Department of Molecular and Cell Biology, University of California, Berkeley, California 94720
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ABSTRACT |
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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 -subunit
(HK
), 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 HK
is highly
resistant to trypsin in the native state (much more than that of the
structurally related Na-K-ATPase
-subunit) and requires denaturation
to expose tryptic sites. Native HK
also resists other proteases,
such as chymotrypsin and V8 protease, which hydrolyze at hydrophobic
and anionic amino acids, respectively. Removal of terminal
-anomeric-linked galactose does not appreciably alter tryptic
sensitivity of HK
. However, full deglycosylation makes HK
much
more susceptible to all proteases tested, including pepsin at pH <2.0.
We propose that 1) intrinsic folding of HK
, 2)
bonding forces between subunits, and 3)
oligosaccharides on HK
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
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INTRODUCTION |
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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 -subunit (HK
), 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
-subunit (HK
) 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). HK
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 HK
may play a role in protecting the holoenzyme from the harsh extracellular environment (8, 12).
Also situated in the extracellular domain of HK 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 HK
from various animal species as well as
within the
-subunit isoforms of the ubiquitous and closely related
Na-K-ATPase (NaK
) (8, 15, 16), considered the
evolutionary ancestor of the H-K-ATPase. Disruption of extracellular disulfide bonds within the extracellular domain of HK
has been shown
to abolish H-K-ATPase activity and to induce structural alterations
within the cytoplasmic domain of the
-subunit (33). Although a defined function has not yet been assigned to HK
, 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 HK may protect the holoenzyme from conditions found
in the extracellular environment. Here, we have examined the relative
susceptibility of gastric HK
and renal NaK
to denaturation and
proteolysis. In particular, the contributions of protein folding, disulfide bonds, glycosylation, and subunit interactions have been evaluated.
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MATERIALS AND METHODS |
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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 -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.
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-Galactosidase treatment of gastric microsomes.
Microsomes solubilized in 1% TX-100 were digested with 5 U/ml
-galactosidase in 100 mM sodium citrate/phosphate buffer (pH 6) for
1 h at 37°C. Both sham-treated and
-galactosidase-treated samples were subsequently subjected to tryptic digestion before analysis by Western blot. To ensure that the treatment with
-galactosidase was sufficient to remove
-anomeric-linked
galactose residues from HK
, samples not subjected to trypsin were
analyzed by Western blots probed with GS-1 lectin. Control experiments
demonstrated that 30 min of treatment with
-galactosidase, as
described above, were sufficient to remove all GS-1-reactive
-galactose residues from HK
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 HK
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 HK or NaK
for 1 h at room temperature. For HK
, we used monoclonal antibody (MAb) 2/2E6 and MAb
2G11, antibodies against specific epitopes in the COOH terminus and
NH2 terminus, respectively (22). For NaK
,
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.
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RESULTS |
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The extracellular domain of HK 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
-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
-subunit, depending on which detergent and MAb
were used to probe the Western blots. In blots probed with MAb 2/2E6,
HK
from intact vesicles appeared to be virtually unaltered by these
tryptic conditions. The same was true for HK
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 HK
was visualized on the 2/2E6 blot
after trypsinolysis. Blots for identically treated samples probed with
MAb 2G11 were negative in the region of HK
except for a trace
remaining with trypsinized intact vesicles. Because the 2/2E6 probe
showed that the majority of HK
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 HK
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 HK
. The 2G11 site has been mapped to the
eight NH2-terminal amino acids of the cytoplasmic domain of
HK
. (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 HK
and tightly interactive with an extracellular loop of
HK
(22). Thus we conclude that the
NH2-terminal cytoplasmic tail of HK
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
HK
are highly resistant to trypsinolysis, unless HK
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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HK is more resistant to trypsinolysis than NaK
in both native
and denatured states.
The similarities in sequence and structure between HK
and NaK
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 HK
with
MAb 2/2E6 or for NaK
using MAb 72M, as shown in Fig.
3A. As expected, HK
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 NaK
were quite different
(Fig. 3B). Even in nondenaturing conditions (TX-100
treated), NaK
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 NaK
band remaining at the 30-min time point. A
summary of several experiments is presented in Fig. 3C.
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HK and NaK
exhibit different tryptic sensitivities following
disulfide bond reduction.
To assess the role of disulfide linkages in contributing to the
structural stability of HK
and NaK
, 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 HK
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 HK
band remained after 1 h of tryptic
digestion. Parallel experiments described in MATERIALS AND
METHODS demonstrated that the disulfide bonds of HK
were reduced by treatment with 0.6 M 2-ME.
