(Received for publication, December 7, 1995)
From the
The Na-dependent glucose transporter (SGLT1)
mediates absorption of luminal glucose by the intestine. However,
available intestinal cell lines that recapitulate a monolayer phenotype
only express SGLT1 at low levels. Thus, to facilitate studies of the
biology of SGLT1 function in epithelial monolayers, we engineered an
epitope-tagged construct containing the YTDIEMNRLGK sequence (from the
vesicular stomatitis virus G protein). The tag was placed at the
carboxyl terminus since this is the least conserved portion of SGLT1.
Transiently transfected COS-1 cells demonstrated surface expression of
the immunoreactive protein and enhanced Na
-dependent
glucose uptake that was phloridzin-sensitive (a specific competitive
inhibitor of SGLT1). However, subsequent detailed analyses of
epitope-tagged SGLT1 using stably transfected clones derived from the
Caco-2 human intestinal epithelial cell line revealed substantial
effects of the epitope on critical functions of SGLT1. When compared
with native SGLT1 transfectants, the apparent K
for sugar transport was increased 23-fold (313 µM to 7.37 mM for native versus epitope-tagged
SGLT1). In contrast, the apparent K
for
epitope-tagged SGLT1 was similar to that for native SGLT1.
Permeabilization studies indicated that the C-terminal epitope tag was
intracellular and thus could not directly disrupt extracellular
ligand-binding sites. Immunolocalization and functional assays designed
to detect polarized surface expression indicated that epitope tagging
resulted in loss of apical targeting and enrichment of basolateral
expression. Functional isolation of the small apical pool of
epitope-tagged SGLT1 (by selective inhibition of basolateral
epitope-tagged SGLT1) revealed that, despite the documented kinetic
alterations in sugar transport, epitope-tagged SGLT1 could promote
absorptive Na
currents. These data show that 1) the C
terminus of SGLT1 is intracellular; 2) disruption of protein structure
by addition of a C-terminal tag leads to selective modifications of
SGLT1 function; 3) the kinetics of sugar transport can be altered
independently of influences on the Na
-binding site of
SGLT1; and 4) the weak basolateral targeting sequence present within
the epitope tag is dominant over endogenous SGLT1 apical targeting
information and can direct polytopic membrane protein localization. The
data also caution that subtle effects of foreign sequences must be
considered when epitope tagging polytopic membrane proteins.
Our initial goal was to develop a system to study
Na-glucose transport across model intestinal
epithelia. Since the ability to analyze the biology of the intestinal
Na
-dependent glucose cotransporter (SGLT1) is limited
by the lack of high affinity antibodies for immunochemical-based
assays, we used the common approach of incorporating an exogenous
epitope into SGLT1, i.e. epitope tagging(1) . This
solution has been successfully applied to analyses of nonmembranous
proteins including cytoskeletal (2, 3) and cytosolic (4, 5) proteins. In contrast, membrane proteins,
particularly polytopic ones with relatively rigid conformational
requirements, might exhibit intolerance to epitope tagging. Such
epitope tags might interfere with synthesis, folding, trafficking, and
function of membrane proteins. To overcome these difficulties, the
epitope tag can be placed at sites considered least likely to interfere
with membrane protein function and global characteristics of the
epitope-tagged construct then assayed to assure retained function.
While reassuring, such assays may fail to detect subtle but critical
effects on essential biologic functions. Transport proteins are ideal
for assessing potential effects of epitope tagging on membrane proteins
since they are precisely targeted within polarized epithelia and
possess defined transport kinetics that can be measured quantitatively.
The Na-dependent glucose transporter (SGLT1) is
responsible for the bulk of intestinal glucose and Na
absorption(6, 7, 8) . SGLT1 is the
prototype of a family of Na
-coupled solute
cotransporters, of which several homologous
Na
-dependent nutrient transporters have been cloned
from intestine and kidney(9) . SGLT1 is apically polarized and
mediates Na
-glucose cotransport across the apical
membrane from the intestinal lumen. Subsequently, the basolateral
GLUT2-facilitated glucose transporter and
Na
-K
-ATPase mediate transport of
cytoplasmic glucose and Na
, respectively, across the
basolateral membrane into the interstitium(6) . The resulting
transepithelial transport of Na
can be recognized
electrically as a transepithelial short-circuit current (I
). (
)SGLT1, as originally cloned
from rabbit, corresponds to a 73-kDa 662-amino acid protein with at
least 11 putative transmembrane domains, an extramembranous N-terminal
tail, and an extramembranous C-terminal tail (6, 7, 8) . Analyses of SGLT1 in isolated
brush-border vesicles or SGLT1 expression by transfected Xenopus oocytes indicate that cotransport involves 2 Na
ions for each sugar molecule, with a corresponding binding site K
value of 0.11 mM (sugar) and a K
value of 32 meq/liter(10) . These
studies have been facilitated by the use of
-methyl glucoside
(
MG), a selective substrate for SGLT1 that accumulates within
cells, since it is neither transported by GLUT2 nor
metabolized(10, 11) . SGLT1-mediated sugar uptake can
be specifically inhibited by phloridzin(12) , thus permitting
an inhibitor-based assay of function as well.
