Species-specific distribution of [alpha]-galactosyl epitopes on the gastric H/K ATPase [beta]-subunit: relevance to the binding of human anti-parietal cell autoantibodies

L. Ann Stewart, Ian R. van Driel, Ban Hock Toh and Paul A. Gleeson1

Department of Pathology and Immunology, Monash University Medical School, Melbourne, Victoria, Australia

Received on September 3, 1998; revised on November 30, 1998; accepted on December 4, 1998

The gastric H/K ATPase [beta]-subunit, an abundant glycoprotein of the secretory membranes of gastric parietal cells, is the major autoantigen recognized by human parietal cell autoantibodies in gastric autoimmunity. Our previous studies demonstrated that the human autoantibodies recognize the H/K ATPase [beta]-subunit from a number of species and that glycosylation of the [beta]-subunit with complex N-glycans is required for autoantibody binding. The N-glycans of the [beta]-subunit contain polylactosamine chains. The lactosamine chains of the rabbit [beta]-subunit are terminated with [alpha]-linked galactosyl residues ([alpha]-galactosyl epitope) (Tyagarajan et al., Biochemistry, 1996, 35, 3238-3246). Here we have investigated the expression of [alpha]-galactosyl epitopes on the H/K ATPase [beta]-subunit from a number of species. Using the [alpha]-galactosyl binding lectin, BS1-IB4, and naturally occurring anti-[alpha]-galactosyl antibodies, we have demonstrated that the rat H/K ATPase [beta]-subunit also contains terminal [alpha]-galactosyl residues, but not the [beta]-subunit from pig, dog, and mouse, indicating species-specific differences in the terminal saccharide sequences of the [beta]-subunit. We also investigated the potential contribution of the [alpha]-galactosyl epitopes to the binding by human sera. The reactivity of human pernicious anemia serum with gastric parietal cells could not be inhibited with saccharide inhibitors and, in addition, no binding was observed with normal human sera. We conclude that the H/K ATPase [beta]-subunit oligosaccharides from rabbit and rat are terminated with [alpha]-galactosyl epitopes, and although the presence of this epitope does not contribute to binding by human parietal cell autoantibodies at the concentrations routinely used, it is recommended that neither rat or rabbit stomachs be used for screening human sera.

Key words: gastric H/K ATPase/[alpha]-galactosyl epitopes/gastric parietal cell/human autoantibodies

Introduction

The gastric H/K ATPase, located in a unique membrane system in parietal cells, is responsible for secretion of acid into the stomach lumen (Forte and Soll, 1989). The H/K-ATPase consists of a heterodimer, a catalytic [alpha]-subunit, and an associated [beta]-subunit (van Driel and Callaghan, 1995; Moller et al., 1996). The [beta]-subunit is a glycoprotein of apparent molecular weight 60-90 kDa consisting of a 33 kDa core protein substituted with 6-7 N-glycan oligosaccharides (Goldkorn et al., 1989; Okamoto et al., 1990; Reuben et al., 1990; Shull, 1990; Toh et al., 1990). Poly-N-acetyllactosamine-specific lectins from Lycopersicon esculentum (tomato) and from Solanum tuberosum (potato) bind the H/K ATPase [beta]-subunit from a number of species (Callaghan et al., 1990; Scarff et al., 1997). Analysis of the rabbit H+/K+ ATPase [beta]-subunit oligosaccharides has confirmed the presence of polylactosamine structures (Tyagarajan et al., 1996) and furthermore found that all branches of lactosamine-type structures were capped with terminal Gal [alpha]1->3 Gal sequences (Tyagarajan et al., 1996, 1997).

The [alpha]- and [beta]-subunits of the gastric H/K ATPase are the major antigens recognized by the circulating autoantibodies found in serum of patients with autoimmune gastritis (Goldkorn et al., 1989; Gleeson and Toh, 1991), an autoimmune disease of the stomach leading to pernicious anemia (Toh et al., 1997). Human pernicious anemia sera recognize the H/K ATPase [beta]-subunit from rabbit, pig, dog, and mouse (Goldkorn et al., 1989). The presence of complex N-glycans is required for autoantibody binding as removal of the N-glycans from the [beta]-subunit results in loss of binding by the human autoantibodies, and the [beta]-subunit expressed in COS cells, which bear only high mannose N-glycans, fails to interact with the human autoantibody (Callaghan et al., 1993).

