Human lactase-phlorizin hydrolase (LPH) is a
digestive enzyme that is expressed in the small intestinal brush-border
membrane. After terminal glycosylation in the Golgi apparatus, the
230-kDa pro-LPH is cleaved into the 160-kDa brush-border LPH
and the 100-kDa profragment (LPH
). Since LPH
is not transport-competent when it is expressed separately from LPH
in COS-1 cells, it was suggested that LPH
functions as an intramolecular chaperone. What
happens to LPH
after cleavage is still unclear.
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INTRODUCTION |
Human lactase-phlorizin hydrolase
(LPH)1 is a disaccharidase
that is localized at the microvillar membrane of epithelial cells in
the small intestine. It is responsible for the hydrolysis of lactose,
the main carbohydrate in mammalian milk.
LPH is synthesized as a 1927 amino acid precursor, prepro-LPH. The
first 19 amino acids of this precursor form the signal sequence that is
cleaved off in the endoplasmic reticulum. The remaining 1908 amino
acids form the pro-LPH, which is complex glycosylated on its way
through the Golgi apparatus. A subsequent cleavage between Arg-734 and
Leu-735 (1) takes place after export from the trans-Golgi
and results in the 160-kDa LPH
and a stretch that is called the
profragment or LPH
(2). LPH
is expressed at the apical membrane,
where it is trimmed by luminal trypsin in the small intestine to the
intestinal form of LPH
. In contrast to LPH
, the fate of LPH
after cleavage is not clear. In Fig. 1,
the processing of LPH is illustrated.

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Fig. 1.
Cartoon of the processing of LPH. LPH is
synthesized as prepro-LPH. Removal of the signal sequence results in
pro-LPH. After complex glycosylation in the Golgi apparatus, pro-LPH is cleaved into LPH (profragment) and LPH (mature LPH). LPH is expressed at the brush border, but the fate of LPH remains unknown. In intestinal cells, LPH is further trimmed by luminal proteases to
intestinal LPH .
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Prepro-LPH consists of four homologous regions between the signal
peptide and the hydrophobic stretch at the C terminus. In Fig.
2, prepro-LPH is schematically drawn. The
first two regions, I and II, are localized in LPH
. None of them
possess the Glu residue that was shown to be essential for the active
sites (3). Indeed, LPH
is enzymatically not active toward lactose
when expressed separately in COS cells or isolated after trypsin
cleavage of the pro-LPH expressed in COS cells (4). Regions III and IV both contain the Glu residue and are responsible for the disaccharidase activity of LPH
(3). Because of the homology between LPH
and
LPH
, and because LPH
comprises 40% of the primary translation product, various functions have been suggested for LPH
(2, 5).

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Fig. 2.
Schematic overview of several structural
features in LPH. The encircled Glu residues depict the essential
residues in the active sites. The homologous regions are denoted by
roman numerals. The introduction site of the VSV tag is
depicted as well. The / cleavage site is located at
Arg-734/Leu-735, intestinal LPH has Ala-869 at its N terminus.
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LPH
has previously been isolated from intestinal biopsy samples
using an antibody directed against the 12 amino acids directly after
the signal peptide (Ser-20-Thr-31) (2). The molecular mass of the
immunoprecipitated profragment in these experiments was ~100 kDa (2).
Because none of the used glucosaminidases (endo-N-acetylglucosaminidase H (endo H) and
endo-N-acetylglucosaminidase F/glycopeptidase F (endo F/GF))
influenced the apparent molecular weight, it was concluded that the 5 consensus N-glycosylation sites were not glycosylated (2),
although some conflicting data exist (1). In addition it was shown that
LPH
does not form a stable complex with LPH
after cleavage in
intestinal biopsy specimens (2). These observations and further
expression studies of the intestinal form of LPH
(from Ala-869 to
the C terminus) in COS-1 cells lead to the hypothesis that LPH
functions as an intramolecular chaperone. Where exactly in the cell the
cleavage takes places as well as what happens to LPH
after cleavage
is still uncertain despite extensive published data (2, 5).
