Gastric H-K-ATPase and acid-resistant surface proteins

Hariharan Thangarajah, Aline Wong, Dar C. Chow, James M. Crothers Jr., and John G. Forte

Department of Molecular and Cell Biology, University of California, Berkeley, California 94720


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Despite the fact that mucus and bicarbonate are important macroscopic components of the gastric mucosal barrier, severe acidic and peptic conditions surely exist at the apical membrane of gastric glandular cells, and these membranes must have highly specialized adaptations to oppose external insults. Parietal cells abundantly express the heterodimeric, acid-pumping H-K-ATPase in their apical membranes. Its beta -subunit (HKbeta ), a glycoprotein with >70% of its mass and all its oligosaccharides on the extracellular side, may play a protective role. Here, we show that the extracellular domain of HKbeta is highly resistant to trypsin in the native state (much more than that of the structurally related Na-K-ATPase beta -subunit) and requires denaturation to expose tryptic sites. Native HKbeta also resists other proteases, such as chymotrypsin and V8 protease, which hydrolyze at hydrophobic and anionic amino acids, respectively. Removal of terminal alpha -anomeric-linked galactose does not appreciably alter tryptic sensitivity of HKbeta . However, full deglycosylation makes HKbeta much more susceptible to all proteases tested, including pepsin at pH <2.0. We propose that 1) intrinsic folding of HKbeta , 2) bonding forces between subunits, and 3) oligosaccharides on HKbeta provide a luminal protein domain that resists gastric lytic conditions. Protein folding that protects susceptible charged amino acids and is maintained by disulfide bonding and hydrophilic oligosaccharides would provide a stable structure in the face of large pH changes. The H-K-ATPase is an obvious model, but other gastric luminally exposed proteins are likely to possess analogous protective specializations.

gastric mucosal barrier; protein stability; glycoproteins


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE STOMACH IS ENDOWED WITH special features collectively referred to as the gastric mucosal barrier. These mechanisms prevent autodigestion of the gastric lining by native denaturants (HCl) and proteases (pepsin), thereby curtailing erosion, gastritis, and degenerative ulceration. Among the macroscopic constituents of this barrier are the mucus layer and bicarbonate secretion, which serve to modulate the accessibility of acid to the luminal surface (1, 30-32). There has been considerable study and much debate regarding the mechanism by which these secretory components afford a protective function (9, 10, 21, 26). Despite the mucus and bicarbonate overlays as protective elements, severe acidic and peptic conditions surely exist at the apical membranes of most gastric glandular cells. Mucus barriers and bicarbonate secretion likely become secondary means of protection for cell surface proteins. It is thus reasonable to conclude that the luminal-facing cell membranes themselves have highly specialized adaptations to oppose external insults. However, characterizing components of the protective barrier on a molecular level has been more difficult.

The heterodimeric H-K-ATPase is the proton pump responsible for gastric acid secretion and has a remarkable ability to resist the harsh conditions on the external surface of the functioning parietal cell. The alpha -subunit (HKalpha ), responsible for ATP-catalyzed exchange of H+ for K+, is a multispanning membrane protein with most of its 114-kDa mass located in the cytoplasm (29, 34). The closely associated beta -subunit (HKbeta ) is a glycoprotein; on the basis of the predicted topology from a single transmembrane segment, 70% of its glycosylated mass is oriented in the extracellular space (6, 23, 28). HKbeta has six or seven N-linked sites of glycosylation (35, 36), although there are reported differences for the nature of the oligosaccharides for different species (2, 11). Previous studies have suggested that glycosylation has some influence on enzymatic activity (17); however, this activity may be secondary to some more basic requirement for proper subunit folding and interaction and/or trafficking through the cell (4). It has also been proposed that the carbohydrate moieties of HKbeta may play a role in protecting the holoenzyme from the harsh extracellular environment (8, 12).

Also situated in the extracellular domain of HKbeta are six cysteine residues that exist as three oxidized disulfide bonds. These linkages have proved to be essential in maintaining the structural and functional stability of the enzyme and have also been shown to be relatively resistant to reduction (7). Interestingly, the positions of these six extracellular cysteine residues are highly conserved within gastric HKbeta from various animal species as well as within the beta -subunit isoforms of the ubiquitous and closely related Na-K-ATPase (NaKbeta ) (8, 15, 16), considered the evolutionary ancestor of the H-K-ATPase. Disruption of extracellular disulfide bonds within the extracellular domain of HKbeta has been shown to abolish H-K-ATPase activity and to induce structural alterations within the cytoplasmic domain of the alpha -subunit (33). Although a defined function has not yet been assigned to HKbeta , it is clear that interactions between both subunits of the H-K-ATPase are essential for the functional and structural stability of the enzyme.

