Carbohydrate Binding Specificity of the Neutrophil-activating Protein of Helicobacter pylori*

(Received for publication, May 5, 1997)

Susann Teneberg Dagger §, Halina Miller-Podraza Dagger , Heather C. Lampert , Doyle J. Evans Jr. , Dolores G. Evans , Dan Danielsson par and Karl-Anders Karlsson Dagger

From the Dagger  Department of Medical Biochemistry, Göteborg University, Medicinaregatan 9A, S-413 90 Göteborg, Sweden, the  Bacterial Enteropathogens Laboratory, Veterans Affairs Medical Center and Baylor College of Medicine, Houston, Texas 77030, and the par  Department of Clinical Microbiology and Immunology, Örebro Medical Center, S-701 85 Örebro, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The possible interaction of the neutrophil-activating protein of Helicobacter pylori with target cell glycoconjugates was investigated by the binding of 125I-labeled recombinant protein to glycosphingolipids from human neutrophils in solid phase assays. Thereby, a distinct binding of the neutrophil-activating protein to four bands in the acid glycosphingolipid fraction from human neutrophils was detected, whereas no binding to the non-acid glycosphingolipids or polyglycosyl ceramides from these cells was obtained. When using glycosphingolipids not present in the cell membrane of human neutrophils, it was found that the neutrophil-activating protein also bound to sulfated glycosphingolipids as sulfatide and sulfated gangliotetraosyl ceramide. Comparison of the binding preferences of the protein to reference glycosphingolipids from other sources suggested that in human granulocytes, the neutrophil-activating protein of H. pylori preferentially recognizes glycoconjugates with a terminally unsubstituted NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta sequence.


INTRODUCTION

The human pathogen Helicobacter pylori is the causative agent of active chronic gastritis (1, 2). Infection with H. pylori is also strongly associated with development of gastric and duodenal ulcers (3). A causal connection between H. pylori infection and gastric malignancies such as gastric adenocarcinoma (4) and lymphoma (5-7) has also been suggested.

A prominent feature in H. pylori-induced gastritis is infiltration of the gastric lamina propria by neutrophil granulocytes, and neutrophils are also seen permeating between the epithelial cells (8). The mechanisms involved in the gastric inflammatory response have been the subject of numerous studies, and it has been demonstrated that H. pylori or cell-bound compounds released from the bacterium can cause chemotaxis (9-13), activate neutrophils to a strong oxidative burst (14), and induce an increased adhesion of neutrophils to endothelial cells (15). Furthermore, neutrophils activated by water extracts of H. pylori promote detachment of endothelial cells (16).

A neutrophil-activating protein from H. pylori was recently identified (17). This 150-kDa protein was isolated from water extracts of the bacterium. The neutrophil-activating protein induces an increased expression of CD11b/CD18 on neutrophils and promotes the adhesion of neutrophils to endothelial cells. By polymerase chain reaction amplification, the gene for the neutrophil-activating protein was found in all H. pylori isolates tested.

Since the activation of human neutrophils might involve lectin-carbohydrate interactions, the binding of radiolabeled neutrophil-activating protein of H. pylori to glycosphingolipids from human neutrophils was investigated using solid phase assays. A selective binding of the neutrophil-activating protein to four compounds in the acid glycosphingolipid fraction of human neutrophils was detected. In addition, the protein bound to sulfatide and sulfated gangliotetraosyl ceramide. Comparison of the binding preferences of the protein to reference glycosphingolipids from other sources suggested that the neutrophil-activating protein of H. pylori preferentially recognizes glycoconjugates with a terminal linear NeuAcalpha 3 Galbeta 4GlcNAcbeta 3Galbeta (4GlcNAcbeta )1 sequence present in the acid glycosphingolipid fraction of human neutrophils (18, 19).


MATERIALS AND METHODS

Glycosphingolipids from Human Neutrophils

Neutrophil leukocytes were isolated from pooled outdated human blood from healthy donors (blood group A) by Ficoll-Paque (Pharmacia, Uppsala, Sweden) centrifugation as described by Bøyum (20), with minor modifications (21).