To track down LPH
after cleavage, we decided to express and analyze
LPH in Caco-2 cells. These cells are able to express LPH endogenously,
and to perform the cleavage between the
and
domain of pro-LPH
(6). Furthermore, we introduced a VSV tag into LPH
to generate
additional recognition possibilities. Immunoprecipitation and
localization studies were performed in Caco-2 cells that were stably
transfected with wild type and VSV-tagged LPH cDNA. The results
strongly suggest that the LPH
profragment is immediately degraded
after cleavage in Caco-2 cells and therefore argue in favor of the
intramolecular chaperone function.
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EXPERIMENTAL PROCEDURES |
Antibodies--
HBB1/909 is an epitope-specific monoclonal
antibody directed against LPH (6). This product of the hybridoma
HBB1/909/34/74 was provided by Hans-Peter Hauri, Biozentrum der
Universität Basel, Switzerland. V496 is a polyclonal antiserum
directed against the first 12 amino acids following the signal sequence
of prepro-LPH (2). P5D4 is a monoclonal antibody against a specific
epitope in VSV-G-protein (7), the hybridoma was provided by Thomas Kreis, Dept. of Cell Biology, University of Geneva, Switzerland.
DNA Constructs--
To insert a VSV epitope tag into the LPH
cDNA, the LPH cDNA (8) was cloned into a pBluescript KS+ vector
in which the SstI site was removed. This construct was
digested with SstI, blunted with T4 DNA polymerase (New
England Biolabs, Beverly, MA) and treated with calf intestinal
phosphatase (Boehringer Mannheim BV, Almere, The Netherlands). For the
construction of a 43-base pair VSV-tag insert, coding for 11 amino
acids of the epitope, two oligonucleotides
(5'-GGAGATCTTATACAGACATAGAGATGAA-3' and
5'-GGGGGATCCCTTTCCAAGTCGGTTCATCTCTATGTCTGTA-3') were annealed and
treated with Klenow (Boehringer Mannheim BV) in the presence of dNTPs
to fill in the 5' overhangs. The double-stranded product was digested
with BamHI and BglII, and the 5' overhangs were
filled in by Klenow treatment. This blunted
BamHI/BglII insert was ligated into the blunted
SstI-digested LPH cDNA so that the reading frame
remained intact. The orientation of the insert was determined by
sequencing. Fig. 3 depicts the sequence
of the insert and the flanking LPH sequences, as well as the amino acid
sequence of the wild type LPH and the tag insert in this region. This
construct, denoted LPHST, was cloned into a modified pSG5 (9)
expression vector containing a puromycin resistance cassette (pSGpuro),
which resulted in the pLPHST plasmid. Wild type LPH cDNA was cloned into pSGpuro as well (pLPHwt).

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Fig. 3.
Alignment of the amino acid and DNA sequences
of wtLPH and LPHST around the introduction site of the VSV tag.
Lines connect the corresponding amino acids. The box is drawn around the VSV tag sequence, which is shown in boldface type. Base
pair numbering is given above the sequence, and amino acid numbering is
given below.
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To generate the pSGpuro vector the SalI fragment of the
pKSpuro vector, which was kindly provided by Peter Laird (University of
Southern California School of Medicine, Norris Comprehensive Cancer
Center, Los Angeles, CA) (10) was inserted into the partially SalI digested pSG8 (a pSG5 vector (9) with a multiple
cloning site).
Cell Lines--
Monkey kidney COS cells (ATCC CRL-1650) were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum and antibiotics. Caco-2 TC7 and PD10 cells (a
gift from Dr. Monique Rousset, Ref. 11) were cultured in Dulbecco's modified Eagle's medium supplemented with 20% heat-inactivated fetal
calf serum, 1% nonessential amino acids and antibiotics (all from Life
Technologies, Inc.). All cell lines were cultured at 37 °C in a
humidified 5% CO2 incubator.