We propose that HKbeta may protect the holoenzyme from conditions found in the extracellular environment. Here, we have examined the relative susceptibility of gastric HKbeta and renal NaKbeta to denaturation and proteolysis. In particular, the contributions of protein folding, disulfide bonds, glycosylation, and subunit interactions have been evaluated.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Trypsin, chymotrypsin, pepsin, V8 protease, 2-mercaptoethanol (2-ME), N-ethylmaleimide, Triton X-100 (TX-100), dodecyltrimethylammonium bromide (DTAB), and EDTA were purchased from Sigma Chemical (St. Louis, MO); SDS and piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) from ICN (Costa Mesa, CA); Tris from Fisher (Pittsburgh, PA); 4-[2-aminoethyl]benzenesulfonylfluoride (Pefabloc) and 3-(N-morpholino)propanesulfonic acid (MOPS) from Boehringer Mannheim (Indianapolis, IN); fluorescein-5-maleimide from Molecular Probes (Eugene, OR); coffee bean alpha -galactosidase and peptide N-glycosidase F (PNGase F) from Oxford Glycosystems (Abingdon, UK); and griffonia simplicifolia lectin (GS-1) from E-Y Labs (San Mateo, CA). All other chemicals were of reagent grade.

Isolation of H-K-ATPase-enriched gastric microsomes. Gastric microsomes were isolated as previously described (24). New Zealand White rabbits were injected with cimetidine and killed 1 h later by an overdose of pentobarbital sodium (Nembutal; intravenous). The stomach was removed, cut open, and washed with ice-cold buffered saline [(in mM): 150 Na+, 4.5 K+, 10 phosphate, and 136 Cl-, pH 7.3]. The mucosa was scraped and homogenized in 125 mM mannitol, 40 mM sucrose, 1 mM EDTA, and 5 mM PIPES (MSEP; pH 6.7) in a Potter-Elvehjem homogenizer with 15 strokes turning at 200 rpm. Crude microsomes were harvested as the pellet sedimented between 10 min at 13,000 g and 1 h at 100,000 g. The pellet was first resuspended in suspending medium (SM: 300 mM sucrose, 0.4 mM EDTA, and 5 mM Tris), then brought to 40% sucrose and overlaid with successive layers of 35% sucrose and 10% sucrose. All sucrose media were made in 5 mM Tris and 0.4 mM EDTA (pH 7.4). After centrifugation at 80,000 g for 4 h, H-K-ATPase-enriched microsomes were collected from the interface between 10 and 35% sucrose and stored at -20°C until use.

The connective tissue capsule and outermost parts of the cortex were sliced from rabbit kidneys and discarded. The remaining cortex and outer medulla were minced in PBS, homogenized in MSEP, and centrifuged to yield a crude microsomal fraction as described above for gastric mucosa. The microsomes were resuspended in SM, directly layered over a 15-45% continuous sucrose gradient, and centrifuged at 100,000 g for 4 h. Western blots of gradient fractions were probed with an antibody against NaKbeta (see Western blotting methods), and fractions richest in Na-K-ATPase were used for further studies.

Treatment of microsomes with ethanol. Gastric microsomes solubilized in 0.75% TX-100 were treated with varying concentrations of ethanol (0-87%) for 20 min, then diluted fivefold with 20 mM MOPS buffer before being subjected to proteolysis.

Proteolytic digestion. Digestion with trypsin or chymotrypsin was carried out at 37°C at pH 7.6-7.8. The proteases were suspended in 25 mM ammonium bicarbonate buffer (pH 7.8) for reaction. Gastric microsomes or renal membranes were solubilized with the indicated concentrations of TX-100 or DTAB (usually 0.5-1% detergent), and the protease was added. In most cases, a protease-to-membrane protein ratio of 1:50 was used; however, in certain instances, as indicated in the legends, the ratio was outside this range. Digestion was allowed for designated times and quenched by the addition of Pefabloc, a serine protease inhibitor, at a ratio of at least 10:1 (inhibitor to protease).

Digestion with V8 protease was carried out in a 50 mM sodium phosphate buffer (pH 7.8) at 37°C. As with tryptic and chymotryptic digests, a protease-to-membrane ratio of 1:50 was used, except where noted. Digestion was quenched by addition of SDS sample buffer and subsequent boiling for 3 min (final concentrations: 1% SDS, 0.4 M urea, 0.7 M 2-ME, 0.25 mM EDTA, 10% glycerol, and 30 mM Tris · HCl, pH 6.8).

Digestion with pepsin was carried out at pH 2.0. Pepsinolysis was inactivated by raising of the sample pH to 8 with 0.28 M Tris, which also contained 1% SDS, 0.4 M urea, 0.7 M 2-ME, 0.25 mM EDTA, and 10% glycerol. For all protease experiments, samples designated as 0 min digestion have the protease inactivated (by addition of inhibitor or denaturation) before addition to the solubilized microsomes.

