(Received for publication, April 21, 1995)
From the
The folding of human intestinal prolactase-phlorizin hydrolase
(pro-LPH) has been analyzed in a cell-free transcription/translation
system. In the presence of the thiol oxidant GSSG, disulfide bond
formation in pro-LPH can be promoted concomitant with the binding of
the molecule to a conformation-specific monoclonal anti-LPH antibody.
Under these conditions, pro-LPH does not bind to the molecular
chaperone BiP. In the absence of GSSG, on the other hand, pro-LPH does
not bind to the monoclonal anti-LPH antibody, but can be
immunoprecipitated with a polyclonal antibody that is directed against
a denatured form of the enzyme. In this case, interaction of pro-LPH
with immunoglobulin heavy chain binding protein can be discerned. The
results demonstrate the existence of intramolecular disulfide bonds
that are essential for the promotion of pro-LPH to a native
conformation. Furthermore, BiP is involved in the folding events of
pro-LPH.
Most proteins in eukaryotic cells are synthesized in the cytosol
and sorted to different cellular compartments or secreted into the
exterior milieu by virtue of specific sorting signals or sorting
patches contained in their primary, secondary, or tertiary structures
(Kornfeld and Mellman, 1989; Mostov et al., 1992). Proteins
destined for the plasma membrane, lysosomes, nuclear envelope, and
endocytic and exocytic membranes possess a signal sequence, a stretch
of at least 6 hydrophobic amino acids, that mediates the integration of
the synthesized protein into the ER ( The pathways leading to the promotion of a
protein to its final native configuration are still not completely
defined. However, a large body of information has proposed that protein
folding ensues by rapid interaction of hydrophobic residues in the
polypeptide chain, formation of secondary structures such as
We are
interested in dissecting the molecular mechanisms implicated in the
generation of transport-competent and biologically active molecules of
human intestinal brush border membrane. Lactase-phlorizin hydrolase
(LPH), an intestinal brush border glycoprotein responsible for the
digestion of lactose in mammalian milk, is one of these molecules. LPH
is synthesized in human intestinal cells as a single chain mannose-rich
precursor (pro-LPH; M
Figure 4:
Effect of reduction and alkylation on the
binding capacity of anti-LPH mAb to LPH. Biopsy samples were
biosynthetically labeled for 6 h with
[
In some
experiments, trypsin treatment of immunoprecipitated pro-LPH was
performed. Here, the immunoprecipitates were washed three times with
dilution buffer and twice with phosphate-buffered saline. The beads
were then resuspended in 40 µl of phosphate-buffered saline and
treated with 50 µg of trypsin at 37 °C for 30 min. The reaction
was arrested by the addition of SDS-PAGE sample buffer and boiling for
5 min. The samples were finally analyzed by SDS-PAGE.
Microsomal membranes were isolated as described by Bulleid
and Freedman(1988). Translocation of the translated proteins was
assessed by proteinase K treatment as described previously (Bulleid and
Freedman, 1988).
Figure 1:
Electrophoretic analysis of human
intestinal brush border LPH. Brush border membranes were purified from
human small intestinal mucosas and solubilized with Triton X-100 and
deoxycholate (0.5% each). The supernatant after a 100,000
Figure 2:
A, cell-free translation of LPH mRNA in
the presence or absence of microsomal membranes. Transcription of the
full-length human LPH cDNA was initiated with SP6 RNA polymerase.
Transcribed RNA was translated using a nuclease-treated rabbit
reticulocyte lysate containing L-[
To further assess the translocation of the translated pro-LPH
products across the microsomal membranes, the susceptibility of the
nascent 200-kDa polypeptide and its glycosylated 215-kDa counterpart to
proteinase K was examined. As depicted in Fig. 2C, only
the nonglycosylated 200-kDa polypeptide was degraded by this treatment,
while glycosylated 215-kDa pro-LPH remained unaffected (lane2). Treatment of the translated product alone (Fig. 2C, lane3) with proteinase K
resulted also in a degradation of this polypeptide (Fig. 2C, lane4). On the other hand,
solubilization of the microsomal membranes with Triton X-100 followed
by proteinase K treatment resulted in a complete degradation of the
215-kDa species (Fig. 2D, lane3).