Approximately 1% of the total IgG in normal human serum is directed against the saccharide Gal[alpha]1->3Gal (Galili, 1993), known as the [alpha]-galactosyl epitope. Since the rabbit H/K ATPase [beta]-subunit is glycosylated with [alpha]-galactosyl terminated oligosaccharides identical to the [alpha]-galactosyl epitope, the question arises whether this epitope plays a role in the binding of human-parietal cell antibodies. Here we have investigated the expression of [alpha]-galactosyl epitopes on the H/K ATPase [beta]-subunit from different species and the potential contribution of this epitope to the binding of human antibodies.

Results

Bandeiraea simplicifolia BS1- IB4 lectin staining of parietal cells in gastric mucosa


Figure 1. Binding of BS1-IB4 lectin to gastric mucosa and kidney tissues.Paraffin-embedded sections (4 µm) of stomach from rat (A, A[prime]), rabbit (B, B[prime]), dog (D), pig (E), and mouse (F) or pig kidney (C, C[prime]) were incubated with biotinylated BS1-IB4, in the absence (A-C, D-F) or presence (A[prime], B[prime], C[prime]) of 0.5 M galactose, followed by streptavidin-horseradish peroxidase. Sections were counterstained with hematoxylin. Binding of lectin is indicated by the presence of a brown precipitate. Scale bar, 73 µm.


Figure 2. Binding of BS1-IB4 lectin to rat and rabbit gastric parietal cells. Paraffin-embedded sections (4 µm) of rat (A) and rabbit (B) stomach were incubated with biotinylated BS1-IB4 followed by streptavidin horseradish-peroxidase. Sections were counterstained with hematoxylin. Binding of lectin is indicated by the presence of a brown precipitate. Preincubation of biotinylated BS1-IB4 with 0.5 M galactose resulting in no staining (not shown). Scale bar, 16 µm.


Figure 3. Binding of human anti-a-galactosyl antibodies to gastric mucosa. Paraffin-embedded sections (4 µm) of mouse (A), pig (B), rat (C, E), and rabbit (D, F) stomach were incubated with either 0.1 mg/ml of affinity-purified anti-[alpha]-galactosyl antibodies (A, B, C, D) followed by anti-human immunoglobulin conjugated to horseradish-peroxidase or the horseradish-peroxidase anti-human immunoglobulin conjugate alone (E, F). Sections were counterstained with hematoxylin. Parietal cells are indicated by arrows. Binding of antibody is indicated by the presence of a brown precipitate. Scale bar, 27 µm

The rabbit H/K ATPase [beta]-subunit has been shown to contain Gal[alpha]1->3Gal[beta]1-> 4GlcNAc extensions on all of its complex N-glycan sequences (Tyagarajan et al., 1996, 1997). To determine whether terminal Gal[alpha]1->3Gal[beta] sequences are a common structural feature of the heavily glycosylated H/K ATPase [beta]-subunit, paraffin-embedded stomach sections from a number of different species were stained with the Gal[alpha]1->3Gal[beta] specific lectin from Bandeiraea simplicifolia, BS1-IB4. As a control for BS1-IB4-peroxidase staining of [alpha]-galactosyl structures, pig kidney was also included as the proximal convoluted tubules and endothelial cells of this tissue have glycoconjugates which bear the [alpha]-galactosyl epitope (Sandrin and McKenzie, 1994). As expected, BS1-IB4 bound strongly to these structures in pig kidney, and this binding was inhibited by 0.5 M galactose (Figure 1C,C[prime]). BS1-IB4 showed strong binding to both rat and rabbit gastric mucosa (Figure 1A,B,A[prime],B[prime]); the lectin-positive cells could be identified based on their size and shape as parietal cells at high magnification (Figure 2). However, BS1-IB4 showed no binding to the gastric mucosa of dog, pig or mouse (Figure 1D-F). Using affinity-purified human anti-[alpha]-galactosyl antibodies at a concentration of 0.1 mg/ml staining of rat and rabbit parietal cells was observed, but not the gastric mucosa of mouse or pig (Figure 3). These findings are consistent with the presence of [alpha]-galactosyl epitopes on the rabbit H/K ATPase [beta]-subunit and also indicate that this epitope may be expressed on the rat [beta]-subunit.