Reduction of disulfide bonds with 2-ME. Gastric microsomes or renal membranes solubilized in 0.5% TX-100 or DTAB were incubated at 44°C with varying concentrations of 2-ME (0.1-0.6 M) for 25-30 min. Experimental control samples had distilled water of appropriate volume in lieu of 2-ME. After the allotted time of treatment, all samples were diluted 10-fold at 0°C to quench 2-ME reduction.

To ensure that treatment with 2-ME was sufficient to reduce beta -subunit disulfide bonds, we assessed the labeling of sulfhydryl groups by fluorescein-5-maleimide (F-M) before and after reduction using a previously described method (7). Briefly, membranes were first solubilized in 0.5% TX-100 and treated with 10mM N-ethylmaleimide to alkylate available SH groups. The sample was then divided in half: one-half treated with 0.6 M 2-ME, and the other serving as the nonreduced control. After separation of membranes from the reagents with a Centricon concentrator (30-kDa pore size spun at 4,000 g for 30 min), both reduced and nonreduced samples were solubilized in 0.5% SDS and labeled with 0.5 mM F-M in 20 mM sodium phosphate (pH 7.1) for 15 min at room temperature, followed by 20 min at 55°C. Sample proteins were separated by SDS-PAGE, and the gels were examined with a BioRad fluorescence imager to ascertain the fluorescent labeling of disulfide bonds that were reduced by our treatment with 2-ME.

alpha -Galactosidase treatment of gastric microsomes. Microsomes solubilized in 1% TX-100 were digested with 5 U/ml alpha -galactosidase in 100 mM sodium citrate/phosphate buffer (pH 6) for 1 h at 37°C. Both sham-treated and alpha -galactosidase-treated samples were subsequently subjected to tryptic digestion before analysis by Western blot. To ensure that the treatment with alpha -galactosidase was sufficient to remove alpha -anomeric-linked galactose residues from HKbeta , samples not subjected to trypsin were analyzed by Western blots probed with GS-1 lectin. Control experiments demonstrated that 30 min of treatment with alpha -galactosidase, as described above, were sufficient to remove all GS-1-reactive alpha -galactose residues from HKbeta oligosaccharides.

PNGase F treatment of gastric microsomes. Microsomes solubilized in either 1% TX-100 or 0.5% SDS were deglycosylated with PNGase F overnight (~18 h) at 37°C. The reaction buffer consisted of 10 mM sodium phosphate and 10 mM EDTA. Samples initially solubilized in SDS were diluted with TX-100 such that final TX-100 concentration was 1% and final SDS concentration was 0.15%. PNGase F was used at a concentration of 1 U/100 µg of microsomal protein. Deglycosylation was stopped by freezing samples at -20°C. Our treatments were sufficient to remove all oligosaccharides from HKbeta solubilized in SDS and about half of the oligosaccharides from TX-100-solubilized material.

