in well-characterized cells. Such glycan-engineered cells are useful tools in many different research areas, e.g., for studies of microbial adhesion to selected carbohydrate structures. Helicobacter pylori (H. pylori) is a gram-negative bacterium associated with gastritis, gastric and duodenal ulcers, and stomach cancer, and binds via adhesins to carbohydrate structures in the host mucosa (Dunn et al. 1997
; Montecucco and Rappuoli 2001; Peek and Blaser 2002). One of its adhesins, BabA, binds to the Lewis b (Leb) carbohydrate determinant, Fuc
2Galβ3(Fuc4)GlcNAc-R (Ilver et al. 1998); BabA binding of Leb is a major risk factor for disease development (Gerhard et al. 1999; Prinz et al. 2001; Rad et al. 2002). One successful strategy for studying H. pylori–host cell interactions in vivo has been to generate glycosyltransferase transgenic mice that express Leb in the stomach (Falk et al. 1995; Guruge et al. 1998).
Cell lines stably expressing Leb may be used as an in vitro model system for H. pylori adhesion. Various cell lines (HT-29, AGS, Kato III, HuTu-80, and HEp-2), as well as primary gastric cells, have been explored (Segal et al. 1996; Chmiela et al. 1997; Simon et al. 1997; Michetti et al. 1999; Nishihara et al. 1999; Clyne and Drumm 2004). However they either do not express Leb or are difficult to culture to confluence. A murine gastric cell line modified to express mucins as a model system for H. pylori adhesion has been generated (Takahashi et al. 2004), but the culture system was reported to be complicated and time-consuming. There are reports on other cell lines expressing carbohydrate determinants based on the type 1 saccharide chain (Galβ3GlcNAc), but some of these cells either express a wide array of carbohydrate epitopes, e.g., Colo-205 (Fernandez-Rodriguez et al. 2001; Pettersen et al. 2003), or epitope expression varies over time, e.g., Caco-2 cells that only express H type 1 and a small amount of Leb during differentiation (Amano and Oshima 1999). Chinese hamster ovary (CHO) cells have several advantages compared to cell lines of gastro-intestinal origin as they (i) rapidly reach confluence, (ii) have a well-defined glycan repertoire (Backstrom et al. 2003; Liu et al. 2005), and (iii) are well characterized from a regulatory point of view as they have been widely used for large-scale production of biotherapeutics (Geisse et al. 1996; Backstrom et al. 2003; Barnes et al. 2003). However, they do not express the Leb epitope and do not support Leb-mediated H. pylori adhesion.
In a previous paper we described the glycan and core chain specificity of glycosyltransferases involved in type 1 chain and Lewis antigen biosynthesis (Holgersson and Löfling, 2006). In order to generate cells carrying Leb determinants on core 3 O-glycans, which is the most likely configuration of the natural Leb receptor for H. pylori in the stomach, CHO-K1 cells were stably transfected with the β1,3-N-acetylglucosaminyltransferase VI (β3GlcNAcT-VI), β1,3-galactosyltransferase V (β3GalT-V), 1,2-fucosyltransferase II (2FucT-II, encoded by FUT2), and 1,3/4-fucosyltransferase III (3/4FucT-III, encoded by FUT3) cDNA. To distinguish between N- and O-linked Lewis determinants, cells were transiently co-transfected with cDNAs encoding the recombinant reporter fusion proteins 1-acid glycoprotein ligand-1/mouse IgG2b (AGP/mIgG2b) (Fournier et al. 2000) which only carries N-glycans and P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b) (Sako et al. 1993) which almost only carries O-glycans. Lewis (Lea, Leb, Lex, and Ley) antigen expression was shown to vary depending on the glycan type (N-, O-linked, and GL), and Leb-expressing CHO cells support adhesion of BabA carrying H. pylori. In addition, GL with the type 2 chain monofucosylated Lex determinant (Galβ4(Fuc3)GlcNAcβ3Galβ4Glcβ-R) was identified in CHO-K1 cells.