Rat H/K ATPase [beta]-subunit has [alpha]-galactosyl epitopes


Figure 4. Colocalization of BS1-IB4 binding sites and H/K ATPase in rat parietal cells by confocal microscopy. Sections (4 µm) of rat stomach were incubated with biotinylated BS1-IB4 lectin (A) and mouse monoclonal antibody to the gastric H/K ATPase [beta]-subunit (B), as described in Materials and methods. Bound lectin was detected with FITC conjugated streptavidin and bound antibody with TRITC labeled rabbit anti-mouse immunoglobulin. (C) Superimposed images of (A) and (B) reveals regions of BS1-IB4 binding sites and H/K ATPase [beta]-subunit colocalization. Note the lack of red fluorescence in (C). Yellow indicates regions where red and green are superimposed. Scale bar, 8 µm.


Figure 5. Lectin blotting of rat gastric H/K ATPase with BS1-IB4. Tomato-lectin purified rat gastric H/K ATPase (0.5 µg/lane) was separated on a 10% polyacrylamide gel under nonreducing conditions and transferred to nitrocellulose membranes. The membranes were blocked and blotted with biotinylated BS1-IB4, in the presence (IB4+ GAL) and absence (IB4) of 0.5 M galactose, or monoclonal antibody 2B6 followed by the appropriate horseradish peroxidase conjugate using a chemiluminescence detection system. An incubation was also carried out with horseradish peroxidase labeled anti-mouse immunoglobulin alone (CONJ).


Figure 6. Binding of pernicious anemia autoimmune sera to the gastric mucosa of different species. Paraffin-embedded sections (4 µm) of formalin fixed stomach from rat (A-C), mouse (D-F), pig (G, H) and rabbit (I, J) stomach and pig kidney (K, L) were incubated with monoclonal antibody 2B6 (A, D), normal human serum (1:50 dilution) (B, E, G, I, K) or pernicious anemia serum (1:50 dilution) (C, F, H, J, L). Bound antibody was detected with either horseradish peroxidase labeled anti-mouse immunoglobulin or biotinylated sheep anti-human immunoglobulin followed by streptavidin labeled horseradish peroxidase. Sections were counterstained with hematoxylin. Binding of lectin is indicated by the presence of a brown precipitate. Scale bar: (A-F) 78 µm, (G-J) 105 µm, (K, L) 76 µm.

To more accurately localize the BS1-IB4 binding sites of rat parietal cells immunofluorescence confocal microscopy was carried out. BS1-IB4 showed an intracellular reticular staining pattern typical of the H/K ATPase-containing secretory membrane network system of these cells (Figure 4A). Further, BS1-IB4 binding sites colocalized extensively with H/K ATPase [beta]-subunit (Figure 4B,C), indicating that the [alpha]-galactosyl residues are present on glycoconjugates of the secretory membranes. To determine if terminal [alpha]-galactosyl residues are present on the rat H/K ATPase [beta]-subunit, the gastric H/K ATPase [alpha]/[beta] complex was isolated from detergent extracts of rat gastric mucosal membranes by binding to tomato-lectin Sepharose 4B and the isolated gastric H/K ATPase analyzed by immuno- and lectin blotting. As expected, the [beta]-subunit specific monoclonal antibody, 2B6, reacted with a 60-90 kDa component of the isolated H/K ATPase (Figure 6). Significantly, BS1-IB4 also showed reactivity with the 60-90 kDa component of the purified rat H/K ATPase and the staining with BS1-IB4 was inhibited by 0.5 M galactose (Figure 6). On the other hand BS1-IB4 did not react with purified H/K ATPase preparations from either pig or mouse (data not shown), consistent with immunoperoxidase staining data. Collectively, these results indicate that the rat [beta]-subunit bears terminal [alpha]-galactosyl residues whereas the mouse and pig [beta]-subunit are devoid of [alpha]-galactosyl residues.