Western blots. Experimental samples were separated by SDS-PAGE using either 4-20% gradient gels or 10% gels and subsequently transferred to nitrocellulose or Immobilon-P (Millipore) transfer membranes using an immersion blotting apparatus (Idea Scientific). Transferred blots were first blocked with 5% nonfat milk in PBS for 1 h at room temperature. After a quick rinse in PBS, blots were probed with monoclonal antibodies against HKbeta or NaKbeta for 1 h at room temperature. For HKbeta , we used monoclonal antibody (MAb) 2/2E6 and MAb 2G11, antibodies against specific epitopes in the COOH terminus and NH2 terminus, respectively (22). For NaKbeta , we used MAb 72M, courtesy of A. McDonough (Univ. of Southern California) and M. Caplan (Yale Univ.). After three washes for 10 min each in PBS, blots were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG for 1 h at room temperature. Lectin probes (GS-1) were directly conjugated to HRP. Finally, blots were developed on film using chemiluminescence reagents (Renaissance, New England Nuclear) as the HRP substrate. Images of the films were taken with a digital camera, and densitometry was carried out with National Institutes of Health Image 1.61 software.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The extracellular domain of HKbeta must be denatured to expose tryptic sites. Intact gastric microsomal vesicles [predominantly oriented cytoplasmic-side out (Tyag)] were treated with trypsin, and the remnant peptides were separated by SDS-PAGE. As demonstrated in Fig. 1, A and B, virtually no intact alpha -subunit remained after this treatment, whether or not the vesicles were solubilized in detergent, consistent with earlier findings from many laboratories. The results were somewhat different for the beta -subunit, depending on which detergent and MAb were used to probe the Western blots. In blots probed with MAb 2/2E6, HKbeta from intact vesicles appeared to be virtually unaltered by these tryptic conditions. The same was true for HKbeta from vesicles solubilized with the nondenaturing detergent TX-100. However, when a denaturing ionic detergent such as DTAB (Fig. 1B) or SDS (not shown) was used, no remnant HKbeta was visualized on the 2/2E6 blot after trypsinolysis. Blots for identically treated samples probed with MAb 2G11 were negative in the region of HKbeta except for a trace remaining with trypsinized intact vesicles. Because the 2/2E6 probe showed that the majority of HKbeta was still present in the trypsinized samples of intact vesicles or TX-100-solubilized vesicles, this suggests that the epitope for MAb 2G11 had been hydrolyzed by trypsin under all conditions tested. Identical results were obtained when the samples were treated with lysyl-endopeptidase C (specific for lysine residues) instead of trypsin; that is, probes with 2G11 were negative, but HKbeta clearly remained when blots of intact and TX-100-solubilized vesicles were probed with 2/2E6 (data not shown). These results can best be interpreted with an understanding of the epitopic sites for MAbs 2G11 and 2/2E6 in HKbeta . The 2G11 site has been mapped to the eight NH2-terminal amino acids of the cytoplasmic domain of HKbeta . (22). Because there is a proximal pair of lysine residues (K7K8) in that region, we would expect that trypsinization of H-K-ATPase, whether solubilized or in intact vesicles, would allow for cleavage in this cytoplasmic tail and a consequent decrease in the 2G11 signal, as seen in Fig. 1. The 2/2E6 epitope has been localized to the region of S226LHY229, which is in the extracellular domain of HKbeta and tightly interactive with an extracellular loop of HKalpha (22). Thus we conclude that the NH2-terminal cytoplasmic tail of HKbeta is sensitive to trypsinolysis, but this site involves a very small portion of the glycopeptide with minimal alterations in molecular size. On the other hand, the transmembrane segment and entire extracellular domain of HKbeta are highly resistant to trypsinolysis, unless HKbeta is treated with denaturing agents, suggesting that the hydrophilic tryptic sites (i.e., lysine and arginine residues) might be buried or protected in the native state.


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Fig. 1.   Sensitivity of alpha - and beta -subunits of H-K-ATPase to trypsin. Intact gastric vesicles and vesicles solubilized with either mild [Triton X-100 (TX-100)] or denaturing [dodecyltrimethylammonium bromide (DTAB)] detergents were treated with trypsin (+) or not (-), as indicated, at a trypsin/membrane protein ratio of 1:50. After 60 min at 37°C, samples were run on SDS-PAGE. Some gels were stained for protein [Coomassie blue (CB)], and others were blotted to nitrocellulose and probed with antibodies against H-K-ATPase beta -subunit (HKbeta ), either a monoclonal antibody (MAb) recognizing the NH2-terminal cytoplasmic tail of HKbeta (2G11) or an MAb specific for the extracellular domain (2/2E6). H-K-ATPase alpha -subunit (HKalpha ), as seen on CB-stained gels, and the NH2-terminal cytoplasmic tail of HKbeta , as seen with 2G11 probing, were highly susceptible to trypsinolysis in all conditions tested. However, the bulk of the HKbeta -subunit, including the transmembrane segment and large extracellular domain, was resistant to trypsinolysis except for conditions in which denaturing detergents were used.

Further support for the interpretation that HKbeta must be denatured for tryptic sensitivity of the extracellular domain comes from experiments, in which TX-100-solubilized microsomes were titrated with ethanol. Ethanol concentrations ranging from 0 to 87% were used to alter noncovalent bonding forces within the protein, and these samples were subsequently trypsinized. Results in Fig. 2 show that increasing ethanol allows for greater susceptibility of HKbeta to trypsinolysis. The similarity in band density of 0% ethanol-treated samples treated with trypsin and control samples (not treated with trypsin) also confirms resistance of the noncytoplasmic bulk of HKbeta to tryptic digestion under nondenaturing conditions.


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Fig. 2.   Denaturation with ethanol increases the tryptic sensitivity of HKbeta . TX-100-solubilized membranes were treated with various concentrations of ethanol (0-87% EtOH), then diluted 1:5 in 3-(N-morpholino)propanesulfonic acid buffer (pH 7.6) before being treated with trypsin for 30 min at 37°C and subsequently run on SDS-PAGE, blotted, and probed for HKbeta with 2/2E6 MAb. A control sample (Ctrl) of membranes not treated with trypsin is shown on the right. MW, molecular wt.