Transfection and Selection--
COS-1 cells were transfected via
electroporation as described before (12). Caco-2 cells were transfected
by LipofectAMINE treatment. One or two days prior to transfection,
0.2 × 106 Caco-2 TC7 cells were seeded per 35-mm
culture dish, so that on the day of transfection the culture was
60-80% confluent but not polarized. During the whole transfection
procedure, no antibiotics were used. On the day of transfection, cells
were washed twice with OptiMEM (Life Technologies, Inc.). Solution A,
consisting of 2 µg of linearized DNA in 200 µl of OptiMEM, was
gently mixed with solution B, consisting of 6 µl of LipofectAMINE
(Life Technologies, Inc.) and 200 µl of OptiMEM. The mixture was
incubated at room temperature for 30 min, and 1600 µl of OptiMEM was
added. The resulting 2 ml. was added to the cells and incubated at
37 °C and 5% CO2 for 6 h. Cells were washed with
normal Caco-2 medium and incubated in this medium for 24 h,
refreshed, and incubated for another 24 h. Cells were trypsinized
and seeded into 96-well plates at a density of 5 × 103 cells per well. After 24 h, selection medium was
added, which consisted of normal Caco-2 medium with an (empirically
determined) puromycin concentration of 13 µg/ml. After 1 week, wells
with only one colony were selected. After another week, these
colonies were trypsinized and cultured further in selection medium.
Metabolic Labeling and Immunoprecipitation--
Caco-2 cells or
transiently transfected COS-1 cells were metabolically labeled with 100 µCi Tran35S-label (ICN Biomedicals) as described by Naim
et al. (8). After the labeling period, the cells were
scraped in lysis buffer (1% Triton X-100, 0.2% bovine serum albumin
in 100 mM phosphate buffer, pH 8.0, containing 1 tablet of
complete protease inhibitor mixture (Boehringer Mannheim BV) per 25 ml)
and lysed at 4 °C for 1 h. For COS cells usually 1 ml of
ice-cold lysis buffer was used for each 100-mm culture dish (about
2-4 × 106 cells). For Caco-2 cells, 0.5 ml was used
per filter. Lysates were stored at
135 °C until use. Detergent
extracts of cells were centrifuged for 1 h at 100,000 × g at 4 °C and the supernatants were immunoprecipitated as
described by Schweizer et al. (13).
SDS-PAGE--
SDS-PAGE was performed according to Laemmli (14),
and the apparent molecular weights were assessed by comparison with
high molecular weight markers (Bio-Rad) run on the same gel. In some experiments, deglycosylation of the immunoprecipitates with endo H and
endo F/GF (also known as PNGase F) (both from New England Biolabs) was
performed prior to SDS-PAGE analysis as described before (15).
Trypsin Sensitivity Assay--
The sensitivity for proteatic
digestion of LPH and LPHST was compared using a trypsin sensitivity
assay. Transfected COS-1 cells were metabolically labeled and lysed.
LPH and LPHST were immunoprecipitated (see above) using the HBB1/909
antibody. Precipitates were divided into several fractions and treated
with 50 µg/ml trypsin at 37 °C for the indicated incubation times.
Samples were subjected to SDS-PAGE on a 8% gel that was analyzed by
fluorography.
Enzyme Activities--
Disaccharidase activities of
immunoprecipitated LPHs were measured according to Dahlqvist (16) using
lactose as a substrate. The method was essentially the same as
described by Naim et al. (8). LPH was immunoprecipitated
using the HBB1/909 antibody from lysates of 165-cm2 culture
flasks of (transfected) Caco-2 cells. The precipitates were dissolved
in 2 ml of phosphate-buffered saline containing 0.3% Triton X-100 of
which 25-µl aliquots were used for the enzyme activity assay. The
amount of precipitated LPH was estimated in 500-, 300-, 200-, and
100-µl aliquots on an SDS-PAGE gel with a bovine serum albumin
concentration standard series run on the same gel; the gel was stained
with Coomassie Brilliant Blue.
Immunofluorescence and Confocal Microscopy--
Cellular
localization of expressed proteins in COS-1 cells and Caco-2 cells was
studied with cells grown on coverslips. Cells were fixed with 3%
paraformaldehyde and permeabilized with 0.1% Triton X-100.