Acid and non-acid glycosphingolipid fractions were isolated by the reported method (22). Briefly, the material was lyophilized and then extracted in two steps in a Soxhlet apparatus with chloroform and methanol (2:1 and 1:9, by volume). The extract was subsequently subjected to mild alkaline hydrolysis and dialysis, and nonpolar lipids were removed by silicic acid column chromatography. Acid and non-acid glycosphingolipids were separated on a DEAE-cellulose column. The non-acid fraction was acetylated (23), separated from alkali-stable phospholipids on a second silicic acid column, and then deacetylated. From 1.6 × 1010 cells of pooled neutrophils, 24.4 mg of acid glycosphingolipids (1.5 × 10-9 mg/cell) and 41.2 mg of non-acid glycosphingolipids (2.6 × 10-9 mg/cell) were obtained.

Polyglycosyl ceramides were isolated as described (24). In brief, the residue from extraction with chloroform and methanol was acetylated and then extracted with chloroform. The extract was filtered, and the residue was washed with water. The material in the chloroform phase was purified by Sephadex LH-20 and Sephadex LH-60 (Pharmacia) chromatography. The main carbohydrate-containing fraction was deacetylated and, after dialysis, suspended in 2-propanol/hexane/water (55:25:20, by volume) and centrifuged. Thus, the polyglycosyl ceramides were obtained in the supernatant, and the yield was approximately 0.3 mg/g of dry weight.

Reference Glycosphingolipids

Acid and non-acid glycosphingolipid fractions were obtained from the sources given in the legend to Fig. 1 and in Table I by standard procedures (22). The individual glycosphingolipids were isolated by repeated chromatography of the native glycosphingolipid fractions or their acetylated derivatives on silicic acid columns. The identity of the purified glycosphingolipids was confirmed by mass spectrometry (25), proton NMR spectroscopy (26-29), and degradation studies (30, 31). For removal of sialic acid, the acid glycosphingolipids were treated with 1% acetic acid at 100 °C for 1 h.


Fig. 1. Binding of 125I-labeled neutrophil-activating protein from H. pylori to mixtures of acid glycosphingolipids separated on thin-layer plates. A, glycosphingolipids detected with anisaldehyde; B, autoradiogram after binding of 125I-labeled neutrophil-activating protein. The glycosphingolipids were separated on aluminum-backed silica gel plates using chloroform/methanol/water (60:35:8, by volume) as the solvent system, and the binding assay was done as described under "Materials and Methods." The lanes contained acid glycosphingolipids of human neutrophils, 40 µg (lane 1); acid glycosphingolipids of human erythrocytes, 40 µg (lane 2); gangliosides of calf brain, 40 µg (lane 3); gangliosides of pig cerebellum, 40 µg (lane 4); acid glycosphingolipids of human meconium, 40 µg (lane 5); acid glycosphingolipids of bovine buttermilk, 40 µg (lane 6); acid glycosphingolipids of calf small intestine, 40 µg (lane 7); acid glycosphingolipids of rabbit thymus, 20 µg (lane 8); acid glycosphingolipids of human hypernephroma, 40 µg (lane 9). Autoradiography was for 3 h.
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Table I. Results from binding of 125I-labeled neutrophil-activating protein from H. pylori to glycosphingolipids on thin-layer chromatographs