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Results |
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Stable expression of Lewis determinants in CHO-K1 cell lines was detected by flow cytometry
Following co-transfection of expression vectors encoding β3GlcNAcT-VI, β3GalT-V,
2FucT-II, and 3/4FucT-III, selected clones were picked, grown, split into duplicate wells, and stained with an anti-Leb antibody. Two Leb positive clones termed 1C5 and 2C2 were further expanded. Flow cytometry showed that both clones expressed Lea and Leb epitopes, while only 2C2 expressed the difucosylated type 2 isomer Ley (Figure 1; Table I). Neither of the clones expressed any Lex detectable by flow cytometry (not shown).
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1C5 and 2C2 express Lewis determinants differently on N- and O-linked glycans
Immunoaffinity-purified AGP/mIgG2b and PSGL/mIgG2b fusion proteins, produced by clones 1C5 and 2C2 following transient transfection, were analyzed by SDS–PAGE and Western blot analysis. The 2C2 cells supported Leb expression on both AGP/mIgG2b and PSGL-1/mIgG2b, while in 1C5 cells only PSGL-1/mIgG2b carried Leb (Figure 2A). On the other hand, both 1C5 and 2C2 cells supported Lea expression on both AGP/mIgG2b and PSGL-1/mIgG2b (Figure 2B). 2C2, but not 1C5, produced Ley on AGP/mIgG2b (Figure 2C). No Lex staining was seen on fusion proteins secreted from 1C5 and 2C2 (not shown). The anti-mouse IgG antibody was used as a negative control to check the amount of fusion protein loaded in each well (Figure 2D).
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2C2, but not 1C5, contains GLs with the Leb determinant
The amounts of total neutral GLs isolated from CHO-K1, 1C5, and 2C2 cells were 11, 10, and 15 µg/mg dry cells, respectively. Following their separation by thin layer chromatography (TLC), chemical detection revealed two major double bands migrating as mono- and diglycosylceramides in all three fractions (Figure 3A). The distinct migration of the double band is presumably due to differing numbers of methylene and/or hydroxyl groups in the ceramide moieties (Holgersson et al. 1991
). These components are most likely glucosylceramide and lactosylceramide as previously reported (Warnock et al. 1993). No obvious difference in the GL patterns of the three different cell types was seen upon chemical detection (Figure 3A). One anti-Leb antibody known to cross-react with H type 1 (clone 96FR2.10) (Larson et al. 1999) detected one band in the 5-sugar region and a double band in the 6-sugar region among GLs from 2C2 cells (Figure 3B). These bands are probably explained by blood group H type 1 pentaglycosylceramides and Leb hexaglycosylceramides, respectively, as only the double band in the 6-sugar region was stained with a Leb-specific antibody (clone 9F6848; not shown). Among GLs isolated from 1C5 cells, the double band in the 6-sugar region was vaguely discerned, while parental CHO-K1 GLs were negative (Figure 3B). The anti-Lex antibody revealed a double band in the >12-sugar region in all three GL fractions, including the one derived from the parental CHO-K1 cells (Figure 3C). The anti-Ley antibody detected a component migrating in the same region, albeit present only in GLs from 2C2 cells (Figure 3D). GLs from all cell types were nonreactive with an anti-Lea antibody (clone 78FR2.3; not shown).
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BabA-mediated cell attachment of H. pylori requires Leb expression
Fluorescein isothiocyanate (FITC)-labeled H. pylori strain 17875/Leb attached to 1C5 and 2C2 to a higher extent than to the CHO-K1 parental cell line (Figure 4A–C). The quantification of adherent bacteria/field area gave average numerical values (±SEM) of 403 ± 107, 2194 ± 590, and 7577 ± 1590 for CHO-K1, 1C5, and 2C2 cell lines, respectively. This verifies that BabA-mediated H. pylori adhesion is dependent on Leb expression, but does not appear to require Leb on N-glycans or GLs. The fact that Leb is required for binding is further supported by the absence of BabA-mediated binding to the parental CHO-K1 cells, and to Lea and H type 1-expressing CHO-K1 cells (data not shown).