Therefore, glycosylated 215-kDa pro-LPH was not accessible to
proteinase K since it was found in the interior of the microsomal
vesicles; solubilization of the membranes led to exposure of this
species and consequently to its degradation by proteinase K.
Altogether, the data obtained clearly demonstrate that the 200-kDa
species is translocated into the microsomal membranes.
Figure 3:
Cell-free translation of LPH mRNA in the
presence of GSSG. A, immunoprecipitation of pro-LPH
synthesized in the presence of increasing concentrations of GSSG.
Translation of pro-LPH was performed in the presence of microsomal
membranes as described for Fig. 2A and in the presence
of varying concentrations of the oxidant GSSG as indicated. The
translation products were immunoprecipitated with anti-LPH mAb and
analyzed by SDS-PAGE on 7% slab gels followed by autoradiography. B, immunoprecipitation of pro-LPH with anti-LPH pAb. Products
of translation in the presence of microsomal membranes and in the
absence (lane1) or presence (lane2) of 2 mM GSSG were immunoprecipitated with
anti-LPH pAb and resolved by SDS-PAGE on 7% slab gels followed by
autoradiography. C, immunoprecipitation of pro-LPH in the
presence of GSSG. Biopsy samples were biosynthetically labeled for 2 h
and solubilized. The detergent extracts were treated with varying
concentrations of GSSG as indicated and immunoprecipitated with
anti-LPH mAb. The immunoprecipitates were analyzed by SDS-PAGE on 7%
slab gels. D, comparison of immunoprecipitated pro-LPH with
total synthesized pro-LPH at different concentrations of GSSG. The
intensity of immunoprecipitated pro-LPH at different GSSG
concentrations (A) was compared with that of total pro-LPH
synthesized in the presence of similar GSSG
concentrations.
On the other hand, it is possible that GSSG may
influence the binding of the mAb to the pro-LPH species at increasing
concentrations of GSSG, thus leading to reduced amounts of
immunoprecipitated pro-LPH at concentrations higher than 2 mM.
To investigate this issue, we labeled biopsy samples biosynthetically
for 2 h, after which time only the 215-kDa mannose-rich pro-LPH species
becomes labeled (Naim et al., 1987; Naim, 1992). The detergent
extracts of the biopsy samples were then treated with various
concentrations of GSSG and immunoprecipitated with anti-LPH mAb. As
shown in Fig. 3C, essentially similar amounts of
labeled pro-LPH were immunoprecipitated at increasing concentrations of
GSSG. Therefore, this result demonstrates that GSSG per se does not affect antibody-antigen binding. Consequently, these and
the scanning data are consistent with the view that the decrease in the
labeling intensity of immunoprecipitated pro-LPH (Fig. 3A) is due to reduced amounts of translated
pro-LPH at high GSSG concentrations. Taken together, the reactivity of
the conformation-specific antibody with pro-LPH generated in the
presence of GSSG, but not in its absence, strongly suggests that the
oxidant facilitates the folding of pro-LPH by formation of disulfide
bonds, whereby pro-LPH ultimately assumes a conformation similar to
that of native intestinal pro-LPH.
The
immunoprecipitation of LPH forms from reduced lysates with anti-LPH pAb
indicated that LPH in these lysates did not undergo substantial
degradation owing to the reduction procedure. To corroborate these
results and to determine whether other proteins are still intact in the
reduced and alkylated lysates, these lysates were immunoprecipitated
with a monoclonal antibody directed against sucrase-isomaltase (Fig. 4, Mab anti-SI), a major brush border membrane
glycoprotein. As shown in Fig. 4, the mannose-rich (SI
Figure 5:
BiP binds pro-LPH in an ATP-dependent
manner. Translations were carried out as described for Fig. 2A either in the presence (lanes2 and 4) or absence (lanes1 and 3) of added GSSG (2 mM). Translations either were
depleted of ATP by incubation with apyrase for 2 min at 37 °C (lanes1 and 2) prior to immunoprecipitation
or were not treated with apyrase and incubated with ATP (1 mM)
present in the immunoprecipitation buffer (lanes3 and 4). All samples were immunoprecipitated with
anti-BiP. The immunoprecipitates were resolved by SDS-PAGE on 7% slab
gels and submitted to fluorography.