Available data indicate that the N terminus of SGLT1 is exposed cytoplasmically, but the topology of the C-terminal tail remains controversial(7, 8, 9) . The N terminus of SGLT1 is highly conserved between species, and mutations in this region of SGLT1 lead to the clinical syndrome of glucose/galactose malabsorption(13, 14, 15) . In contrast, the C-terminal third of SGLT1 shows the greatest interspecies diversity(9) , appears to be resilient to mutation, and thus represents a potentially ideal site for epitope tagging.
To develop
a system to study Na-glucose transport across model
intestinal epithelia, we employed the Caco-2 cell line. This cell line
was selected since it grows as polarized monolayers, expresses and
correctly sorts hydrolases to the apical membrane, and represents a
model for studies of villus absorptive enterocytes, the subtype of
intestinal epithelial cell responsible for nutrient absorption (16, 17, 18, 19, 20) .
Since parent Caco-2 cell lines (like other available polarized
intestinal epithelial cell models) exhibit barely detectable levels of
transepithelial Na
-dependent glucose transport,
apparently due to an isolated deficiency of SGLT1 expression (21, 22) , we sought to stably transfect Caco-2 with
SGLT1. To facilitate immunochemical identification of SGLT1, we also
created stable transfectants expressing an epitope-tagged SGLT1
protein. For epitope tagging, we used an 11-amino acid epitope of the
vesicular stomatitis virus G protein (VSV-G) for which a well
characterized monoclonal antibody is available(23) . As
predicted, we found that C-terminal tagging by VSV-G did not interfere
with the ability of the protein to be surface-expressed or with its
ability to transport substrates (Na
and glucose) and
interact with specific inhibitors (phloridzin), criteria that
reasonably confirm that the tagged protein functions as the native one.
However, upon more detailed analyses, we found that this commonly used
epitope tag both altered protein targeting and selectively influenced
transport kinetics for one substrate. Nonetheless, apically expressed
epitope-tagged SGLT1 was capable of generating transepithelial
absorptive Na
currents. These results not only have
implications regarding SGLT1 polarization, transport function, and
topology-transport relationships, but also illustrate that subtle
effects of an apparently successful effort at epitope tagging can
disrupt biologic function. The data also illustrate critical pitfalls
in the use of epitope tags in situations where protein function cannot
be sensitively measured by independent means.
Measurement of polarized uptake in cultured monolayers was similar
to that described above, but [C]
MG was
alternatively placed either apically or basolaterally or on both sides
of the monolayer. Duplicate controls with 0.5 mM phloridzin
were performed for each of these conditions, and phloridzin-inhibitable
apical or basolateral uptake was calculated as a fraction of measured
total uptake.
Figure 1:
Functional and immunofluorescent
detection of epitope-tagged SGLT1 in transiently transfected COS-1
cells. A, transfection increased phloridzin-inhibitable
MG uptake substantially above that of mock-transfected COS-1
cells. Each bar represents the difference between the mean of
two independent measures of
MG uptake in the absence or presence
of phloridzin. B and C, both surface and
intracellular epitope-tagged SGLT1 were detectable by confocal
immunofluorescence with the anti-epitope monoclonal antibody
(immunofluorescence image (B) and phase contrast image (C)).
Figure 2:
A, first-order kinetics of sugar transport
in Caco-2 cells transfected with native or epitope-tagged SGLT1. Shown
is the concentration dependence of sugar transport. Native SGLT1
() displayed an apparent K
of 0.31
mM (R
= 0.96). In contrast, the
apparent K
for epitope-tagged SGLT1
(
) was 7.37 mM (R
= 0.96).
Each point represents an individual measurement of
MG
uptake after subtraction of mean nonspecific
MG uptake, i.e. uptake in the presence of phloridzin. Data were fit to
Michaelis-Menten first-order kinetics, J = v
([S]/([S] + K
)). B, Na
dependence of glucose transport in Caco-2 cells transfected with
native or epitope-tagged SGLT1. Both native and epitope-tagged SGLT1
display nearly identical dependence on extracellular
[Na
]. Apparent K
values of 43.2 and 69.8 meq/liter were calculated for native
SGLT1 (
) and epitope-tagged SGLT1 (
), respectively. Hill
coefficients were 1.96 and 1.86 for native SGLT1 (R
= 0.87) and epitope-tagged SGLT1 (R
= 0.98), respectively. Each point represents an
individual measurement of
MG uptake after subtraction of mean
nonspecific
MG uptake, i.e. uptake in the presence of
phloridzin. Data were fit to the Hill equation, J = J
[Na]
/([Na]
+ K
).