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Discussion |
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We have shown that expression of β3GlcNAcT-VI, β3GalT-V,
2FucT-II, and 3/4FucT-III in CHO-K1 cells enables synthesis of Leb on O-glycans, N-glycans, and GLs (Figures 1–3). Our aim was to engineer CHO-K1 cells expressing Leb on O-glycans for two reasons. First, H. pylori is found mainly in the mucus layer of the stomach in infected individuals and H. pylori strains carrying the BabA adhesin binds the mucin MUC5AC in a Leb-dependent manner (Linden et al. 2002). Mucins carry predominantly O-glycans. Second, carbohydrate determinant expression on O-glycans in mucin domains can be expected to yield a higher density of the desired determinant than if carried on N-glycans and GLs alone (Gustafsson and Holgersson 2006). Such high expression densities are envisioned to contribute to a tighter, more easily detected binding of H. pylori to the engineered cells. Biosynthesis of the Leb antigen on O-glycans in CHO cells requires the sequential action of a number of glycosyltransferases (Brockhausen 1999; Hanisch 2001; Holgersson and Löfling 2006). First, β3GlcNAcT-VI (Iwai et al. 2002) is needed to produce the core 3 structure, GlcNAcβ3GalNAc-Ser/Thr. The GlcNAc residue is elongated by β3GalT-V to generate the type 1 core chain, Galβ3GlcNAc-R. Finally, fucosylation of the type 1 chain by 2FucT-II produces the H type 1 and 3/4FucT-III generates Leb. The action of the glycosyltransferases above was also shown to produce the desired epitope on N-glycans and GLs. To our knowledge, the expression dependence of the protein/lipid backbone and the mechanisms mediating this selectivity have not been studied before. Interestingly, the 1C5 clone expresses Leb on O-glycans, but not on N-glycans as revealed by transient expression of reporter proteins PSGL-1/mIgG2b and AGP/mIgG2b, and barely on GLs. The other clone, 2C2, expresses Leb on O- and N-glycans as well as on GLs. There is also a difference in the flow cytometry staining between the two Leb-expressing clones in that 1C5 does not express Ley, whereas 2C2 does (Figure 1), a finding that was corroborated by the Ley staining of N-glycans and GLs of 2C2 (Figures 2C and 3D). Both clones supported Lea expression on N- and O-glycans, while GLs isolated from the cells were nonreactive with Lea-specific antibodies (not shown), which indicates that the Lea epitope is only located on glycoproteins.
Although the 2C2 and 1C5 cells are derived from single colonies, they may still not be completely monoclonal as suggested by the flow cytometric analysis indicating a nonhomogenous cell population with regard to Leb expression for the 1C5 clone (Figure 1). The difference between 2C2 and 1C5 with regard to their glycosylation phenotype is, however, not merely quantitative because (i) the Leb-staining on N-glycans is completely absent in 1C5 (Figure 2), (ii) 1C5 cells express no Ley epitopes (Figures 1–3), and (iii) the intensity of the Leb-staining among GLs isolated from 2C2 is at least 50 times higher than the Leb-staining in the GLs from 1C5 (Figure 3). These observations indicate a qualitative difference in the presentation of Leb and Ley determinants between 1C5 and 2C2. Moreover we found that the glycosyltransferases tested had indeed varying specificity for different core chains, such as 3/4FucT-III that acted on the type 1 precursor to produce Lea on N- and O-glycans albeit not on the GL backbone. However, the transferase was able to act on H type 1 to produce Leb on all three backbones and on H type 2 to produce Ley on N-glycans and GL. Selectivity of 3FucT-IV for GLs and 3FucT-VII for glycoprotein substrates has been reported (Huang et al. 2002), but besides this, limited comparative studies of glycosyltransferase selectivity on N- and O-glycans and GLs have been performed.
Unexpectedly, the 1C5 and 2C2 clones as well as the parental CHO-K1 cells were found to express Lex GLs (Figure 3C). In addition to the Lex antibody used in Figure 3C, the Lex finding was repeated for 2C2 with an anti-CD15 antibody which detected the same >12-sugar GL (not shown). Unfortunately the limited amount of 1C5 and parental CHO cell GL fractions did not permit repeated testing with this antibody. As the anti-Lex antibody-reactive band was not stained by anti-H type 1 and 2 or anti-Lea, -Leb, or -Ley antibodies, antibody cross-reactivity can be excluded. Parental CHO-K1 cells are reported to lack 3FucT activity, but contain at least two silent 3FucT genes that can be activated upon transfection with DNA (Potvin et al. 1990). The Lex epitope was only observed on GLs and not by flow cytometry of the intact cells or on glycoproteins. This may explain why it has not been previously reported, since limited attention has been paid to CHO cell GLs.