We also demonstrated that the complex
formed between pro-LPH and BiP can be dissociated in the presence of
ATP. Pro-LPH translation products synthesized in the presence or
absence of GSSG were immunoprecipitated with antibodies to BiP either
in the presence or absence of 1 mM ATP present in the
immunoprecipitation buffer. The results shown in Fig. 5reveal
that coimmunoprecipitation of pro-LPH occurs only when GSSG and ATP are
absent from the translation reactions (lane1) and
that this complex can be dissociated in the presence of ATP (lane3). This shows that the association of BiP with pro-LPH
is a specific interaction and confirms previous results that
demonstrate the ATP-dependent interaction of BiP with malfolded
proteins (Hurtley et al., 1989). To provide further
evidence that the conformation of pro-LPH bound to BiP is different
from that generated in the presence of 2 mM GSSG, we performed
protease sensitivity assays. It is known that native pro-LPH undergoes
intracellular cleavage to the brush border membrane LPHm species
(Danielsen et al., 1984; Hauri et al., 1985; Naim et al., 1987; Naim, 1992). Furthermore, the site of cleavage
has been proposed to lie between Arg
Figure 6:
Trypsin sensitivity assays. Pro-LPH was
synthesized in the presence of microsomal membranes and in the absence (lanes1 and 2) or presence (lanes3 and 4) of 2 mM GSSG. The translation
products were immunoprecipitated with anti-LPH pAb (lanes1 and 2) or anti-LPH mAb (lanes3 and 4). The immunoprecipitates (IP) were treated
with trypsin (lanes2 and 4) or were not
treated (lanes1 and 3). The samples were
finally prepared for SDS-PAGE analysis on 7% slab
gels.
In this paper, we analyzed the folding state of pro-LPH in a
cell-free transcription/translation system with particular emphasis on
the formation of intramolecular disulfide bonds. The pro-LPH molecule
contains 19 cysteine residues, 13 of which are found in a large domain
at the N-terminal end ( In intestinal
biopsy samples, LPH is synthesized as a mannose-rich polypeptide
(pro-LPH) that undergoes intracellular cleavage to LPHm. Our data
demonstrate that anti-LPH mAb (HBB1/909) recognizes the pro-LPH and
LPHm species under conditions that do not disrupt the native
conformation of LPH forms. When the native conformation of pro-LPH and
LPHm is altered upon reduction of the biopsy lysates with DTT, anti-LPH
mAb fails to bind pro-LPH and LPHm. By contrast, a control polyclonal
antibody, which binds denatured forms of LPH on Western blots,
immunoprecipitates the LPHm species only after the biopsy lysates have
been reduced with DTT. Moreover, the binding capacity of this antibody
to pro-LPH increases substantially upon DTT reduction, most likely due
to unfolding of pro-LPH. These findings indicate that anti-LPH mAb (HBB
1/909) specifically binds regions in pro-LPH that implicate disulfide
bonds in their secondary structure, while the polyclonal antibody binds
the unfolded or intermediate forms of LPH. This underscores, on the
other hand, the important role played by disulfide bonds in the
generation of a native conformation of LPH. The translated nascent
polypeptide chain of pro-LPH as well as its glycosylated form obtained
in the presence of microsomal membranes reveal similar apparent
molecular weights compared with their counterparts in intestinal cells.