As expected, uptake of MG by both native and epitope-tagged
SGLT1 was dependent on the extracellular Na
concentration (Fig. 2B). The data fit to the Hill
equation, J = J
[Na]
/(K
+ [Na]
), with apparent K
values of 43.2 and 69.8 meq/liter for native
and epitope-tagged SGLT1, respectively. These data are consistent with
the reported value of 32 meq/liter for SGLT1 expressed in Xenopus oocytes(10) . Hill coefficients of 1.96 and 1.86 were
obtained for native SGLT1 (R
= 0.87) and
epitope-tagged SGLT1 (R
= 0.98),
respectively, similar to the reported Hill coefficient for SGLT1
expressed in Xenopus oocytes(10) . These data are in
agreement with previous reports analyzing native SGLT1 and also
indicate that, like native SGLT1, epitope-tagged SGLT1 appears to
contain two Na
-binding sites(6, 27) .
Thus, in contrast to sugar transport, Na
transport
appears to be unaffected by the C-terminal epitope tag.
To further
evaluate the effect of the epitope tag on sugar transport, the
substrate specificities of native and epitope-tagged SGLT1 were
compared by competition with similar monosaccharides. The rank order of
sugar specificity for both native and epitope-tagged SGLT1 is D-glucose > D-galactose > MG >
3-O-methylglucose
L-glucose, mannitol (Fig. 3). This is consistent with previous reports describing
sugar specificities for native SGLT1(10) . However, the degrees
of inhibition of
MG uptake by the competing sugars D-glucose, galactose, and 3-O-methylglucose were
significantly less with epitope-tagged SGLT1 than with native SGLT1 (Fig. 3). Taken together, these data show that the addition of
the epitope tag to the C terminus of SGLT1 alters protein conformation
in a subtle yet functionally important fashion.
Figure 3:
Specificity of sugar transport by native
and epitope-tagged SGLT1. To evaluate the substrate specificities of
native and epitope-tagged SGLT1, uptake of 100 µM MG
in the presence of 10 mM competing sugar was performed. The
concentration of phloridzin was 0.5 mM. Each measurement
represents the mean ± S.D. of two independent measurements. Data
were normalized to uptake in the absence of competing sugars (Control).
Figure 4: The epitope tag is localized to the cytoplasmic face of the plasma membrane. Caco-2 cells transfected with epitope-tagged SGLT1 were grown on coverslips and fixed with paraformaldehyde. Some preparations were then permeabilized with acetone or Triton X-100. Immunostaining for the viral epitope demonstrates inaccessibility of the epitope to extracellular antibody in nonpermeabilized cells (B) relative to permeabilized cells (A) and verifies that the epitope tag is intracellular (cytoplasmic). Confocal xy-plane sections through the mid-portion of the cells are shown. Both surface membrane (also shown in Fig. 5) and cytoplasmic staining are apparent.
Figure 5:
Maintenance of apical and basolateral
membrane domains in Caco-2 cells transfected with epitope-tagged SGLT1.
Caco-2 cells transfected with epitope-tagged SGLT1 were grown on
permeable supports, fixed, permeabilized, and immunostained for the
VSV-G epitope tag (A), ZO-1 (B), 5`-nucleotidase (C), or Na-K
-ATPase (D). Confocal xz-plane images are
shown.
To quantitatively analyze the apical and
basolateral expression of epitope-tagged SGLT1 and to directly compare
this expression with that of native SGLT1, apical and basolateral
phloridzin-inhibitable MG uptake were evaluated in polarized
monolayers expressing native or epitope-tagged SGLT1 (Fig. 6).
Two separate clones transfected independently with native SGLT1
exhibited apically polarized phloridzin-inhibitable uptake. In
contrast, two clones transfected independently with epitope-tagged
SGLT1 exhibited predominantly basolateral uptake. These functional data
provide direct evidence that the VSV-G epitope tag results in
disruption of the steady-state distribution of SGLT1 from predominantly
apical to predominantly basolateral membrane domains.
Figure 6:
Polarity of SGLT1-mediated sugar transport
in Caco-2 cells transfected with native or epitope-tagged SGLT1. To
evaluate the polarity of functional SGLT1 expression, transfected cells
were grown on permeable supports. Uptake of MG was measured
following incubation with apical, basolateral, or apical and
basolateral
MG. Uptake of
MG in the presence of phloridzin
was also measured under each of these conditions.
Phloridzin-inhibitable uptake of
MG applied only to the apical or
basolateral surface was normalized to phloridzin-inhibitable uptake of
MG applied to apical and basolateral surfaces. Data are the means
± S.D. of two monolayers for each
condition.