In conclusion, we have by stably transfecting CHO-K1 cells with four different glycosyltransferases produced two CHO-K1 cell clones, 1C5 and 2C2, expressing the Leb epitope. Both clones mediated strong binding of BabA-positive H. pylori, and may thus serve as an in vitro model for molecular and cell biological studies on host cell and bacterial responses to Leb-mediated H. pylori attachment. Because the Lewis antigen expression in the 1C5 and 2C2 clones differed between N- and O-linked glycans and GLs, they should be useful tools in attempts to dissect the role of carrier glycan for H. pylori adhesion.
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Material and methods |
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Construction of vectors
Fusion proteins
The expression plasmid encoding the PSGL-1/mIgG2b fusion protein was constructed as described (Liu et al. 1997
). The AGP-coding sequence was PCR amplified, excluding the leader peptide and the stop codon from a human liver cDNA library. The forward primer for amplification of AGP was cgc ggg gct agc cca gat ccc att gtg tg and the reverse primer was cgc ggg gga tcc gat tcc ccc tcc tcc tg. The AGP cDNA was fused in frame with the cDNA encoding the CD5 leader sequence upstream and the Fc portion of mouse IgG2b downstream using the NheI and BamHI sites in the expression cassette. The same vector backbone was used for both fusion protein constructs.
β3GlcNAcT-VI
The core 3 synthase cDNA was PCR amplified from human stomach cDNA using cgc ggg aag ctt acc atg gct ttt ccc tgc cgc as forward and cgc ggg tct aga tca gga gac ccg gtg tcc as reverse primer and subcloned into CDM8 using HindIII and XbaI.
β3GalT-V
The cDNA for β3GalT-V (Zhou et al. 1999; Isshiki et al. 2003) was amplified by PCR using genomic DNA from human placenta as a template with cgc ggg aag ctt acc atg gct ttc ccg aag atg as forward and cgc ggg cgg ccg ctt tag aca ggc gga caa tct tc as reverse primer and subsequently subcloned into the CDM8 expression plasmid using HindIII and NotI.
2FucT-II
The FUT2 (Secretor gene) cDNA was amplified and subcloned as described (Lofling et al. 2002).
3/4FucT-III
The FUT3 (Lewis gene) cDNA (Kukowska-Latallo et al. 1990) expression plasmid was a kind gift of Professor Brian Seed, Dept. of Molecular Biology, MGH, Boston, MA.
The vectors used to generate stable transfectants were bidirectional having the CMV promoter upstream of a polylinker identical to the one in CDM8, a splice donor and acceptor site, and the bidirectional poly(A) addition signal of SV40. A second transcription unit consisting of the HSV TK promoter followed by the coding sequences for the guanosine phosphoribosyl transferase (Gpt), the hygromycin b, the zeocin, or the neomycin resistance genes were utilizing the poly(A) signals from the opposite direction (J. Holgersson and B. Seed, unpublished). The cDNAs encoding glycosyltransferases were swapped into the vector for stable expression using the restriction enzymes described above. The core 3 synthase gene-containing plasmid carried the zeocin resistance gene, the β3GalT-V plasmid carried the Gpt gene, the FUT2 plasmid had the neomycin resistance gene, and the FUT3 plasmid contained the hygromycin resistance gene.
Cell culture
CHO-K1 cells (ATCC CCL-61) and stable transfectants were cultured in Dulbecco's Modified Eagle Medium (Gibco, Invitrogen; Paisley, UK) supplemented with 10% heat inactivated fetal bovine serum (FBS, Gibco, Invitrogen) and glutamine (Gibco, Invitrogen) as described (Lofling et al. 2002).