Trivial name or abbreviation Glycosphingolipid structure Bindinga Source

 1. Sulfatide SO3Galbeta 1Cer +++ Human meconium
 2. Lactosyl ceramide Galbeta 4Glcbeta 1Cer  - Dog intestine
 3. Globotriaosyl ceramide Galalpha 4Galbeta 4Glcbeta 1Cer  - Human erythrocytes
 4. Globoside GalNAcbeta 3Galalpha 4Galbeta 4Glcbeta 1Cer  - Human erythrocytes
 5. Lactotetraosyl ceramide Galbeta 3GlcNAcbeta 3Galbeta 4Glcbeta 1Cer  - Human meconium
 6. Neolactotetraosyl ceramide Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer (+) Human granulocytes
 7. Gangliotetraosyl ceramide Galbeta 3GalNAcbeta 4Galbeta 4Glcbeta 1Cer (+) Mouse intestine
 8. Fuc-gangliotetraosyl ceramide Fucalpha 2Galbeta 3GalNAcbeta 4Galbeta 4Glcbeta 1Cer (+) Mouse intestine
 9. Sulf-gangliotetraosyl ceramide SO3Galbeta 3GalNAcbeta 4Galbeta 4Glcbeta 1Cer +++ Mouse intestine
10. GM3 NeuAcalpha 3Galbeta 4Glcbeta 1Cer  - Human granulocytes
11. GM1 Galbeta 3GalNAcbeta 4(NeuAcalpha 3)Galbeta 4Glcbeta 1Cer  - Human brain
12. GD1a NeuAcalpha 3Galbeta 3GalNAcbeta 4(NeuAcalpha 3)Galbeta 4Glcbeta 1Cer  - Human brain
13. NeuAcalpha 3neolactotetraosyl ceramide NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer  - Human granulocytes
14. NeuAcalpha 6neolactotetraosyl ceramide NeuAcalpha 6Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer  - Human meconium
15. NeuGcalpha 3neolactotetraosyl ceramide NeuGcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer  - Rabbit thymus
16. NeuGcalpha 3lactotetraosyl ceramide NeuGcalpha 3Galbeta 3GlcNAcbeta 3Galbeta 4Glcbeta 1Cer  - Rabbit thymus
17. NeuAcalpha 3-Lea NeuAcalpha 3Galbeta 3(Fucalpha 4)GlcNAcbeta 3Galbeta 4Glcbeta 1Cer  - Human bile bladder tumor
18. Disialylneolactotetraosyl ceramide NeuAcalpha 8NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer  - Human erythrocytes
19. Galbeta 4GlcNAcbeta 6(NeuAcalpha 6Galbeta 4GlcNAcbeta 3)Galbeta 4Glcbeta 1Cer  - Bovine buttermilk
20. NeuGcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer +++ Rabbit thymus
21. NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer +++ Human erythrocytes
Human placenta
22. NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Gal beta 4Glcbeta 1Cer +++ Human erythrocytes
23. NeuAcalpha 3Galbeta 4GlcNAcbeta 6(NeuAcalpha 3Galbeta 4GlcNAcbeta 3)Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer  - Human placenta
24. Fucalpha 2Galbeta 4GlcNAcbeta 6(NeuAcalpha 3Galbeta 4GlcNAcbeta 3)Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer  - Human erythrocytes
25. Galalpha 3Galbeta 4GlcNAcbeta 6(NeuAcalpha 3Galbeta 4GlcNAcbeta 3)Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer  - Bovine erythrocytes
26. Desialo 20 and 21 Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer  - Rabbit thymusb
Human erythrocytesb

a +++ marks a significant darkening on the autoradiogram when 2 µg of the glycosphingolipid was applied on the thin-layer chromatogram, (+) marks an occasional binding when 4 µg was applied, whereas - marks no binding even at a level of 4 µg.
b Glycosphingolipid 26 was obtained by mild acid treatment of glycosphingolipid 20 from rabbit thymus and 21 from human erythrocytes.

Production of Recombinant Neutrophil-activating Protein from Helicobacter pylori

The napA gene, GenBankTM accession number U16121 (32), was cloned into the BamHI site of plasmid pTrxFus in Escherichia coli host strain Gi724 so that a thioredoxin-HPNAP2 fusion protein was expressed under control of the bacteriophage lambda  promotor. Expression of the tryptophan-inducible thioredoxin HPNAP fusion protein and its recovery in lysozyme EDTA whole cell lysates were done using the ThioFusion Expression System (Invitrogen Corp., San Diego, CA 92121). The fused protein was purified by the same method as described for the native neutrophil-activating protein of H. pylori (17). The N-terminal thioredoxin did not diminish the biological activity of the neutrophil-activating protein, as determined using the nitro blue tetrazolium dye reduction assay (17).

The protein was concentrated to 1 mg/ml on a Filtron 10K Microsep membrane (Filtron Scandinavia AB, Bjärred, Sweden). Aliquots of 100 µl were labeled with 125I by the Iodogen method (33), yielding on average 5 × 103 cpm/µg.