HKbeta is more resistant to trypsinolysis than NaKbeta in both native and denatured states. The similarities in sequence and structure between HKbeta and NaKbeta made comparative studies between the two proteins unavoidable. We thus sought to evaluate the resistance of both proteins to proteolytic digestion in the native and denatured states. Gastric microsomes or renal membranes were solubilized in 0.75% TX-100 or DTAB, subjected to a time course of trypsinolysis, and respectively probed for HKbeta with MAb 2/2E6 or for NaKbeta using MAb 72M, as shown in Fig. 3A. As expected, HKbeta remained fairly resistant to tryptic digestion when solubilized in TX-100 but was more rapidly degraded when solubilized with the denaturing detergent DTAB. The results with NaKbeta were quite different (Fig. 3B). Even in nondenaturing conditions (TX-100 treated), NaKbeta showed a marked sensitivity to trypsinolysis, with only 20% of the original band density remaining after 2 h of digestion. After DTAB solubilization, digestion was even more rapid, with only a faint NaKbeta band remaining at the 30-min time point. A summary of several experiments is presented in Fig. 3C.


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Fig. 3.   HKbeta is more resistant to trypsin than the beta -subunit of Na-K-ATPase. The respective membrane preparations were solubilized in TX-100 or DTAB, subjected to trypsin for varying periods of time, then blotted and probed for either beta -subunit. A: single experiment with gastric vesicles and probed for HKbeta . B: single experiment with renal vesicles and probed for NaKbeta . Lane assignments: 1: TX-100, 0 time trypsin; 2: DTAB, 0 time trypsin; 3: TX-100, 30 min trypsin; 4: DTAB, 30 min trypsin; 5: TX-100, 60 min trypsin; 6: DTAB, 60 min trypsin; 7: TX-100, 120 min trypsin; 8: DTAB, 120 min trypsin. C: summary data for 3 experiments (±SE) comparing HKbeta and NaKbeta .

HKbeta and NaKbeta exhibit different tryptic sensitivities following disulfide bond reduction. To assess the role of disulfide linkages in contributing to the structural stability of HKbeta and NaKbeta , we tested the tryptic sensitivity of solubilized and reduced membranes. Gastric microsomes or renal membranes were solubilized in 0.5% TX-100 (or 0.5% DTAB) and treated with graded concentrations of 2-ME at 44°C for 30 min. Samples were then digested with trypsin for 1 h and analyzed by SDS-PAGE and Western blot. Results from a typical experiment are shown in Fig. 4. Reducing conditions up to 0.6 M 2-ME apparently did not alter the tryptic sensitivity of HKbeta solubilized in TX-100 (Fig. 4A). Only when a denaturing detergent was used (DTAB) was there significant reduction in band density. Indeed, as the quantitative summary data show (Fig. 4C), even under severe reducing conditions of 0.6 M 2-ME, >80% of the original HKbeta band remained after 1 h of tryptic digestion. Parallel experiments described in MATERIALS AND METHODS demonstrated that the disulfide bonds of HKbeta were reduced by treatment with 0.6 M 2-ME.


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Fig. 4.   HKbeta and NaKbeta exhibit different tryptic sensitivities following disulfide bond reduction. H-K-ATPase and Na-K-ATPase membranes were solubilized in either 0.5% TX-100 or DTAB, as indicated, and incubated at 44°C with 2 mercaptoethanol (2-ME; 0-0.6 M) for 30 min. Samples were then diluted 10-fold, then subjected to trypsinolysis for 1 h and analyzed by Western blot. A: single experiment probed for HKbeta . B: single experiment probed for NaKbeta . C: summary data for 3 experiments (±SE) comparing HKbeta and NaKbeta .

The tryptic sensitivity of NaKbeta after 2-ME reduction differed markedly from that of HKbeta , although both have the same three disulfide bonds. As the concentration of 2-ME was increased, NaKbeta became much more susceptible to tryptic digestion. For the sample treated with 0.6 M 2-ME, only a minute fraction of the original NaKbeta band density remained after 1 h of digestion (Fig. 4B). Hence, even under harsh reducing conditions, HKbeta maintained a remarkable resistance to tryptic digestion. Because the same phenomenon is not seen with NaKbeta , the greater protection of HKbeta would seem to involve other specialized features.

Removal of terminal alpha -galactose sugars has little effect on tryptic sensitivity. As mentioned above, rabbit HKbeta and the renal isoform of NaKbeta used here differ in their respective glycosylation properties. The former maintains seven sites of N-glycosylation, whereas only three such sites are found on the latter. It has previously been proposed that glycosylation of HKbeta provides protection against acidic and autodigestive extracellular conditions (8, 22). Experiments were designed to selectively remove either terminal sugars or entire oligosaccharides before digestion with protease.

In one test, gastric microsomes solubilized in 1% TX-100 were first digested with the exoglycosidase, alpha -galactosidase. In control experiments, we found that 1 h of treatment with 5 U/ml of alpha -galactosidase was sufficient to remove all terminal alpha -anomeric-linked galactose residues from HKbeta oligosaccharides (data not shown). Accordingly, alpha -galactosidase-treated samples were subsequently subjected to a time course of trypsinolysis and analyzed with SDS-PAGE and Western blot. Figure 5 shows that removal of terminal alpha -galactose residues did not affect the sensitivity of HKbeta to tryptic digestion, with similar rates of degradation seen for HKbeta during the 2-h time course with and without alpha -galactosidase pretreatment. Thus the terminal alpha -galactose capping of beta -subunit oligosaccharides does not play a significant role in protecting HKbeta against tryptic digestion.