Transfections and drug selection
To generate stable transfectants, plasmids were linearized with AvrII or Spe1 and subsequently transfected into CHO-K1 cells using Lipofectamine 2000 according to the manufacturer (Invitrogen; Paisley, UK). Twenty-four hours following transfection, cells in each T-flask were split into five 100-mm petri dishes and incubated in the selection medium. The concentrations of the different selection drugs were 400 µg/mL, 200 µg/mL, and 0.5 mg/mL for zeocin, hygromycin B, and G418, respectively. Gpt expressing cells were selected by growth in a medium containing mycophenolic acid (25 µg/mL), xanthine (13.6 µg/mL), and hypoxanthine (0.25 mg/mL). The selection medium was changed every third day. Drug resistant clones were seen after approximately 2 weeks, identified under the microscope, and handpicked using a pipetman. Selected colonies were cultured in 96-well plates in the presence of selection drugs for 2 weeks.
Growing cells were split into duplicate wells and stained with anti-Leb antibodies. Two positive clones, 1C5 and 2C2, were further expanded. CHO-K1 and Leb expressing cells were transiently transfected with Lipofectamine 2000 (Invitrogen; Paisley, UK) and 19.5 µg of plasmids encoding the AGP/mIgG2b or PSGL-1/mIgG2b fusion proteins according to the instructions of the manufacturer. Fusion proteins were secreted into the culture medium and affinity-purified by use of anti-mouse IgG agarose beads (Lofling et al. 2002).
Antibodies
Primary antibodies used are listed in Table II. Secondary antibodies used were Alexa 488-conjugated (Molecular Probes; Eugene, OR) goat anti-mouse IgM and IgG for flow cytometry (1:2,000–4,000 dilution), goat anti-mouse IgM HRP (Pierce; Rockford, IL) and goat anti-mouse IgG-FAB HRP (Sigma; St Louis, MO) for Western blot analysis (1:80,000–160,000 dilution), and goat anti-mouse Ig (Sigma) for TLC (1:500 dilution).
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Flow cytometry analysis
Cells were washed twice with PBS and incubated in 1% EDTA for 5–10 min or trypsinized for 5 min. Detached cells were washed twice in PBS with intervening centrifugations at 200 x g for 5 min and incubated with the primary antibody diluted in PBS with 1% bovine serum albumin (BSA, Sigma) at 4°C for 30 min. After washing twice in PBS, cells were incubated with the secondary antibody at 4°C for 30 min. Subsequently, cells were washed twice in PBS and kept at 4°C until flow cytometry analysis. A FACSort flow cytometer (Becton Dickinson; Palo Alto, CA) was used and 10,000 events were collected in each analysis. Negative control staining was performed with the secondary antibody alone.
SDS–PAGE and Western blot of O- and N-linked glycoproteins
Recombinant fusion proteins affinity-purified on anti-mouse IgG agarose beads were washed twice in PBS. Beads were mixed with 100 µL of 2x LDS sample buffer (Invitrogen; Paisley, UK) and incubated for 10 min at 70°C. Samples of 10 µL were loaded on a 4–12% NUPAGE gel (Invitrogen). Electrophoresis was run at 200 V for approximately 60 min. For Western blots, samples were blotted onto 0.2 µm nitrocellulose membranes (Invitrogen) at 40 V for 2 h and membranes were blocked with 3% BSA in PBS supplemented with 0.05% Tween (PBST) overnight at 4°C. Membranes were incubated with the primary antibody for 1 h, washed three times followed by incubation with the secondary antibody for 1 h and again washed three times. Antibodies were diluted in 3% BSA in PBST and membranes were developed as previously described (Lofling et al. 2002).
GL isolation and characterization
Total neutral GLs were isolated from CHO-K1, 1C5, and 2C2 cells (80, 70, and 100 mg dry weight, respectively) as described (Breimer et al. 1981; Karlsson 1987). In short, cells were extracted with methanol and chloroform/methanol mixtures followed by alkaline methanolysis, acetylation, separation on silica and DEAE-cellulose columns, and deacetylation.