Thin-layer Chromatography

Thin-layer chromatography was performed on glass- or aluminum-backed silica gel 60 HPTLC plates (Merck, Darmstadt, Germany) using chloroform/methanol/water (60:35:8, by volume) as the solvent system. For separation of gangliosides, a solvent system composed of chloroform/methanol/water with 0.25% KCl (50:40:9, by volume) was used, and chloroform/methanol/water with 0.2% CaCl2 (50:55:19, by volume) was utilized for separation of polyglycosyl ceramides. Chemical detection was done with anisaldehyde (34) or the resorcinol reagent (35).

Chromatogram Binding Assay

Binding of radiolabeled recombinant neutrophil-activating protein from H. pylori to glycosphingolipids separated on thin-layer plates was done as described previously for the binding of bacteria (36). Mixtures of glycosphingolipids (20-40 µg/lane) or pure compounds (1-4 µg/lane) were separated on aluminum-backed silica gel 60 PHTLC plates. After drying, the chromatography plates were treated with 0.5% (w/v) polyisobutylmethacrylate (Plexigum P28, Röhm, GmbH, Darmstadt, Germany) in diethyl ether for 1 min. The plates were then soaked in phosphate-buffered saline (PBS), pH 7.3, containing 2% (w/v) bovine serum albumin and 0.1% (w/v) NaN3 (Solution 1) for 2 h at room temperature. A suspension of 125I-labeled protein (diluted in Solution 1 to approximately 2 × 103 cpm/µl) was sprinkled over the plates, and the plates were incubated for 2 h at room temperature and then washed six times with PBS. Autoradiography was performed for 6-24 h using XAR-5 x-ray film (Eastman Kodak Co.) with an intensifying screen.

Microtiter Well Assay

The microtiter well binding assay was performed as described previously (37). In short, serial dilutions (each dilution in triplicate) of pure glycosphingolipids in methanol were applied in microtiter wells (Cooks M24, Nutacon, Holland). When the solvent had evaporated, the wells were blocked for 2 h with 200 µl of Solution 1. Then 50 µl of radiolabeled neutrophil-activating protein diluted in Solution 1 (approximately 2 × 103 cpm/µl) was added per well and incubated for 4 h at room temperature. After washing six times with Solution 1, the wells were cut out, and the radioactivity was counted in a gamma counter.

Hemagglutination Assay

Human erythrocytes (blood group ARh+ and ORh+) were washed three times with 0.9% NaCl (w/v) and suspended in PBS to 3% (by volume). The erythrocyte suspension (25 µl) was added to serial dilutions (in PBS) of the neutrophil-activating protein from H. pylori in microtiter wells (50 µl/well, starting at 1 mg/ml), and the resulting dilutions were incubated for 30 min at room temperature then visually inspected (38). As a control, the hemagglutination of the lectin from Erythrina cristagalli (39) was tested in parallel.


RESULTS

Chromatogram Binding Assay

Radiolabeled HPNAP was incubated with separated glycosphingolipids on thin-layer chromatography plates, and bound protein was visualized by autoradiography. A distinct binding to four bands in the acid glycosphingolipid fraction isolated from human granulocytes was detected (Fig. 1, lane 1). The binding-active compounds migrated below sialylneolactotetraosyl ceramide. Desialylation of the acid glycosphingolipids from human neutrophils abolished the binding of the neutrophil-activating protein (Fig. 2, lane 6). No binding of the protein to the non-acid glycosphingolipid fraction (Fig. 2, lane 4) or polyglycosyl ceramides (not shown) from human granulocytes was obtained.