[alpha]-Galactosyl epitopes do not contribute to parietal cell binding of human autoimmune serum

The H/K ATPase [beta]-subunit is a major autoantigen recognized by circulating human autoantibodies from patients with pernicious anemia. Previously, we have shown that complex N-glycans are required for autoantibody binding as removal of the [beta]-subunit N-glycans results in loss of binding by the human autoantibodies, and the [beta]-subunit expressed in COS cells, which bear only high mannose N-glycans, fails to interact with the autoantibody (Callaghan et al., 1993). Normal human sera contains a high titer of antibodies (1% of total IgG) against the Gal[alpha]1->3Gal[beta] epitope (Galili, 1993). Since the rabbit and rat H/K ATPase [beta]-subunit bears [alpha]-galactosyl terminated oligosaccharides, the question arises whether the [alpha]-galactosyl epitope plays a role in the binding of human-parietal cell autoimmune sera.

To determine if [alpha]-galactosyl epitope contributes to reactivity of human anti-parietal cell autoantibody staining, the binding of normal human serum and pernicious anemia serum to gastric mucosa of different species was evaluated. Serum from pernicious anemia patients at 1:50 dilution shows strong binding to parietal cells of rat, mouse, pig, and rabbit paraffin-embedded stomach sections (Figure 6C,F,H,J) with a staining pattern similar to that obtained with the H/K ATPase [beta]-subunit specific monoclonal antibody 2B6 (Figure 6A,D). The binding of a number of normal human sera to rat, mouse, pig and rabbit stomach and to pig kidney sections was also investigated using a 1:10-1:50 dilution of sera. No specific parietal cell staining was seen with eight different normal human sera on either stomach or kidney sections. Figure 6 shows an example of one of the normal sera (Figure 6B,E,G,I,K). There was some variation in the level of background staining between the different normal sera; however, this background staining was not reduced by prior incubation of the sera with 0.5 M galactose for 1 h at room temperature (not shown). Also pernicious anemia serum showed no staining of pig kidney sections (Figure 6L), a tissue with high levels of the [alpha]-galactosyl epitope. Serum dilutions of 1:10 to 1:20 are routinely used to screen for parietal cell autoantibodies. Higher concentrations of serum cannot be examined due to a high level of nonspecific background. Similar results were obtained using frozen sections rather than paraffin-embedded sections. These results demonstrated that at the serum dilution routinely used, human antibodies show no reactivity with tissue associated [alpha]-galactosyl epitopes by immunocytochemistry.

Discussion

The [beta]-subunit is essential for the function of the heterodimeric gastric H/K ATPase, the enzyme responsible for acid secretion by the stomach (Gottardi and Caplan, 1993). Autoimmune disease of the gastric mucosa is associated with a specific autoantibody response to both the [alpha]- and the [beta]-subunits of the H/K ATPase (Gleeson and Toh, 1991; Gleeson et al., 1996a; Toh et al., 1997). Circulating parietal cell autoantibodies associated with human chronic atrophic gastritis recognize the H/K ATPase [beta]-subunit from several animal species and can be detected by either immunofluorescence or immunoblotting. The binding of these human autoantibodies to the [beta]-subunit is influenced by both the carbohydrate and protein moieties of the autoantigen as treatment with N-glycanase or reduction of disulfide bonds reduced autoantibody binding (Goldkorn et al., 1989). In addition, autoantibody binding is critically dependent on the presence of a full complement of N-linked glycans as partially deglycosylated proteins, and recombinant [beta]-subunit expressed in COS cells bearing only high mannose N-glycans failed to bind the autoantibody (Callaghan et al., 1993). On identifying the presence of [alpha]-galactosyl epitopes on the rabbit [beta]-subunit, Tyagarajan et al. (1996) have raised the possibility that human sera may bind to the gastric H/K ATPase [beta]-subunit via these [alpha]-galactosyl epitopes.

Here we have assessed whether the H/K ATPase [beta]-subunit from a number of species bear [alpha]-galactosyl epitopes. In addition to the rabbit [beta]-subunit, we have demonstrated that [alpha]-galactosyl epitopes are also found on the [beta]-subunit from the rat, whereas the lactosamine chains of the [beta]-subunit from mouse and pig do not appear to be capped with [alpha]-galactosyl residues. Hence there is a species-specific difference in glycosylation of this gastric glycoprotein. It is known that both mouse and pig have the relevant [alpha]1,3-galactosyltransferase activity in various tissues (Joziasse et al., 1992; Vanhove et al., 1996), although the activity of this enzyme in the gastric mucosa has not been investigated. Thus, the difference in the glycosylation of the [beta]-subunit between species may reflect differences in tissue specific expression of the [alpha]1,3 galactosyltransferase or alternatively differences in site specific processing of the N-glycans due to the influence of the polypeptide chain.