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Fig. 5.   Removal of alpha -galactose from terminal N-glycosylation branch sites had minimal effect on the tryptic sensitivity of HKbeta . Gastric vesicles were solubilized in TX-100; 1 aliquot was treated with alpha -galactosidase for 1 h, another was not. The preparations were then subjected to trypsinolysis over a time course, as indicated, and finally probed for remaining HKbeta . Separate blots probed with GS-1 lectin (not shown) revealed that the conditions of treatment with alpha -galactosidase were sufficient to remove all terminal alpha -galactose from the N-linked glycans on HKbeta .

Complete deglycosylation of beta -subunit greatly enhances trypsinolysis. To test whether the same resistance to proteolysis would be maintained when the protein was completely deglycosylated, we treated solubilized microsomes with PNGase F, then digested with trypsin. Deglycosylation by PNGase F was a function of which detergent was to solubilize microsomes. When the vesicles were solubilized in denaturing detergents, efficient deglycosylation occurred; all N-linked oligosaccharides were removed. For nondenatured HKbeta , in TX-100, deglycosylation was less efficient, as noted by many others. However, on Western blots, we could clearly differentiate the 32-kDa deglycosylated core peptide from the 60-80 Mr intact HKbeta ; therefore, it was convenient to assess the relative rates of proteolysis of both the fully glycosylated and deglycosylated forms on the same blot. Figure 6 compares the time course of trypsinolysis for the normal glycosylated HKbeta and the HKbeta core peptide that was deglycosylated by PNGase overnight in TX-100. The fully glycosylated HKbeta was resistant to trypsin, as was seen in other experiments. In sharp contrast, the HKbeta core peptide was completely digested within minutes. An even more rapid trypsinolysis occurred in the case of HKbeta that was denatured and fully deglycosylated in SDS (data not shown). Thus removal of oligosaccharides converts HKbeta into a highly trypsin-sensitive form, even when carried out in nondenaturing detergents. Studies with NaKbeta showed essentially the same response to deglycosylation as HKbeta . That is, after complete removal of N-linked sugars with PNGase, the core NaKbeta became more highly susceptible to trypsinolysis than the glycosylated form (data not shown).


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Fig. 6.   Total deglycosylation renders HKbeta very sensitive to tryptic hydrolysis. H-K-ATPase-rich vesicles were solubilized in TX-100 and treated overnight with PNGase as described in MATERIALS AND METHODS. Samples were then subjected to trypsin over a time course and analyzed for remaining HKbeta . Because of the high sensitivity of deglycosylated HKbeta to proteolysis, the trypsin-to-membrane protein ratio was 1:500. Both the fully glycosylated (60-80 kDa) and core protein (34 kDa) are readily apparent after the deglycosylation treatment (at 0 time), but the core peptide completely disappears after a brief trypsinolysis. A: single experiment showing that the core HKbeta peptide is much more sensitive to trypsin than the fully glycosylated moiety. The bands at <22 kDa are tryptic breakdown products of the core peptide. B: mean (±SE) of 3 experiments comparing tryptic sensitivity of glycosylated and deglycosylated HKbeta .

Sensitivity to other proteases. To determine whether the resistance of HKbeta was specific for trypsin, we employed several other proteases whose hydrolytic specificity is directed at different amino acids. The results of exposing detergent-solubilized H-K-ATPase to chymotrypsin and V8 protease are shown in Fig. 7. As with trypsin, when membranes were solubilized in TX-100, they remained relatively resistant both to chymotrypsin (Fig. 7A), which cleaves at hydrophobic residues, and to V8 protease (Fig. 7B), which cleaves at negatively charged residues, whereas proteolysis of the beta -subunit by chymotrypsin and V8 protease was very much enhanced when the denaturing detergent DTAB was used (Fig. 7, A and B). We did find that the deglycosylated core beta -subunit showed different sensitivities to these enzymes (Fig. 7C). The core peptide was effectively hydrolyzed by chymotrypsin in 15 min but was relatively resistant to V8 protease unless exposed to denaturants.


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Fig. 7.   Denaturation or deglycosylation makes HKbeta sensitive to chymotrypsin and V8 protease. A and B: H-K-ATPase-rich vesicles were solubilized in 1% TX-100 or DTAB and treated with either chymotrypsin (A) or V8 protease (B) over a time course as indicated; finally, blots were probed for HKbeta . C: H-K-ATPase-rich vesicles were solubilized in TX-100, treated with PNGase, as described in Fig. 6 and MATERIALS AND METHODS, then exposed to either chymotrypsin or V8 protease as indicated, and finally probed to show remnant HKbeta , both the fully glycosylated (gly beta ) and deglycosylated core peptide (core beta ). In A and B, protease-to-membrane protein ratio is 1:50; in C, ratio is 1:300.