In the absence of GSSG, these polypeptides reacted with a polyclonal
anti-LPH antibody, but not with a monoclonal anti-LPH antibody,
indicating that under these conditions, pro-LPH has not yet assumed its
native conformation. This is further corroborated by the following two
observations. (i) Pro-LPH binds the molecular chaperone BiP, which is
known to stabilize unfolded or partially folded protein molecules
(Gething and Sambrook, 1992) in an ATP-dependent manner; and (ii)
pro-LPH generated in the absence of GSSG undergoes complete degradation
upon trypsin treatment, while pro-LPH obtained in the presence of GSSG
is cleaved to two distinct polypeptides, one of which most likely
corresponds to the mannose-rich counterpart of LPHm. Upon addition
of GSSG to microsomal membranes, a dose-dependent reactivity of pro-LPH
with the conformation-specific monoclonal anti-LPH antibody can be
manifested. In view of the binding specificity of anti-LPH mAb, this
result is indicative of a native conformation of pro-LPH being
facilitated by the formation of disulfide bonds. The failure of pro-LPH
to bind BiP strongly favors the notion that pro-LPH has assumed a
native conformation under these conditions. The profile of
monoclonal anti-LPH antibody binding to pro-LPH at varying
concentrations of GSSG parallels most likely the formation of disulfide
bonds. This reaction appears to reach equilibrium at a GSSG
concentration of 2 mM, at which the binding of pro-LPH to the
monoclonal antibody is maximal. At lower GSSG concentrations, the
intensity of pro-LPH immunoprecipitated with anti-LPH mAb is markedly
reduced. This could be explained by the presence of folding
intermediates of pro-LPH that do not efficiently bind the
conformation-specific antibody. Alternatively, concentrations lower
than 2 mM GSSG promote the formation of disulfide bonds of
only a proportion of existing pro-LPH molecules, which ultimately bind
to the monoclonal antibody. Since a protein can refold in vitro without the presence of other protein components, it is
anticipated that secondary and tertiary structures other than disulfide
bridges form in pro-LPH regardless of the absence or presence of GSSG.
These structures, however, do not endow pro-LPH per se with a
native configuration since pro-LPH does not bind the
conformation-specific antibody in the absence of GSSG. At
concentrations higher than the optimum, GSSG inhibits protein synthesis
as shown previously (Scheele and Jacoby, 1982; Marquardt et
al., 1993). Finally, it remains to be determined whether
disulfide bond formation has directly affected the folding state of
pro-LPH by stabilizing folding intermediates of the molecule or has
secondary effects, for example by influencing the glycosylation state,
which in turn alters the configuration of pro-LPH. In fact, evidence
has been obtained to show that disulfide bond formation and the
glycosylation state of several proteins are closely linked events
(Bulleid et al., 1992).
)(von Heijne, 1986).
Several modification reactions are initiated during entry of the
extending polypeptide chain into the ER lumen and may facilitate the
folding of the protein into native conformation. These include signal
sequence cleavage, attachment of mannose-rich carbohydrate chains, and
disulfide bond formation. In addition, a number of ER resident
proteins, most notably, protein chaperones, such as BiP/GRP78 and
calnexin, and enzymes, such as protein-disulfide isomerase, are thought
to be crucial in protein folding, protein-protein interactions in
oligomeric structures, and elimination of malfolded polypeptides
(Sanders et al., 1992; Ou et al., 1993) (for reviews,
see Hurtley and Helenius (1989), Gething and Sambrook(1992), and Doms et al. (1993)).
-helices and
-sheets, and finally formation of disulfide
bonds or other covalent interactions to stabilize particular regions of
the protein (for a review, see Gething and Sambrook(1992)).
= 215,000) that
acquires complex glycosylated sugars in the Golgi apparatus prior to
proteolytic cleavage and targeting to the brush border membrane as
mature LPH (LPHm; M
= 160,000). The subunit
structure of LPHm revealed one single polypeptide under completely
denaturing conditions (Naim et al., 1987). Although the
quaternary structure of LPH has not been analyzed in detail, the
presence of dimeric LPH forms has been suggested (Danielsen, 1990) (
)The complete amino acid sequence of LPH has been deduced
from cDNA cloning and revealed structural features of a type I protein
that is synthesized with a cleavable signal sequence and that has a
transmembranous orientation (Mantei et al., 1988). Of note is
the presence of a large profragment (LPH
; 868 amino acids) at the
N-terminal end that precedes brush border LPHm. Recent identification
of LPH
in intestinal biopsy samples (Naim et al., 1994)
and analysis of its possible function have led to the hypothesis that
LPH
may play a crucial role in the folding of pro-LPH, perhaps as
an intramolecular chaperone (Oberholzer et al., 1993; Naim et al., 1994). The LPH
profragment is rich in hydrophobic
amino acids and contains 13 cysteine residues, while mature LPH (1061
amino acids) contains, in comparison, only 6 cysteine residues. It is
not known whether these cysteine residues are involved in disulfide
bond formation. In fact, analysis of pro-LPH as well as cleaved LPHm
under reducing and nonreducing conditions did not reveal differences in
their electrophoretic mobilities (see ``Results''). However,
it is possible that slight variations in the apparent molecular weights
cannot be detected by SDS-PAGE analysis. In this paper, we investigated
the folding of pro-LPH with particular emphasis on disulfide bond
formation.