Our data demonstrates that human parietal cell autoantibodies are clearly not dependent on the [alpha]-galactosyl epitope for binding as this epitope is absent from the [beta]-subunit of pig and mouse. Furthermore, as the H/K ATPase [beta]-subunit autoantibodies do not show cross-reactivity with other tissue glycoproteins it is unlikely that the autoantibodies interact solely with the [beta]-subunit N-glycans. The epitopes recognized by the autoantibodies may include both carbohydrate and protein moieties, or the native carbohydrate structures may be important in maintaining the correct conformation of the [beta]-subunit polypeptide.

In spite of the presence of naturally occurring antibodies to the [alpha]-galactosyl epitope in normal human serum, no binding of a number of normal human sera was observed to the gastric mucosa of rabbit or rat. Approximately 1% of the total IgG in normal human serum is directed against the [alpha]-galactosyl epitope (Galili, 1993). In previous reports the binding of anti-[alpha]-galactosyl antibody to either cells or tissue sections have used affinity purified antibodies at an equivalent concentration to undiluted normal serum (Galili et al., 1988; Oriol et al., 1993). We also found that affinity purified anti-[alpha]-galactosyl antibodies effectively stained rat gastric mucosa (or pig kidney) but only at these high concentrations. Thus, it is likely that the anti-[alpha]-galactosyl antibodies are of low affinity, and this would explain why no reactivity of normal human serum with either rat or rabbit gastric mucosa is observed at dilutions routinely used for screening anti-parietal cell autoantibodies. Nonetheless there have been reports of the staining of parietal cells of rat stomach sections with a small proportion of human sera, due to the presence of heteroantibodies (Muller et al., 1971; Strickland and Hooper, 1972; Hawkins et al., 1977). It is likely that these heteroantibodies are anti-[alpha]-galactosyl antibodies. Therefore, as gastric tissue is used in the routine screening for parietal cell autoantibodies (Gleeson et al., 1996b), neither rat nor rabbit stomach sections should be used.

Materials and methods

Lectins and antibodies

Affinity-purified naturally occurring human anti-[alpha]-galactosyl antibodies were kindly supplied by Dr. M.Sandrin, Austin Research Institute, Melbourne. Bandeiraea simplicifolia BS1-IB4 lectin was purchased from Sigma. Human sera were from the departmental clinical immunology laboratory. Monoclonal antibody 2B6 is specific for the H/K ATPase [beta]-subunit (Mori et al., 1989).

Tissue sections

Fresh tissues were fixed in 10% formalin in 0.1 M sodium phosphate buffer, pH 7.2 (10% buffered formalin), and then processed overnight in an automated tissue processor Hypercentre II (Shandon). Dehydration was carried out through increasing grades of ethanol, washing in Histosol (Interpath Services, Australia), and embedding in paraffin. Sections (4-6 µm) were cut and dewaxed by immersion in Histosol then ethanol, rehydrated by immersing in H2O, and stored in phosphate-buffered saline (PBS).

For frozen sections, tissue was mounted in Tissue-Tek II OCT embedding compound (Miles Inc., Elkart, IN) and snap-frozen in isopentane cooled by liquid nitrogen.

Immunofluorescence

For dual labeling experiments, formalin-fixed paraffin-embedded rat stomach sections were incubated with monoclonal antibody 2B6 (10 µg/ml) for 30 min at room temperature, washed, and incubated with biotinylated BS1-IB4 (10 µg/ml) (Sigma) for 30 min at room temperature. After further washing, sections were incubated with tetramethyl rhodamine isothiocyanate (TRITC)-labeled rabbit anti-mouse immunoglobulin (1:50 diluted in 1% bovine serum albumin (BSA)/PBS) (Dako, cat. no. R0270) and fluorescein isothiocyanate (FITC)-labeled streptavidin (1:100 diluted in 1% BSA/PBS; Amersham Life Sciences, cat. no. RPN1232) for 30 min at room temperature. After a final wash, sections were mounted in Mowiol and examined by confocal microscopy using a Bio-Rad MRC 1024 imaging system.