Immunolabeling was carried out using (as primary antibodies) monoclonal
antibodies HBB1/909 against human LPH, P5D4 against the VSV epitope
tag, and the polyclonal antibody V496 against the 12 amino acids
directly after the signal sequence of pro-LPH. The secondary antibodies
employed fluorescein isothiocyanate-conjugated goat anti-mouse or swine
anti-rabbit IgG, Texas Red-conjugated goat anti-mouse or anti-rabbit
IgG (all were from Boehringer Mannheim BV). Surface localization of
proteins was assessed in transfected cells that were not fixed nor
permeabilized. Labeling was carried out at 4 °C. Label was
visualized using a Bio-Rad MRC1000 confocal scanning laser microscope
using a double channel for fluorescein isothiocyanate and Texas Red or
on a routine fluorescence microscope.
Immunoelectron Microscopy--
Ultrastructural localization
studies were performed on transfected Caco-2 TC7 clones grown on a
filter that had been confluent for 5 days. They were fixed with 1%
paraformaldehyde and 0.1% glutaraldehyde in phosphate buffer (pH 7.3)
for 1 h and stored until use in 1% paraformaldehyde. Filters were
stacked in 10% gelatin and fixed in 1% paraformaldehyde for 24 h. Ultrathin cryosectioning was performed as described before (13, 17).
Sections were incubated with the monoclonal antibodies HBB1/909 against
human LPH or P5D4 against the VSV epitope tag, followed by a rabbit polyclonal serum against mouse IgG (Dako A/S, Glostrup, Denmark) and
protein A complexed with 10 nm gold (17). Electron microscopy was
performed with a JEOL 1010 electron microscope.
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RESULTS AND DISCUSSION |
Processing of wtLPH and LPHST in COS-1--
To examine the
influence of the introduction of the VSV tag into LPH, the processing,
localization, and protease sensitivity of both wtLPH and LPHST were
compared in COS-1 cells. Immunoprecipitations using the HBB1/909
antibody directed against pro-LPH and mature LPH resulted in
precipitation of high mannose (~215 kDa) and complex glycosylated
(~230 kDa) LPH (Fig. 4, first
four lanes). No mature form (~160 kDa) could be observed, due to
the absence in these cells of the protease that is responsible for the
/
cleavage (1, 2, 8, 18). However, both LPHST and wtLPH were
present at the cell surface as shown by immunofluorescence labeling
(see below). The results clearly show that introduction of the VSV tag
did not affect the transport competence of the mutant.

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Fig. 4.
Immunoprecipitation of wtLPH and LPHST from
transfected COS-1 cells using the antibodies HBB1/909, P5D4, and
V496. Transfected COS-1 cells were metabolically labeled with
Tran35S-label for 1 h, and chased for 0 or 4 h.
The aliquots were divided and precipitated with the indicated
antibodies. Samples were analyzed by SDS-PAGE followed by fluorography.
In the P5D4 lane 0, a faint background band is visible.
Neither pro-LPHwt nor pro-LPHST are cleaved.
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P5D4, the antibody against the VSV epitope tag, did not recognize wild
type pro-LPH but did precipitate the tagged LPH construct LPHST (Fig.
4, middle lanes). The V496 antibody directed against an
epitope of LPH
precipitated both constructs (Fig. 4, last four
lanes). The pro-LPH species precipitated by the V496 antibody corresponds to the mannose-rich precursor as was determined by endo H
treatment of the precipitates (not shown).
Trypsin Sensitivity Assay--
To compare sensitivities toward
protease treatment of both LPH and LPHST proteins, they were
precipitated from transfected COS-1 cells by the HBB1/909 antibody
against LPH. The precipitates were treated for several time intervals
with trypsin. In Fig. 5 is shown that
insertion of the tag did not alter the sensitivity toward trypsin since
both proteins show exactly the same pattern. Trypsin treatment results
already after 75 s in a complete cleavage of the high mannose and
complex forms of wtLPH and LPHST into two bands of around 140 kDa that
remain trypsin insensitive for at least 1 h.

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Fig. 5.