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Removal of terminal -galactose sugars has little effect on
tryptic sensitivity.
As mentioned above, rabbit HK
and the renal isoform of NaK
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 HK
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.
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Complete deglycosylation of -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
HK
, 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 HK
; 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 HK
and the HK
core
peptide that was deglycosylated by PNGase overnight in TX-100. The
fully glycosylated HK
was resistant to trypsin, as was seen in other
experiments. In sharp contrast, the HK
core peptide was completely
digested within minutes. An even more rapid trypsinolysis occurred in
the case of HK
that was denatured and fully deglycosylated in SDS (data not shown). Thus removal of oligosaccharides converts HK
into
a highly trypsin-sensitive form, even when carried out in nondenaturing
detergents. Studies with NaK
showed essentially the same response to
deglycosylation as HK
. That is, after complete removal of
N-linked sugars with PNGase, the core NaK
became more highly susceptible to trypsinolysis than the glycosylated form (data
not shown).
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Sensitivity to other proteases.
To determine whether the resistance of HK 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
-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
-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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DISCUSSION |
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Being one of the most predominant protein domains exposed on the
external surface of the functioning parietal cell, HK 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
-subunit of H-K-ATPase
is much more resistant to a variety of proteases than its partner
-subunit, which has its mass predominantly exposed to the
intracellular surface. To some extent, the same could be said for the
- and
-subunits of the structurally and functionally related
Na-K-ATPase, except that direct comparison revealed that HK
was
proteolyzed at an even slower rate than NaK
. Not surprisingly, the
sensitivity to proteolysis for both
-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 HK
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 HK
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 HK. We had
earlier shown that the N-linked complex oligosaccharides on HK
were somewhat unusual, being totally devoid of sialic acid. Instead, the branched polylactosamine structures in some species were
terminated exclusively by
-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
-linked
galactose residues did not appreciably affect the tryptic sensitivity
of rabbit HK
. Also, HK
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 HK
.
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 HK and HK
in HEK-293 cells, Asano et al. (4)
showed that deletion of any one of the seven N-linked sites
on HK
did not appreciably alter holoenzyme activity or
/
-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
HK
affect the stability of the
-subunit.
Comparison of HK with the closely related NaK
is instructive.
Both in the native and denatured states, HK
was proteolyzed more
slowly than NaK
. This suggests properties within the structure of
the gastric proton pump that provide greater stability and resistance
to proteolysis. Both
-subunits have three disulfide bonds at
identical positions within the extracellular domain. Reduction of these
disulfide bonds in HK
did not cause a large change in the tryptic
sensitivity, although equivalent reducing conditions did increase
trypsinolysis of NaK
. Earlier, it was shown that these three
disulfide bridges in HK
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 HK
distally located at the cytoplasmic surface (33).
Thus, whereas previous work implicated disulfide bonds as being
integral to holoenzyme function and protecting the companion HK
subunit from trypsin, they do not appear to play as prominent a role in
protecting HK
against trypsinolysis. There are several basic
structural differences between HK
and NaK
that might contribute
to differential tryptic sensitivity. HK
has seven N-linked
glycosylation sites, whereas the renal NaK
1 isoform used
in this study contains only three sites (8). Also, there
are considerably fewer tryptic sites (Arg and Lys) in HK
than in
NaK
(28 vs. 46) and, interestingly, fewer negatively charged amino
acids (Asp and Glu) in HK
than in NaK
(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.
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ACKNOWLEDGEMENTS |
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We thank C. Fan and M. Ushigome for excellent assistance in experimental procedures.
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FOOTNOTES |
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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.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Allen, A,
Flemstrom G,
Garner A,
and
Kivilaakso E.
Gastroduodenal mucosal protection.
Physiol Rev
73:
823-857,
1993
2.
Appelmelk, BJ,
Simoons-Smit I,
Negrini R,
Moran AP,
Aspinall GO,
Forte JG,
DeVries T,
Quan H,
Verboom T,
Maaskant JK,
Ghiara P,
Kuipers E,
Bloemena E,
Tadema TM,
Townsend RR,
Tyagarajan K,
Crothers JM, Jr,
Monteiro MA,
Savio A,
and
DeGraaff J.
Potential role of molecular mimicry between Helicobacter pylori lipopolysaccharide and host Lewis blood group antigens in autoimmunity.