Immunoperoxidase staining

Formalin-fixed, paraffin-embedded sectionswere incubated in 0.3% H2O2 in methanol for 30 min on ice to inactivate endogenous peroxidase activity and washed in PBS. For antibody staining, sections where incubated with serum or monoclonal antibody for 60 min at room temperature and unbound antibody removed by three 5 min washes in PBS. Sections were then incubated with either rabbit anti-mouse immunoglobulin-horseradish peroxidase (1:100 in 1% BSA/PBS; Dako, cat. no. P0161) or biotinylated sheep anti-human immunoglobulin (1:500 in 1% BSA/PBS; Amersham Life Sciences, cat. no. RPN1003) for 30 min, washed, and then incubated with streptavidin-horseradish peroxidase conjugate (1:400 in 1% BSA/PBS; Silenus, cat. SAH). Following three 5 min washes with PBS, bound horseradish peroxidase was detected by incubation with PBS containing 0.5 mg/ml diaminobenzidine (Sigma), 0.3 mg/ml NiCl2 and 0.2% H2O2 for 20 min.

For lectin staining, sections were incubated with biotinylated BS1-IB4 lectin (10 µg/ml in PBS/1% BSA) for 30-60 min at room temperature. Unbound lectin was removed by three 5 min washes in PBS and the sections were then incubated with streptavidin-horseradish peroxidase (1:250 dilution in PBS/1% BSA; Silenus, cat. SAH) for 45 min. Following three 5 min washes with PBS, bound horseradish peroxidase was detected by incubation with PBS containing 0.5 mg/ml diaminobenzidine, 0.3 mg/ml NiCl2, and 0.2% H2O2 for 15 min. To determine whether BS1-IB4 binding was specific, incubations were also carried out where the BS1-IB4 lectin was incubated with the inhibitor 0.5 M galactose for 60 min prior to addition to the sections.

All sections were washed in water, counterstained in hematoxylin, dehydrated, and mounted and examined by light microscopy.

Gastric H/K ATPase preparation

Pig, rat and mouse gastric mucosal membranes were isolated and the gastric H/K ATPase was purified by tomato-lectin chromatography as described previously (Callaghan et al., 1990).

Immuno- and lectin blotting

Proteins in samples were separated by SDS-PAGE under nonreducing conditions and transferred to nitrocellulose which was blocked in 3% fish skin gelatin in PBS/0.05% Tween 20 overnight at 4°C. Nitrocellulose membrane strips were incubated with either the H/K ATPase [beta]-subunit specific monoclonal antibody, 2B6 (10 µg/ml in PBS/0.05% Tween 20 containing 3% fish skin gelatin) or biotinylated BS1-IB4 lectin (50 µg/ml in PBS/0.05% Tween 20 containing 3% fish skin gelatin) for 1 h at room temperature. Control incubations were carried out where biotinylated BS1-IB4 lectin had been pre-incubated with 0.5 M galactose for 60 min at room temperature. Strips were washed four times for 20 min in PBS/0.05% Tween 20 and then incubated with either rabbit anti-mouse immunoglobulin-horseradish peroxidase conjugate (1:1000 in PBS/0.05% Tween 20 containing 3% fish skin gelatin; Dako) or streptavidin-horseradish peroxidase (1:400 in PBS/0.05% Tween 20 containing 3% fish skin gelatin; Silenus) for 60 min at room temperature. After washing, bound horseradish peroxidase was detected using enhanced chemiluminescence (ECL; Amersham Life Sciences) and exposed on Fuji x-ray film.

Acknowledgments

We thank the National Health and Medical Research Council of Australia for support for this work. A.S. is supported by an Australian postgraduate research award. We thank Dr. M.Sandrin (Austin Research Institute) for the anti-[alpha]-galactosyl antibodies.

Abbreviations

FITC, fluorescein isothiocyanate; TRITC, tetramethyl rhodamine isothiocyanate; BS1-IB4, Bandeiraea simplicifolia agglutinin isolectin B4.

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1To whom correspondence should be addressed at: Department of Pathology and Immunology, Monash University Medical School, Commercial Road, Prahran, Victoria 3181, Australia


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