GL fractions were separated on aluminium-backed silica gel 60 high performance thin-layer chromatography (HPTLC) plates (Merck; Darmstadt, Germany) using chloroform/methanol/water, 60/35/8 (by volume) as a solvent system. GLs were chemically detected by the anisaldehyde reagent or immunostained using monoclonal antibodies as described previously (Karlsson 1987). Total neutral and purified blood group active GLs based on type 1 and type 2 carbohydrate chains were used as reference substances (Breimer et al. 1981; Lindstrom et al. 1992; Ulfvin et al. 1993; Strokan et al. 1998).
H. pylori adhesion assay
Cells were cultured on 8-well Lab-TekTM II Chamber SlideTM System glass slides (Nunc; Roskilde, Denmark), fixed in a 1% aqueous paraformaldehyde solution or 30% acetone in methanol (v/v), washed in PBS, and kept at 4°C until use. Prior to bacterial cell adhesion experiments, slides were washed for 3 min with 70% ethanol, 3 min with 50% ethanol, 5 min with running water, and finally for 3 x 5 min with PBS. Slides were subsequently blocked with 1% BSA in PBST for 1 h.
The Leb-binding H. pylori strain 17875/Leb was cultured for 24 h at 37°C in 5% O2, 10% CO2 on blood agar (Mahdavi et al. 2002). Bacteria were labeled with FITC (Boren et al. 1993), adjusted to an OD600 of 0.2, and added to the slides. After 2 h incubation, slides were washed by manual dipping 150 times in PBST (buffer change every 50 dips) and mounted. Bacterial adhesion was quantified as described (Aspholm-Hurtig et al. 2004). In brief, slides were examined under 200x magnitude using a Leica DC 200 color video camera (Leica Microsystems AB; Solna, Sweden), and the number of adherent bacteria was quantified by LEICA Qwin Image Processing and Analysis system version 2.8 (Leica Microsystems).
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Funding |
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TopAbstractIntroductionResultsDiscussionMaterial and methodsFundingConflict of interest statementReferences |
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The Swedish Research Council no. 11612 (to M.E.B.), 11218 (to T.B.), and 13031 and 15356 (to J.H.), L-E Gelin Memorial Foundation (to M.E.B, M.D., J.H.), the Swedish Cancer Society (to T.B.) and the program "Glycoconjugates in Biological Systems" (to J.H., J.L.) financed by the Swedish Foundation for Strategic Research.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterial and methodsFundingConflict of interest statementReferences |
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Dr. Lofling and Dr. Holgersson have filed a patent application on the Leb-expressing CHO-K1 cells. This patent application has been assigned to Recopharma AB, a company of which Dr. Holgersson is a shareholder.
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Acknowledgements |
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The authors thank Drs. Cecilia Ehrnfelt and Carolina Holmén for technical assistance, Dr. Brian Seed for kindly sharing expression plasmids and Dr. Agneta Shanwell at the Blood bank, Karolinska University Hospital, Huddinge, for providing antibodies.
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Abbreviations |
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AGP/mIgG2b,
1-acid glycoprotein/mouse IgG2b; BabA, blood group antigen binding adhesin A; BSA, bovine serum albumin; CHO, chinese hamster ovary; Core 3, GlcNAcβ1,3GalNAc-Ser/Thr; FITC, fluorescein isothiocyanate; 2FucT, 1, 2Fucosyltransferase; 3/4FucT, 1,3/4Fucosyltransferase; GL, glycolipid; β3GlcNAcT, β1,3-N-acetylglucosaminyltransferase; β3GalT, β1,3-galactosyltransferase; H. pylori, Helicobacter pylori; H Type 1, fuc2Galβ3GlcNAc-R; H Type 2, fuc2Galβ4GlcNAc-R; Lea (Lewis a), Galβ3(Fuc4)GlcNAc-R; Leb (Lewis b), fuc2Galβ3(Fuc4)GlcNAc-R; Lex (Lewis x), galβ4(Fuc3)GlcNAc-R; Ley (Lewisy), fuc2Galβ4(Fuc4)GlcNAc-R; PBS, phosphate-buffered saline; PSGL-1/mIgG2b, P-selectin glycoprotein ligand-1/mouse IgG2b; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TLC, thin layer chromatography; Type 1, galβ3GlcNAc-R; Type 2, Galβ4GlcNAc-R |
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