Fig. 2. Thin-layer chromatograms of separated acid and non-acid mixtures of glycosphingolipids after detection with anisaldehyde (A), and autoradiogram after binding of 125I-labeled neutrophil-activating protein from H. pylori (B). The glycosphingolipids were separated on aluminum-backed silica gel plates using chloroform/methanol/water (60:35:8, by volume) as the solvent system and further treated as described under "Materials and Methods." The lanes contained non-acid glycosphingolipids of human blood group AB erythrocytes, 40 µg (lane 1); gangliosides of calf brain, 40 µg (lane 2); acid glycosphingolipids of human erythrocytes, 40 µg (lane 3); non-acid glycosphingolipids of human neutrophils, 40 µg (lane 4); acid glycosphingolipids of human neutrophils, 40 µg (lane 5); acid glycosphingolipids of human neutrophils after de-sialylation, 40 µg (lane 6). Autoradiography was for 6 h.
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The amount of acid glycosphingolipids obtained from pooled granulocytes from healthy donors was judged to be too low to permit an isolation of the binding-active gangliosides. Therefore, a number of reference glycosphingolipids of known structure was utilized in chromatogram binding assays in an attempt to circumstantially identify the structures of the compounds responsible for binding of the neutrophil-activating protein. The results are summarized in Table I. The protein bound to sulfatide (Table I, 1; Fig. 1, lane 5) and sulfated gangliotetraosyl ceramide (Table I, 9) with a detection level at 1 µg and also to neolactotetraosyl ceramide (Table I, 6), gangliotetraosyl ceramide (Table I, 7) and fucosyl-gangliotetraosyl ceramide (Table I, 8). The binding to the latter three compounds was, however, sporadic, and the detection level was 4 µg. Several of the gangliosides tested were consistently nonbinding. Thus, no binding to GM3 (Table I, 10), GM1 (Table I, 11), GD1a (Table I, 12), NeuAc/NeuGcalpha 3neolactotetraosyl ceramide (Table I, 13 and 15), NeuAcalpha 6neolactotetraosyl ceramide (Table I, 14), sialyllactotetraosyl ceramide (Table I, 16), NeuAcalpha 3Lea hexaglycosyl ceramide (Table, 17), or disialylneolactotetraosyl ceramide (Table, 18), was detected. However, a consistent binding to the gangliosides NeuGcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer (Table I, 20; Fig. 1, lane 8) and NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer (Table I, 21; Fig. 3, lanes 3 and 4) was obtained, with a detection level at 1 µg. De-sialylation of these two gangliosides (Table I, 26) abolished the binding (not shown). NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer (Table I, 22) was also bound by the neutrophil-activating protein (not shown). However, the related gangliosides, NeuAcalpha 3Galbeta 4GlcNAcbeta 6(NeuAcalpha 3Galbeta 4GlcNAcbeta 3)Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer (Table I, 23; Fig. 3, lane 5), Fucalpha 2Galbeta 4GlcNAcbeta 6(NeuAcalpha 3Galbeta 4GlcNAcbeta 3)Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer (Table I, 24), and Galalpha 3Galbeta 4GlcNAcbeta 6(NeuAcalpha 3Galbeta 4GlcNAcbeta 3)Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer (Table I, 25), having branches at the 6 position of the terminal Gal of a neolactotetraosyl ceramide core structure, were nonbinding.


Fig. 3. Binding of 125I-labeled neutrophil-activating protein from H. pylori to glycosphingolipid mixtures and purified compounds on thin-layer chromatogram. Glycosphingolipids were detected with anisaldehyde (A), and the autoradiogram was obtained after binding of 125I-labeled neutrophil-activating protein (B). The glycosphingolipids were separated on aluminum-backed silica gel plates using chloroform/methanol/water (60:35:8, by volume) as the solvent system. For details of the binding assay, see "Materials and Methods." The lanes contained acid glycosphingolipids of human erythrocytes, 40 µg (lane 1); acid glycosphingolipids of human neutrophils, 40 µg (lane 2); NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer and NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer of human placenta, 8 µg (lane 3); NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer of human placenta, 4 µg (lane 4); NeuAcalpha 3Galbeta 4GlcNAcbeta 6(NeuAcalpha 3Galbeta 4GlcNAcbeta 3)Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer of human placenta, 4 µg (lane 5). Autoradiography was for 12 h.
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Microtiter Well Assay

To further define the binding specificities and compare the relative binding affinities, the binding of radiolabeled HPNAP to selected glycosphingolipids was evaluated in the quantitative microtiter well assay. As shown in Fig. 4A, the protein bound to NeuGcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer with half-maximal binding occurring at 2 ng/well. Binding to SO3-Galbeta 3GalNAcbeta 4Galbeta 4Glcbeta 1Cer and sulfatide (Fig. 4B) was also obtained, with halfmaximal binding occurring at 2 and 30 ng/well, respectively.