Materials
SP6 RNA polymerase, nucleotides,
nuclease-treated rabbit reticulocyte lysate, amino acids minus
methionine, RNasin ribonuclease inhibitor, and dog pancreas microsomal
membranes were purchased from Promega. Restriction enzymes, proteinase
K, and endoglycosidase H were purchased from Boehringer Mannheim. trans-[S]Methionine was purchased from
ICN.
Cell-free Transcription
The vector pGEM-4Z
containing the complete cDNA for human LPH, pLPH (Naim et al.,
1991), was linearized with SalI. Transcription was initiated
with SP6 RNA polymerase and was carried out essentially as described
(Gurevich et al., 1991). Subsequently, the mixture was
extracted once with phenol/chloroform and twice with chloroform. After
ethanol precipitation, the RNA was resuspended in 50 µl of
RNase-free water.Cell-free Translation
Transcribed RNA was
translated using a nuclease-treated rabbit reticulocyte lysate. The
reaction mixture comprised 18 µl of reticulocyte lysate, 1 µl
of 1 mM amino acids (minus methionine), 15 µCi of L-[S]methionine, and 3 µl of
transcribed RNA. Where indicated, the samples were supplemented with 1
µl of nuclease-treated microsomal membranes. Translations were
performed in the absence or presence of GSSG at 30 °C for 60 min.
Reduction and Alkylation of Solubilized Biopsy
Specimens
Human small intestinal mucosas (5-10 mg,
wet weight) were obtained from patients biopsied for diagnostic
purposes. They appeared normal when examined by light microscopy and
expressed normal levels of brush border disaccharidase activities.
Biosynthetic labeling was performed according to Naim et
al.(1987). Briefly, the biopsy specimens were placed on stainless
steel grids in organ culture dishes, washed three times in
methionine-free RPMI 1640 medium containing 10% dialyzed fetal calf
serum, and incubated in the same medium for 2 h at 37 °C in a
CO
+ O
(5:95, v/v) incubator. The biopsy
samples were labeled with trans-[
S]methionine at 100
µCi/biopsy sample. After labeling, the specimens were washed three
times in RPMI 1640 medium and homogenized at 4 °C with a
Teflon-glass homogenizer in 1 ml of 25 mM Tris-HCl, pH 8.1,
supplemented with 50 mM NaCl and a mixture of protease
inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml
pepstatin, 5 µg/ml leupeptin, 1 µg/ml aprotinin, and 17.4
µg/ml benzamidine). Thereafter, the homogenates were solubilized
with 1% Triton X-100 for 1 h at 4 °C and centrifuged at 100,000
g for 1 h at 4 °C. The supernatant was treated
with 20 mM dithiothreitol at 37 °C for 2 h, cooled on ice,
and alkylated with 80 mM iodoacetamide for 4 h at 4 °C.
The reduced and alkylated lysates were then dialyzed in the cold
against 25 mM Tris-HCl, pH 8.1, containing 50 mM NaCl
and 1% Triton X-100. Usually, 1 ml of the lysates was dialyzed against
500 ml of dialysis buffer, which was changed four times within 48 h. As
a control, labeled biopsy specimens were processed similarly as
described above, except that no DTT or iodoacetamide was added. Reduced
and alkylated lysates as well as untreated lysates were
immunoprecipitated with anti-LPH mAb or anti-LPH pAb. Control
immunoprecipitations were performed with monoclonal antibody against
intestinal sucrase-isomaltase of the brush border membrane.