Infect Immun
64:
2031-2040,
1996[Abstract].
3.
Arnold, U,
Schierhorn A,
and
Ulbrich-Hofmann R.
Influence of the carbohydrate moiety on the proteolytic cleavage sites in ribonuclease B.
J Protein Chem
17:
397-405,
1998[ISI][Medline].
4.
Asano, S,
Kawada K,
Kimura T,
Grishin AV,
Caplan MJ,
and
Takeguchi N.
The roles of carbohydrate chains of the beta-subunit on the functional expression of gastric H+,K+-ATPase.
J Biol Chem
275:
8324-8330,
2000
5.
Baudys, M,
Uchio T,
Mix D,
Wilson D,
and
Kim SW.
Physical stabilization of insulin by glycosylation.
J Pharm Sci
84:
28-33,
1995[ISI][Medline].
6.
Canfield, VA,
Okamoto CT,
Chow D,
Dorfman J,
Gros P,
Forte JG,
and
Levenson R.
Cloning of the H,K-ATPase beta subunit. Tissue-specific expression, chromosomal assignment, and relationship to Na,K-ATPase beta subunits.
J Biol Chem
265:
19878-19884,
1990
7.
Chow, DC,
Browning CM,
and
Forte JG.
Gastric H+-K+-ATPase activity is inhibited by reduction of disulfide bonds in -subunit.
Am J Physiol Cell Physiol
263:
C39-C46,
1992
8.
Chow, DC,
and
Forte JG.
Functional significance of the -subunit for heterodimeric P-type ATPases.
J Exp Biol
198:
1-17,
1995
9.
Chu, S,
Tanaka S,
Kaunitz JD,
and
Montrose MH.
Dynamic regulation of gastric surface pH by luminal pH.
J Clin Invest
103:
605-612,
1999
10.
Copeman, M,
Matuz J,
Leonard AJ,
Pearson JP,
Dettmar PW,
and
Allen A.
The gastroduodenal mucus barrier and its role in protection against luminal pepsins: the effect of 16,16 dimethyl prostaglandin E2, carbopol-polyacrylate, sucralfate and bismuth subsalicylate.
J Gastroenterol Hepatol
9, Suppl1:
S55-S59,
1994[ISI][Medline].
11.
Crothers, JM, Jr,
Appelmelk BJ,
Tyagarajan KT,
Townsend RR,
and
Forte JG.
Lewis y (Ley) antigen is prominently expressed on the -subunit of human gastric H,K-ATPase (Abstract).
Gastroenterology
110, Suppl:
A85,
1996[ISI].
12.
Forte, TM,
and
Forte JG.
Histochemical staining and characterization of glycoproteins in acid-secreting cells of frog stomach.
J Cell Biol
47:
437-452,
1970
13.
Imoto, T,
Yamada H,
and
Ueda T.
Unfolding rates of globular proteins determined by kinetics of proteolysis.
J Mol Biol
190:
647-649,
1986[ISI][Medline].
14.
Imperiali, B,
and
O'Connor SE.
Effect of N-linked glycosylation on glycopeptide and glycoprotein structure.
Curr Opin Chem Biol
3:
643-649,
1999[ISI][Medline].
15.
Kawamura, M,
and
Nagano K.
Evidence for essential disulfide bonds in the -subunit of Na++K+-ATPase.
Biochim Biophys Acta
774:
188-192,
1984[ISI][Medline].
16.
Kirley, TL.
Determination of three disulfide bonds and one free sulfhydryl in the -subunit of (Na,K)-ATPase.
J Biol Chem
264:
7185-7192,
1989
17.
Klaassen, CH,
Fransen JA,
Swarts HG,
and
De Pont JJ.
Glycosylation is essential for biosynthesis of functional gastric H+,K+-ATPase in insect cells.
Biochem J
321:
419-424,
1997[ISI][Medline].
18.
Lis, H,
and
Sharon N.
Protein glycosylation. Structural and functional aspects.
Eur J Biochem
218:
1-27,
1993[Abstract].
19.
Loomes, KM.
Structural organisation of human bile-salt-activated lipase probed by limited proteolysis and expression of a recombinant truncated variant.
Eur J Biochem
230:
607-613,
1995[Abstract].
20.
Loomes, KM,
Senior HE,
West PM,
and
Roberton AM.
Functional protective role for mucin glycosylated repetitive domains.
Eur J Biochem
266:
105-111,
1999
21.
Matuz, J.