Fig. 4. Binding of 125I-labeled neutrophil-activating protein of H. pylori to serial dilutions of glycosphingolipids in microtiter wells. The assay was done as described under "Materials and Methods." Data are presented as mean values of triplicate determinations after subtraction of background values. The sources of the glycosphingolipids were the same as given in Table I.
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Hemagglutination Assay

Serial dilutions of HPNAP in microtiter wells were tested for ability to agglutinate human erythrocytes. However, no agglutination was obtained, even at the highest protein concentration used (1 mg/ml). The lectin from E. cristagalli, used as a control, caused hemagglutination at approximately 10 µg/ml.


DISCUSSION

The gangliosides from human neutrophils, the target cells of H. pylori neutrophil-activating protein, are a complex mixture. The main components are the GM3 ganglioside and sialylneolactotetraosyl ceramide. The larger gangliosides are based on repetitive N-acetyllactosamine units and in many cases carry Fucalpha residues linked to the 3 position of GlcNAc. In addition, NeuAc may be alpha 3- or alpha 6-linked to the terminal Gal of neolactotetraosyl ceramide and the longer glycosphingolipids with repetitive N-acetyllactosamine units (18, 19, 40). The ceramide part is composed of sphingosine and mainly nonhydroxy 16:0, 24:0, and 24:1 fatty acids. This distribution of the fatty acids makes each glycosphingolipid appear as a double band on thin-layer chromatograms.

In the present study, four bands with HPNAP-binding activity were detected in the acid fraction of human neutrophils. When a number of reference gangliosides from other sources were tested for binding activity, most of them were nonbinding. However, HPNAP bound distinctly to NeuAc- and NeuGcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer, which comigrated with the uppermost binding-active double band in the acid fraction of human neutrophils (Fig. 3, lanes 3 and 4), and to NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer, which migrated in the region of the lower binding-active double band (not shown). NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer and NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer have also been identified in human neutrophils (18, 19, 40).

When the terminal sialic acid of NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer was removed, the binding of HPNAP was abrogated. In addition, NeuAcalpha 3neolactotetraosyl ceramide (Table I, 13) was nonbinding. Taken together, this indicates that NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta , or possibly NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta , is minimally required for binding of the neutrophil-activating protein.

A further notation is that HPNAP bound to both NeuAc- and NeuGcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer and thus accepted the presence of an acetyl group (-NHCOCH3) as well as an N-glycolyl group (-NHCOCH2OH) at C-5 of the terminal sialic acid. However, N-glycolylneuraminic acid has not been found in normal human tissues (41).

From the results of this study, it cannot be excluded that HPNAP also recognized NeuAcalpha 6Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer. However, there is conflicting evidence about the presence of this glycosphingolipid in human neutrophils. Although previously reported as one of the human neutrophil gangliosides (18), this compound was not found in the recent thorough study of gangliosides of human neutrophils (19, 40). NeuAcalpha 6Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer is present in human erythrocytes (42), but this compound was, unfortunately, not available in pure form. A large number of monosialoganglioside fractions from human erythrocytes were, however, tested for binding of the neutrophil-activating protein, but the only binding-active compounds detected were NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer and NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer.

No binding of HPNAP to human neutrophil polyglycosyl ceramides was obtained. When using the extraction procedures of this study, the more complex gangliosides are mainly found in the polyglycosyl ceramide fraction. In human neutrophils, these complex gangliosides have a poly-N-acetyllactosamine core structure with terminal NeuAcalpha 3 and alpha 3-linked Fuc at one or several of the internal GlcNAc residues (19, 40). The absence of binding to these compounds indicates that the alpha 3-linked fucose residues interferes with the binding process.