Immunoprecipitation of LPH and LPHST from
three Caco-2 clones: WT2 and ST54, two clones transfected with wtLPH
and LPHST, respectively, and PD10, which expresses LPH
endogenously. Confluent Caco-2 clones were metabolically labeled
for 1 h, and chased for 0, 4, and 24 h. LPH was
immunoprecipitated from lysates using the HBB1/909 monoclonal antibody.
Precipitates were treated with endo H (+ lanes) or left
untreated ( lanes). Precipitates were analyzed by SDS-PAGE
followed by fluorography. The processing pattern of transfected wtLPH
and LPHST is essentially the same as the pattern of endogenous LPH. In
the ST54 lanes, a persistent, endo H insensitive background band is
marked with an asterisk (*). This band is also observed in
immunoprecipitations of other proteins (sucrase-isomaltase, DPPIV) from
Caco-2 clones, and is also faintly visible in lanes 1 and
7.
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After 75 s, a band of about 100 kDa can be observed that gradually
disappears after 15 min. In the same time, a band of about 69 kDa
appears. Most likely these bands comprise LPH
as proposed earlier
(1, 2).
Since differently folded molecules are expected to behave differently
toward the same protease, in this case trypsin, the results lend strong
support to the notion that the folding of LPH and LPHST in COS-1 cells
is similar.
Processing and Enzymatic Activity of wtLPH and LPHST in
Caco-2--
A Caco-2 clone with an undetectable endogenous LPH
expression (TC7) (11) was transfected with the pLPHST and pLPHwt
constructs. Stable transfectants were selected with puromycin and
screened using immunofluorescence microscopy with antibodies directed
against LPH
. From each transfection, the clone with the highest
expression was chosen for further studies. For the LPHST construct,
this clone is called ST54, the wtLPH clone is WT2. As a control, a clone with an endogenous LPH expression (PD10) (11) was used.
Immunoprecipitation of pro-LPH and mature LPH from pulse-chased WT2
cells shows that LPH is synthesized as a 215-kDa single-chain polypeptide (Fig. 6, lanes 1 and 2), which becomes complex glycosylated to 230 kDa and
therefore endo H insensitive after 4 h of chase (lanes
3 and 4). At this point, LPH
appears as a faint band
of 160 kDa. This band is more intense after 24 h of chase when the high mannose form has disappeared (lanes 5 and
6). This processing pattern matches the pattern observed in
PD10 cells (lanes 13-18). In intestinal explants, the
processing appears to be somewhat faster (2) but is essentially the
same.

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Fig. 6.
Trypsin sensitivity assay of LPH and LPHST
expressed in COS-1 cells. Transfected COS-1 cells were labeled
with Tran35S-label for 4 h. LPH was precipitated using
the HBB1/909 antibody. Precipitates were treated with 50 µg/ml
trypsin for the indicated time intervals and analyzed by SDS-PAGE
followed by fluorography.
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Introduction of a VSV tag in LPH did not influence the processing since
LPHST was processed in ST54 cells (Fig. 6, lanes 7-12) in a
comparable fashion to wild type LPH in both WT2 and PD10 cells.
Sometimes a band of about 180 kDa is observed in Fig. 6 (lanes
1, 3, 7, 9, 11, and
13). This is a nonspecific background band that can be
observed in other immunoprecipitations as well, like with
anti-sucrase-isomaltase antibodies from not only ST54 but from
other Caco-2 cells as well (data not shown).
The enzymatic activity of immunoprecipitated lactase from a
165-cm2 culture flask wtLPH or LPHST transfected Caco-2
cells was determined. The lactase precipitated from both cell lines was
capable of cleaving 59 and 61 nmol of lactose per minute, respectively.
The protein quantities as determined by Coomassie staining of an
SDS-PAGE gel with the immunoprecipitates were essentially the same.
Therefore we conclude that both constructs display comparable lactase
activities and that the introduction of the tag has no influence on
this activity.
Immunoprecipitation of LPH
--
V496 is an antibody directed
against the 12 amino acids following the signal sequence of pro-LPH
(2). In previous studies, a 100-kDa band could be observed after
immunoprecipitations from intestinal explants using this antibody,
which was suspected to be LPH
(2). A band of this size was never
observed in immunoprecipitation studies from Caco-2 clones in this
study. Only the high mannose pro-LPH could be precipitated by the V496
antibody from both Caco-2 WT2 and ST54 cells. No complex glycosylated
bands were observed even after a chase period of 24 h (not shown).