Analogous results were obtained when fully glycosylated and deglycosylated HKbeta were treated with pepsin. After treatment with PNGase F in TX-100, as described above, samples were transferred to pH 2.0 buffer containing pepsin. It must be underscored that digestion with pepsin requires a low pH, and this environment will undoubtedly alter the conformation of the substrate protein. Nonetheless, the time course for the disappearance of HKbeta shows that pepsinolysis was also greatly accelerated by deglycosylation (Fig. 8).


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Fig. 8.   N-linked glycosylation protects HKbeta from degradation by pepsin. H-K-ATPase-rich vesicles were solubilized in TX-100 and treated with PNGase to remove oligosaccharides from N-linked sites. Other samples served as the glycosylated control. All samples were treated with pepsin at pH 2.0; aliquots were removed at timed intervals as shown, run on SDS-PAGE, and blotted with MAb 2/2E6. The band densities of the fully glycosylated 60- to 80-kDa HKbeta and the deglycosylated 34-kDa core peptide were measured. Data are shown as means (±SE) of 4 separate experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Being one of the most predominant protein domains exposed on the external surface of the functioning parietal cell, HKbeta is here hypothesized to have specialized features that allow it to resist the acidic and autodigestive conditions found in the gastric milieu. The results presented here clearly show that the beta -subunit of H-K-ATPase is much more resistant to a variety of proteases than its partner alpha -subunit, which has its mass predominantly exposed to the intracellular surface. To some extent, the same could be said for the alpha - and beta -subunits of the structurally and functionally related Na-K-ATPase, except that direct comparison revealed that HKbeta was proteolyzed at an even slower rate than NaKbeta . Not surprisingly, the sensitivity to proteolysis for both beta -subunits was increased when the protein was denatured.

Of special concern is whether the observed characteristics of proteolytic sensitivity/resistance have any functional role in adaptation to the external environment occupied by these extracellular protein domains. In particular, do the folding and stability of HKbeta serve to protect the enzyme and the parietal cell from lytic external conditions, and what are the structural features that provide the stability? It is clear that total protein unfolding, i.e., denaturation, markedly changes the accessibility to a variety of proteases. In fact, Imoto and his colleagues (13, 39) developed a strategy to determine physicochemical transition states and unfolding rate constants for proteins using digestion with proteases as an end point. We can generally assume that the folded form of HKbeta is kinetically stable, possibly due to the high free-activation energy change required for its unfolding.

The most pronounced change in sensitivity to proteases was rendered on removal of the N-linked oligosaccharides on HKbeta . We had earlier shown that the N-linked complex oligosaccharides on HKbeta were somewhat unusual, being totally devoid of sialic acid. Instead, the branched polylactosamine structures in some species were terminated exclusively by alpha -anomeric galactose linkages (e.g., rabbit, pig; see Refs. 35 and 36) and in others were terminated as Lewis x and y type structures (e.g., human, mouse; see Refs. 2 and 11). At that time, we proposed that there might be some special protective feature for the oligosaccharides. Here, we found that complete removal of the terminal alpha -linked galactose residues did not appreciably affect the tryptic sensitivity of rabbit HKbeta . Also, HKbeta with high mannose core glycosylation had about the same sensitivity to trypsin as the fully mature form. Thus it appeared that the sugar composition of the oligosaccharides was less important than the absolute presence of the N-linked glycans themselves, and probably their locations along the polypeptide, in maintaining the folding and structural integrity of HKbeta .

Glycosylation is recognized as an important influence on both the protein folding process and the stability of the native glycoprotein conjugate. Lis and Sharon (18) propose that cotranslational glycosylation aids in folding the nascent polypeptide chain, and several groups have demonstrated that removal of N-linked (or O-linked) glycoforms destabilizes a variety of proteins and tends to make them more susceptible to aggregation, especially at low pH (14, 37, 38). Glycosylation has also been shown to favorably influence the stability of several gastrointestinally active enzymes subjected to the lytic conditions of the gut. Glycosylated forms of pancreatic RNase, called RNase B, were found to be thermodynamically more stable and resistant to pronase than the unglycosylated RNase A form (3, 25). The functional activity of human gastric lipase (HGL) was reduced by total deglycosylation or by specific removal of the Asn308 glycosylation site (19). In this same study, the digestion of HGL by pepsin at low pH was markedly increased by deglycosylation. This is especially significant as HGL and other related lipases (lingual and esophageal) must operate in the acidic and peptic milieu of the stomach (27). Loomes et al. (20) showed that another enzyme that functions in the infant stomach, human milk cholesterol esterase, was resistant to acid denaturation and digestion by pepsin at low pH when its glycosylated COOH terminus was intact even if this COOH-terminal region was replaced by a glycosylated segment of a stomach mucin (Muc6).