The non-binding of NeuAcalpha 3Galbeta 4GlcNAcbeta 6(NeuAcalpha 3Galbeta 4GlcNAcbeta 3)Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer is probably due to steric hindrance imposed by the beta 6-linked branches. This could have implications for the interaction of the protein with glycoproteins and polyglycosyl ceramides. Although the NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta sequence is often present in the carbohydrate chains of these glycoconjugates, it is mainly found on multiply branched structures and thus may be nonbinding. This suggestion was supported by the observed inability of HPNAP to cause agglutination of human erythrocytes. HPNAP consists of 10 15-kDa subunits and thus is multimeric. Still, no agglutination of human erythrocytes was obtained, indicating that the N-linked polylactosamine chains of band 3 of adult human erythrocytes having terminal NeuAcalpha 3 but with beta 6-linked N-acetyllactosamine units at internal Gal residues, Galbeta 4GlcNAcbeta 6(NeuAcalpha 3Galbeta 4GlcNAcbeta 3)Galbeta 4GlcNAcbeta 3 (43), was not recognized by HPNAP. The binding-active NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer and NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4GlcNAcbeta 3Galbeta 4Glcbeta 1Cer are also present in human erythrocytes (42, 44). However, the density of these minor gangliosides may not be enough to induce hemagglutination. No binding activity was detected in the total acid fraction of human erythrocytes as shown in Fig. 1, lane 2 and Fig. 3, lane 1.

HPNAP also bound to sulfatide and SO3-Galbeta 3-GalNAcbeta 4Galbeta 4Glcbeta 1Cer. If this binding is a true recognition process or due to complementary ionic interactions remains to be investigated. However, since sulfated glycosphingolipids have not been convincingly identified in human neutrophils, the biological implications of this binding property are, at present, elusive.

The results from this study do not exclude the possibility that HPNAP interacts with some granulocyte glycoproteins. However, when proteins from human neutrophils were extracted (45) and binding of H. pylori neutrophil-activating protein was tested on blotting membranes, no specific binding was obtained (data not reproduced). Still, most of the reported oligosaccharides of human neutrophil glycoproteins also have repetitive N-acetyllactosamine cores (46). However, the majority of these glycoproteins have terminal NeuAcalpha 6, and the internal GlcNAc residues are substituted with Fucalpha 3. The concluded binding-active NeuAcalpha 3Galbeta 4GlcNAcbeta 3Galbeta (4GlcNAcbeta ) sequence has, however, been proposed to exist in unsubstituted form in some glycoproteins of human neutrophils, as e.g. leukosialin (47).

The molecular mechanisms by which H. pylori colonization of the gastric epithelium leads to diseases of the stomach and duodenum are largely unknown. The pathogenesis of H. pylori-induced gastroduodenal diseases is likely to be a complex process, and no single event or phenomenon may be solely responsible for all its clinical manifestations. The major known virulence factors of the bacterium are the urease (48, 49) and the VacA cytotoxin (50). The newly identified neutrophil-activating protein is likely to be involved in the early stages of the disease process, as in gastritis, which is characterized by an abundant accumulation of neutrophils in the superficial gastric mucosa.

In this study, a HPNAP-binding carbohydrate sequence present in human neutrophils was identified by utilizing reference glycosphingolipids from other sources than the target cells. Current studies aim to isolate the binding-active glycosphingolipids from human neutrophils to conclusively establish their structures and obtain more information on the binding epitope. Inhibition studies have now been initiated, and preliminary results indicate that the phagocytosis of the neutrophil-activating protein by preactivated human neutrophils may be partly inhibited by synthetic carbohydrate analogues of the proposed binding-active sequence.3


FOOTNOTES

*   This work was supported by Swedish Medical Research Council Grants 3967, 10435, and 16X-04723, a grant from the Department of Veterans Affairs (to D. J. E.), and National Institutes of Health Public Health Service Grant AI28837 (to D. G. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed: Dept. of Medical Biochemistry, Göteborg University, Medicinaregatan 9A, S-413 90 Göteborg, Sweden. Tel.: 46 31 773 34 92; Fax: 46 31 413 190.
1   The glycosphingolipid nomenclature follows the recommendations by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN for Lipids, Eur. J. Biochem. (1977) 79, 11-21, J. Biol. Chem. (1982) 257, 3347-3351, and J. Biol. Chem. (1987) 262, 13-18). It is assumed that Gal, Glc, GlcNAc, GalNAc, NeuAc and NeuGc are of the D-configuration, Fuc is of the L-configuration, and all sugars present are in the pyranose form.
2   The abbreviations used are: HPNAP, Helicobacter pylori neutrophil-activating protein; PBS, phosphate-buffered saline.
3   D. Danielsson, unpublished data.

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