Apart from this high mannose band, no other specific band was visible.
This lower affinity of V496 for complex glycosylated LPH was found in
COS-1 cells as well.
P5D4 did not precipitate any specific protein from WT2 cells but
precipitated both high-mannose and complex glycosylated form from ST54
cells (not shown). No other specific band could be found. Overall no
specific LPH
band could be precipitated by the different antibodies
and from the different cellular models.
Localization of wtLPH and LPHST in COS-1 Cells--
HBB1/909 is a
monoclonal antibody that recognizes pro-LPH and mature LPH (6). In
immunofluorescence studies on permeabilized wtLPH and LPHST-transfected
COS-1 cells, this antibody labeled the cell surface and an
intracellular network, probably the endoplasmic reticulum and the Golgi
apparatus (Fig. 7, panel A and
D). On nonpermeabilized cells, only the surface was labeled
(not shown). The V496 antibody labeled an intracellular network,
probably the endoplasmic reticulum and the endoplasmic reticulum-Golgi
intermediate compartment (Fig. 7, panel B and E).
On nonpermeabilized cells, no labeling could be observed (not shown),
indicating that V496 was not able to label LPH at the cell surface. By
contrast, P5D4 did not only show labeling of an intracellular network
but also surface labeling on permeabilized cells that were transfected with LPHST (Fig. 7, panel F). P5D4 was also able to label
the cell surface of nonpermeabilized LPHST cells (not shown). P5D4 but
not V496 recognizes complex glycosylated pro-LPHST, indicating that
only complex glycosylated LPHST reaches the cell surface, as was
already shown by Sterchi et al. (1). Cells transfected with
wtLPH were not labeled by P5D4 (Fig. 7, panel C).

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Fig. 7.
Immunofluorescence microscopy of
permeabilized COS-1 (panels A-F) and Caco-2 cells
(panel G-L), transfected with wtLPH (panels A, B, C,
G, H, and I) or LPHST (panels D, E, F, J,
K, and L) using antibodies against pro-LPH and mature
LPH (HBB1/909), LPH (V496), or the VSV epitope tag (P5D4).
Bar, 25 µm.
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Localization of wtLPH and LPHST in Caco-2 Cells--
In
immunofluorescence studies using HBB1/909 on permeabilized 5 day
confluent Caco-2 WT2 and ST54 cells, a patchy labeling pattern was
visible on some clusters of cells, whereas others did not show label
(Fig. 7, panel G and J). After subcloning using a
limiting dilution protocol, the same distribution was observed (not
shown). A comparable mosaic expression has been described for in
vivo expression of LPH and for other transfections in Caco-2 cells
before (11, 19). Labeling of nonpermeabilized cells also showed a
patchy labeling pattern, indicating that mainly the cell surface was
labeled (Fig. 8, panel A and
D). This surface was shown to be the apical membrane by
confocal microscopy (Fig. 9).

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Fig. 8.
Immunofluorescence microscopy of
nonpermeabilized Caco-2 WT2 (A and B) and ST54
(C and D) cells labeled with HBB1/909
(A and C) or P5D4 (B and
D). Bar, 25 µm.
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Fig. 9.
Confocal microscopy on permeabilized Caco-2
LPHST cells showed that only the apical surface of the cells was
labeled. LPH was labeled with HBB1/909 and rabbit
anti-mouse-fluorescein isothiocyanate, and the nuclei were stained
using propidium iodide. On the line across panel
A, a vertical section was made that is represented in panel
B. Bar, 25 µm.
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Immunoelectron microscopy studies on WT2 and ST54 cells confirmed the
immunofluorescence data. Some cells did not have any label, whereas
others were clearly labeled the brush border (Fig. 10). At the basolateral membrane (Fig.