Thus the importance of oligosaccharides in conveying thermodynamic stability and resistance to proteolysis of the glycoprotein conjugate is well established. The specific mechanism for this protection remains to be established. It must be recognized that these glycoforms can be rather large structures (2,000-3,500 Å3) and could represent a significant fraction of the total glycoprotein mass (14). Thus one view would offer a steric mechanism for protease sensitivity. However, an unfolding of the glycoprotein renders it more sensitive to proteases even with its glycoconjugates intact, and an abundance of evidence clearly shows that the presence of oligosaccharides contributes to the folding stability. Therefore, a simple steric mechanism limiting protease accessibility is not the total answer, but there may be some more complex steric role in which the glycoform might limit the conformational space available for the peptide. Also, the carbohydrate may offer solvation properties to maintain compact peptide conformation even in the face of variable salt and low pH (36).

Complete absence of N-linked oligosaccharides totally abolishes H-K-ATPase activity (17) and, as shown here, greatly destabilizes the protein structure. Using a coexpression system for HKalpha and HKbeta in HEK-293 cells, Asano et al. (4) showed that deletion of any one of the seven N-linked sites on HKbeta did not appreciably alter holoenzyme activity or alpha /beta -assembly but that there was progressive impairment of these activities with multiple deletions. It will be of interest to test how specific modifications of N-linked glycosylation sites in HKbeta affect the stability of the beta -subunit.

Comparison of HKbeta with the closely related NaKbeta is instructive. Both in the native and denatured states, HKbeta was proteolyzed more slowly than NaKbeta . This suggests properties within the structure of the gastric proton pump that provide greater stability and resistance to proteolysis. Both beta -subunits have three disulfide bonds at identical positions within the extracellular domain. Reduction of these disulfide bonds in HKbeta did not cause a large change in the tryptic sensitivity, although equivalent reducing conditions did increase trypsinolysis of NaKbeta . Earlier, it was shown that these three disulfide bridges in HKbeta were important for the catalytic function of the H-K-ATPase (7) and further that reduction of these extracellular disulfide bonds greatly increased the tryptic sensitivity of HKalpha distally located at the cytoplasmic surface (33). Thus, whereas previous work implicated disulfide bonds as being integral to holoenzyme function and protecting the companion HKalpha subunit from trypsin, they do not appear to play as prominent a role in protecting HKbeta against trypsinolysis. There are several basic structural differences between HKbeta and NaKbeta that might contribute to differential tryptic sensitivity. HKbeta has seven N-linked glycosylation sites, whereas the renal NaKbeta 1 isoform used in this study contains only three sites (8). Also, there are considerably fewer tryptic sites (Arg and Lys) in HKbeta than in NaKbeta (28 vs. 46) and, interestingly, fewer negatively charged amino acids (Asp and Glu) in HKbeta than in NaKbeta (26 vs. 39). These structural differences may contribute to the observed differences in susceptibility to proteolysis.

The protective effect of oligosaccharides and/or disulfide bonds in the extracellular domains of H-K- and Na-K-ATPases suggests that certain chemical modifications may generally protect biomolecules facing an extracellular medium. At the same time, the differences between these closely related proteins, including altered amino acid sequences, suggest a branching evolution of different specific protective measures for different extracellular conditions, possibly including the harsh luminal conditions along the digestive tract.

Practical implications of the protective function of the carbohydrate moieties on glycoproteins through understanding the mechanism of how they protect the glycoprotein are numerous. In fact, the stabilizing effect of glycosylations has already been used in many fields. For example, human insulin is being modified by the covalent attachment of monosaccharide moieties to specific amino groups to improve both the pharmaceutical stability and biological response (5) of the hormone. Furthermore, many diseases are believed to result from the malfunctioning of glycosylation machinery, and a better understanding of the functions of glycans may aid in preventing or curing these diseases.


    ACKNOWLEDGEMENTS

We thank C. Fan and M. Ushigome for excellent assistance in experimental procedures.


    FOOTNOTES

This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-10141 and DK-38972 (to J. G. Forte).

Present address for D. C. Chow: Dept. of Microbiology and Immunology, Fairchild Bldg., Rm. D325, Stanford Univ., Stanford, CA 94305.

Address for reprint requests and other correspondence: J. G. Forte, Dept. of Molecular & Cell Biology, 245 LSA, #3200, Univ. of California, Berkeley, CA 94720-3200 (E-mail: jforte{at}uclink.berkeley.edu).

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.

First published February 20, 2002;10.1152/ajpgi.00399.2001

Received 10 September 2001; accepted in final form 2 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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