Immunoprecipitation
Mouse monoclonal anti-human
LPH antibody (anti-LPH mAb) was generously provided by Dr. Hans-Peter
Hauri (Biocenter, Basel, Switzerland) (Hauri et al., 1985).
Polyclonal anti-LPH antibody (anti-LPH pAb) was prepared by
intramuscular injection of electrophoretically purified LPH into
rabbits (Naim et al., 1994). The specificity of the antibody
was examined by Western blotting using whole brush border membranes as
an antigen (see Fig. 4, lane 1). Monoclonal anti-BiP
antibody was a generous gift from Dr. Linda Hendershot (St. Jude
Children's Hospital, Memphis, TN) (Bole et al., 1986).
To deplete the translation mixtures of ATP for coprecipitation
experiments with anti-BiP antibody, the samples were incubated for 2
min at 37 °C with apyrase (100 units/ml). Prior to
immunoprecipitations, the antibodies were conjugated to protein
A-Sepharose beads as follows. 1 µl of anti-LPH mAb, 1 µl of
anti-BiP antibody, or 5 µl of anti-LPH pAb was mixed with 30 µl
of protein A-Sepharose and 1 ml of sodium phosphate buffer, pH 8.0, for
2 h at 4 °C. Thereafter, the beads were washed once with sodium
phosphate buffer and used in the immunoprecipitations. The translation
mixtures were diluted in 50 mM Tris-HCl, pH 7.4, containing
150 mM NaCl, 5 mM EDTA, 0.25% gelatin, 0.05% Nonidet
P-40, and 0.02% sodium azide and immunoprecipitated with the
corresponding Immunobeads for 6 h at 4 °C. To study the ATP
dependence of the binding of LPH to BiP, ATP (1 mM) was
included in the immunoprecipitation buffer. The pelleted Sepharose
beads were washed three times in dilution buffer and resuspended in 40
µl of SDS-PAGE sample buffer (62.5 mM Tris-HCl, pH 6.8, 2%
(w/v) SDS, 10% (w/v) glycerol, and bromphenol blue).
S]methionine, homogenized, and solubilized with
Triton X-100. The detergent extracts were treated for 2 h at 37 °C
with 20 mM DTT and alkylated for 4 h at 4 °C with
iodoacetamide, and excess DTT and iodoacetamide were removed by
dialysis. Reduced and alkylated lysates as well as untreated lysates
were immunoprecipitated with anti-LPH mAb (lanes2 and 3) or anti-LPH pAb (lanes4 and 5). Immunoprecipitation with a monoclonal
anti-sucrase-isomaltase antibody (Mab anti-SI) served as a
control (lanes6 and 7). Lane1 is a Western blot (W.B.) of brush border membranes with
anti-LPH pAb to confirm the specificity of this antibody in recognizing
denatured forms of brush border LPH (M
=
160,000). SI
, mannose-rich
sucrase-isomaltase; SI
, complex
glycosylated sucrase-isomaltase.
Other Methods
Highly purified brush border
membranes were prepared, solubilized, and immunoprecipitated with
monoclonal anti-human LPH antibody as described by Naim et
al.(1987). Endoglycosidase H treatment of immunoprecipitates or
translation products was performed as described (Naim et al.,
1987).SDS-PAGE
Samples were prepared for electrophoresis
by mixing with 5 volumes of SDS-PAGE sample buffer in the presence of
50 mM DTT for reducing conditions and in the presence of 100
mM iodoacetamide for nonreducing conditions and boiled for 5
min. SDS-PAGE was performed by the method of Laemmli(1970). After
electrophoresis, gels were processed for autoradiography and exposed to
Kodak X-Omat AR film.
Electrophoretic Analysis of Human Intestinal
LPH
To examine the presence of intermolecular or intramolecular
disulfide bonds in brush border LPH, brush border membranes were
solubilized and immunoprecipitated with monoclonal anti-LPH antibodies. Fig. 1demonstrates that under completely denaturing conditions, i.e. in the presence of SDS and DTT, brush border LPH is
composed predominantly of one single polypeptide of M = 160,000 (lane2). When DTT was omitted
from the electrophoresis sample buffer, the same protein band pattern
was obtained (Fig. 1, lane1). In addition, a
high molecular weight band (M
> 202,000, the
molecular weight of the standard protein myosin) was detected. This
band has been also previously detected and suggested to correspond to
dimeric forms of brush border LPH (Sterchi et al., 1990).