Although the
majority of epitope-tagged SGLT1 is expressed basolaterally, both
functional and morphologic assays show that apical expression still
occurs. Nonetheless, a glucose-induced phloridzin-inhibitable I was not detectable. One possibility to explain
such data is that apically expressed epitope-tagged SGLT1 is unable to
sustain sufficient Na
transport to generate a
measurable I
, perhaps due to the subtle effects
of the epitope tag on sugar transport. Alternatively, in the presence
of actively transporting basolateral epitope-tagged SGLT1,
Na
cycling across the basolateral membrane (uptake via
basolateral epitope-tagged SGLT1, exit via basolateral
Na
-K
-ATPase) might be substantial
enough to interfere with detection of transepithelial Na
movement. To differentiate these possibilities, basolateral
epitope-tagged SGLT1 was selectively inhibited by the addition of
basolateral phloridzin, thus blocking the basolateral Na
recycling described above and affording the opportunity to
functionally isolate apical epitope-tagged SGLT1. Under these assay
conditions, monolayers of epitope-tagged SGLT1 transfectants were able
to generate glucose-dependent phloridzin-inhibitable I
(Table 1, part B). Thus, when basolateral epitope-tagged
SGLT1 is inhibited, apical epitope-tagged SGLT1 can initiate
phloridzin-inhibitable I
. These data indicate
that epitope-tagged SGLT1, if appropriately polarized, can sustain a
transepithelial absorptive Na
current despite
perturbations in the kinetics of sugar transport.
The aim of this work was to create an epitope-tagged construct of a polytopic transport protein, SGLT1. Since the C terminus is not highly conserved(9) , it seemed a logical location for placement of an epitope tag. Initial studies of epitope-tagged SGLT1 using common approaches confirmed surface expression, global transport function, and sensitivity to specific inhibitors. These data suggested that epitope tagging was successful, and we proceeded with stable transfection into polarized epithelial cells. Studies of stable transfectants showed that, despite the initial characterization, two apparently unrelated functional characteristics of the transporter were disrupted by the epitope tag. These subtle biochemical changes resulted in significant alterations of biologic function.
We initially observed that
polarized monolayers of Caco-2 cells transfected with epitope-tagged
SGLT1 did not generate phloridzin-inhibitable I.
We hypothesized that this might be due to missorting of epitope-tagged
SGLT1 and demonstrated both functionally and morphologically that
epitope-tagged SGLT1 is missorted. We then sought to verify that
missorting, and not the altered K
, was responsible
for the failure of monolayers expressing epitope-tagged SGLT1 to
generate phloridzin-inhibitable I
. Monolayers
were created with apically polarized expression of epitope-tagged SGLT1
through use of the monolayer-impermeant SGLT1 inhibitor phloridzin.
Addition of phloridzin to the basolateral medium selectively inhibited
basolateral epitope-tagged SGLT1. Under these conditions, monolayers
expressing epitope-tagged SGLT1 were able to generate glucose-dependent I
inhibitable by apical phloridzin. This result
confirms that missorting, and not altered K
,
explains the initial inability of epitope-tagged SGLT1 to generate
phloridzin-inhibitable I
.
On the basis of
sequence homology, sites including the extracellular loop that
separates the third and fourth membrane-spanning domains,
membrane-spanning region 5, and the segment between membrane-spanning
regions 9 and 11 have been suggested as possibly involved in solute, e.g. glucose, binding(8) . In contrast, the current
studies suggest that modifications of the C terminus influence both the
kinetics and hierarchy of sugar binding, without affecting the kinetics
or stoichiometry of Na binding. These observations
indicate that the tertiary structure that determines sugar transport
can be influenced independently of the Na
-binding site
and raise the possibility that determinants of the sugar transport site
may reside preferentially in the C terminus, while those determining
Na
binding may reside preferentially in an extended
N-terminal domain.
The basolateral targeting sequences characterized to date are all derived from monotopic proteins(32, 33, 34, 35, 36) . Assays characterizing these sequences have also depended on monotopic proteins, e.g. hemagglutinin. Thus, the ability of these sequences to accurately target polytopic proteins has not been previously analyzed. Our data demonstrating the ability of the VSV-G sequence to target SGLT1 to the basolateral membrane show that basolateral targeting sequences from monotopic proteins can direct targeting of polytopic proteins. Furthermore, since the full-length SGLT1 protein is present within the epitope-tagged construct, the data also show that the VSV-G basolateral sequence is dominant over endogenous apical targeting information. The identity of endogenous apical targeting information within SGLT1 is not known. It is possible that the SGLT1 apical targeting information is contained within an extracellular (luminal) domain, as has been suggested for dipeptidyl peptidase IV(35) .