Role of mucus in mucosal protection through ethanol and pepsin damage models.
Acta Physiol Hung
80:
189-194,
1992[Medline].
22.
Okamoto, CT,
Chow DC,
and
Forte JG.
Interaction of - and
-subunits in native H-K-ATPase and cultured cells transfected with H-K-ATPase
-subunit.
Am J Physiol Cell Physiol
278:
C727-C738,
2000
23.
Okamoto, CT,
Karpilow JM,
Smolka A,
and
Forte JG.
Isolation and characterization of gastric microsomal glycoproteins. Evidence for a glycosylated -subunit of the H+/K+-ATPase.
Biochim Biophys Acta
1037:
360-372,
1990[ISI][Medline].
24.
Reenstra, WW,
and
Forte JG.
Isolation of H+,K+-ATPase-containing membranes from the gastric oxyntic cell.
Methods Enzymol
192:
151-165,
1990[Medline].
25.
Rudd, PM,
Joao HC,
Coghill E,
Fiten P,
Saunders MR,
Opdenakker G,
and
Dwek RA.
Glycoforms modify the dynamic stability and functional activity of an enzyme.
Biochemistry
33:
17-22,
1994[ISI][Medline].
26.
Schade, C,
Flemstrom G,
and
Holm L.
Hydrogen ion concentration in the mucus layer on top of acid-stimulated and -inhibited rat gastric mucosa.
Gastroenterology
107:
180-188,
1994[ISI][Medline].
27.
Sheriff, S,
Du H,
and
Grabowski GA.
Characterization of lysosomal acid lipase by site-directed mutagenesis and heterologous expression.
J Biol Chem
270:
27766-27772,
1995
28.
Shull, GE.
cDNA cloning of the beta-subunit of the rat gastric H,K-ATPase.
J Biol Chem
265:
12123-12126,
1990
29.
Shull, GE,
and
Lingrel JB.
Molecular cloning of the rat stomach H+-K+-ATPase.
J Biol Chem
261:
16788-16791,
1986
30.
Slomiany, BL,
and
Slomiany A.
Role of mucus in gastric mucosal protection.
J Physiol Pharmacol
42:
147-161,
1991[Medline].
31.
Synnerstad, I,
Johansson M,
Nylander O,
and
Holm L.
Intraluminal acid and gastric mucosal integrity: the importance of blood-borne bicarbonate.
Am J Physiol Gastrointest Liver Physiol
280:
G121-G129,
2001
32.
Szabo, S.
Mechanisms of gastric mucosal injury and protection.
J Clin Gastroenterol
13, Suppl2:
S21-S34,
1991[ISI][Medline].
33.
Tyagarajan, K,
Chow DC,
Smolka A,
and
Forte JG.
Structural interactions between - and
-subunits of the gastric H,K-ATPase.
Biochim Biophys Acta
1236:
105-113,
1995[ISI][Medline].
34.
Tyagarajan, K,
Forte JG,
and
Townsend RR.
Topology of Membrane Proteins in Native Membranes Using Matrix-assisted Laser Desorption Ionization/Mass Spectrometry. Techniques in Protein Chemistry VIII. New York: Academic, 1996, p. 533-542.
35.
Tyagarajan, K,
Lipniunas PH,
Townsend RR,
and
Forte JG.
The N-linked oligosaccharides of the -subunit of rabbit gastric H,K-ATPase: site localization and identification of novel structures.
Biochemistry
36:
10200-10212,
1997[ISI][Medline].
36.
Tyagarajan, K,
Townsend RR,
and
Forte JG.
The -subunit of the rabbit H,K-ATPase: a glycoprotein with all terminal lactosamine units capped with
-linked galactose residues.
Biochemistry
35:
3238-3246,
1996[ISI][Medline].
37.
Wang, C,
Eufemi M,
Turano C,
and
Giartosio A.
Influence of the carbohydrate moiety on the stability of glycoproteins.
Biochemistry
35:
7299-7307,
1996[ISI][Medline].
38.
Wyss, DF,
and
Wagner G.
The structural role of sugars in glycoproteins.
Curr Opin Biotechnol
7:
409-416,
1996[ISI][Medline].
39.
Yamada, H,
Ueda T,
and
Imoto T.
Thermodynamic and kinetic stabilities of hen-egg lysozyme and its chemically modified derivatives: analysis of the transition state of the protein unfolding.
J Biochem (Tokyo)
114:
398-403,
1993[Abstract].