Since this band is mainly detected in the nonreduced sample, we
conclude that it represents dimers that are covalently associated by
intermolecular disulfide bonds. However, the extent of such dimers in
total brush border LPH is very low since the M
= 160,000 polypeptide is the main LPH species detected
under reducing and nonreducing conditions. Furthermore, we were not
able to detect shifts in the mobilities of the brush border LPH species
under reducing and nonreducing conditions. A shift in the mobility
would be indicative of the presence of intramolecular disulfide bonds.
It is therefore obvious that the presence of possibly existing
disulfide bonds in brush border LPH cannot be assessed by this
approach.
g centrifugation was immunoprecipitated with monoclonal anti-LPH
antibody. The immunoprecipitates were analyzed by SDS-PAGE on 5% slab
gels under reducing (lane2) or nonreducing (lane1) conditions. The gel was stained with Coomassie Blue.
Apparent molecular masses of standard proteins (lane 3) are
indicated on the right.
Cell-free Synthesis of Pro-LPH
In view of the
results shown above, we sought to investigate the presence of disulfide
bonds by utilizing a cell-free transcription/translation system. Here,
a full-length LPH cDNA containing the coding sequence for human LPH in
the pGEM-4Z vector (denoted pF1; see Naim et al.(1991)) was
transcribed with phage SP6 RNA polymerase. The resulting RNA was
translated in a cell-free translation/translocation system consisting
of rabbit reticulocyte lysate supplemented with dog pancreas microsomal
vesicles (Fig. 2A). The major polypeptide synthesized
in the absence of added microsomal vesicles had an apparent M of 200,000 (Fig. 2A, lane1), consistent with the molecular weight of prepro-LPH
calculated from the deduced amino acid sequence (Mantei et
al., 1988). In the presence of microsomal membranes, an additional
polypeptide of M
= 215,000 was generated (Fig. 2A, lane2). To assess whether
the 215-kDa species was translocated into the interior of the
microsomal membranes, two different approaches were carried out. In the
first approach, the microsomal membranes were separated from the
reticulocyte lysates by centrifugation through a sucrose cushion. As
shown in Fig. 2B (lane2), the
215-kDa species was the predominantly labeled polypeptide in the
microsomal membranes. Furthermore, the 215-kDa polypeptide was
glycosylated, and its glycosylation was of the mannose-rich type since
it was converted by endoglycosidase H treatment to a band of 200 kDa (Fig. 2B, lane3), similar to the
apparent molecular mass of the nascent polypeptide (lane1). The recovery of a glycosylated 215-kDa polypeptide in
the microsomal membranes indicates that the 200-kDa translation product
was exposed to the glycosylation machinery in the interior of the
microsomal membranes, leading to the generation of the 215-kDa species.
S]methionine. Translation was
performed in the absence (lane1) or presence (lane2) of microsomal membranes (MM). The
samples were analyzed by SDS-PAGE on 7% slab gels and autoradiography. B, endoglycosidase H (Endo H) treatment of LPH
synthesized in a cell-free system. LPH was synthesized in the presence (lanes2 and 3) or absence (lane1) of microsomal membranes. The microsomal membranes were
isolated by centrifugation through a sucrose cushion (lanes2 and 3) and treated with endoglycosidase H (lane3) or were not treated (lane2). The samples were finally analyzed by SDS-PAGE on 7%
slab gels followed by autoradiography. C, treatment of
translation products with proteinase K. Translation of LPH was carried
out as described for A. Parts of the LPH translation products
in the presence (lanes1 and 2) or absence (lanes3 and 4) of microsomal membranes were
treated with proteinase K (Prot. K) (lanes2 and 4). The samples were analyzed by SDS-PAGE on 7% slab
gels and autoradiography. D, treatment of translation products
with proteinase K in the presence or absence of Triton X-100.