10, arrow), no labeling was observed. Some gold particles
could be found in the endoplasmic reticulum. The labeling pattern of
WT2 (Fig. 10, panel A) and ST54 (panel B) was
essentially the same.

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Fig. 10.
Electron microscopy on Caco-2 wtLPH
(A) and LPHST (B) cells shows immunogold
staining at the brush border (BB). No labeling could
be detected at the basolateral membrane (arrows) Bar, 200 nm.
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Despite the fact that V496 labeled some intracellular structures in
transfected COS-1 cells in immunolocalization studies, no labeling
could be found under the same conditions in transfected Caco-2 cells
(Fig. 7, panels H and K). This is a consequence
of the differences in the steady state in COS-1 and Caco-2 cells. In
transiently transfected COS-1 cells, the protein synthesis level is
much higher then in stably transfected Caco-2. Therefore relatively
more LPH in COS-1 cells is present in its high mannose glycosylated
form, and recognized by V496. P5D4 against the VSV tag was able to show
a faint label at the surface of a few (less then 0.1%) Caco-2 cells
expressing tagged LPH (Fig. 7, panel L; Fig. 8, panel
D). This surface staining is most likely labeling of complex
glycosylated pro-LPH, which could be precipitated in surface
immunoprecipitation experiments on Caco-2 ST54 and WT2 cells using the
HBB1/909 antibody (Fig. 11). From the
apical membrane, LPH
and complex glycosylated pro-LPHST could be
precipitated (lane 1). From the basolateral membrane, some
pro-LPHST but not LPH
could be precipitated (lane 2).
Expression of pro-LPH at the cell surface has been found before in
biopsy samples (20). In transfected Madin-Darby canine kidney cells, it
was found at both membrane domains as well (21, 22). The ultimate fate of pro-LPH on the basolateral membrane, degradation, or transport to
the brush border needs further analysis.

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Fig. 11.
Surface immunoprecipitation of Caco-2 ST54
cells. Confluent Caco-2 ST54 cells on filter were metabolically
labeled for 24 h. At 4 °C, HBB1/909 was added to the
basolateral or the apical compartment of the filter. Cells were lysed,
and the antibody-bound LPH was precipitated. The immunoprecipitates
were analyzed on SDS-PAGE followed by fluorography. At the apical
membrane (Ap), complex glycosylated pro-LPH can be found as
well as LPH (mature). At the basolateral side
(Bl), only complex glycosylated pro-LPH is present. WT2
cells showed essentially the same pattern (not shown).
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Concluding Remarks--
Many proteins undergo proteolytic
processing after translation. Among these proteins are lysosomal (23),
secretory (24, 25), and some plasma membrane proteins. Most
disaccharidases are plasma membrane proteins that are cleaved into
their subunits (26). LPH is an example of a disaccharidase that
undergoes proteolytic cleavage. Unlike other examples LPH has a
C-terminal instead of a N-terminal transmembrane region, and both its
active sites are located on one cleavage product. Furthermore, it
consists of four homologous regions instead of two, which are probably
derived from a double gene duplication (20). Two of these regions, III and IV, contain the active sites and are both localized on the same
cleavage product, LPH
, which is expressed on the brush-border membrane. The second cleavage product, LPH
, possesses the other two
homologous regions, I and II, but does not show any activity toward
disaccharides (2, 4). Two separate studies have reported a role for
LPH
as an intramolecular chaperone (2, 5). An additional function
could be hypothesized because of the internal homologies and because
LPH
is relatively large (714 residues). Therefore we developed a
model in which we could study LPH
in more detail. This model
consists of Caco-2 cells expressing wild type or modified LPH
containing a VSV epitope tag in its LPH
domain for additional
recognition possibilities. Immunoprecipitation studies from these cells
using both an antibody against LPH
and an antibody against the
LPH
-inserted tag did not result in a specific profragment band.
Furthermore, localization studies in Caco-2 cells did not result in
specific labeling in any LPH-positive cell. We suggest that in Caco-2
cells LPH
is degraded soon after cleavage since no intra- or
extracellular accumulation could be observed. Our results do not
ascertain whether degradation of LPH
occurs on its way to the brush
border or at the brush border itself.