Translation of LPH was carried out as described for A. The LPH
translation products in the presence of microsomal membranes (lanes2 and 3) were treated with proteinase K after
solubilization with Triton X-100 (TX-100) (lane3) or without prior solubilization (lane2). The samples were analyzed by SDS-PAGE on 7% slab gels
and autoradiography. Lane 1 represents LPH synthesized in the
absence of microsomal membranes and without treatment with Triton X-100
or proteinase K.
Formation of Disulfide Bonds Is an Essential Event in the
Folding of Pro-LPH
To investigate the presence of
cotranslationally formed disulfide bonds, the translation reaction was
carried out in the presence of GSSG. GSSG oxidizes existing thiol
groups and therefore facilitates the formation of disulfide bonds. It
was required in the reaction mixture to compensate for the presence of
the reducing agent dithiothreitol in the commercially prepared
reticulocyte lysates. Varying concentrations of GSSG were added to the
reticulocyte lysates before initiation of translation to obtain a
lysate that was competent in forming disulfide bonds in the newly
synthesized protein. A change in the thiol/disulfide redox status of
the translation products has been shown to influence the migration
pattern of many proteins (Goldenberg and Creighton, 1984). However,
this could not be assessed electrophoretically for brush border LPHm (Fig. 1), LPH isolated from biopsy samples (see Fig. 4),
or LPH synthesized in a cell-free system (data not shown). We therefore
sought to determine at the immunochemical level whether GSSG induced
conformational alterations in pro-LPH reminiscent of the formation of
native disulfide bonds. Here, we used two antibodies directed against
LPH: a conformation-specific monoclonal anti-LPH antibody (anti-LPH
mAb) (Hauri et al., 1985) and a polyclonal antibody directed
against the denatured form of LPH (anti-LPH pAb). In the absence of
GSSG, immunoprecipitation of the translation products with anti-LPH mAb
did not reveal molecular species corresponding to pro-LPH (Fig. 3A, lane1), although the
translation products contained pro-LPH molecules (Fig. 3B, lane1). In fact, anti-LPH
pAb clearly recognized pro-LPH species that were translated in the
presence or absence of GSSG (Fig. 3B). On the other
hand, in the presence of GSSG, a dose-dependent reactivity of pro-LPH
with anti-LPH mAb was manifested. The intensity of pro-LPH (M = 215,000) increased when the
concentration of GSSG was raised from 1 mM (Fig. 3A, lane2) to 2 mM (lane3), but decreased at higher concentrations
(up to 4 mM) (lanes4 and 5).
Comparison of the labeling intensities by scanning densitometry of
immunoprecipitated pro-LPH (Fig. 3A) and total pro-LPH
in the translation products (data not shown) revealed that the decrease
in the intensity of immunoprecipitated pro-LPH was due to inhibition of
protein synthesis by GSSG and not due to unfolding of pro-LPH. In fact, Fig. 3D demonstrates that the ratio of
immunoprecipitated pro-LPH at a certain GSSG concentration versus total synthesized pro-LPH at the same GSSG concentration increased
when the concentration of GSSG was raised from 1 to 2 mM, but
reached a plateau at higher GSSG concentrations. This result therefore
indicates that anti-LPH mAb immunoprecipitates similar proportions of
pro-LPH from total pro-LPH synthesized in the presence of 2, 3, and 4
mM GSSG. This strongly suggests that the epitope recognized by
the mAb in the pro-LPH molecule does not undergo significant
conformational alterations at GSSG concentrations between 2 and 4
mM.
The Epitope Recognized by the Monoclonal Antibody HBB
1/909 Contains Disulfide Bonds
The observation that anti-LPH mAb
(HBB 1/909) binds LPH forms obtained in the presence of GSSG suggests
that the epitope recognized by the antibody contains disulfide bonds.
To examine this possibility, it was necessary to reduce possibly
existing disulfide bonds in LPH and subject the reduced forms to
immunoprecipitation with anti-LPH mAb. For this purpose, biopsy samples
were biosynthetically labeled for 6 h, after which time the
mannose-rich LPH precursor polypeptide, pro-LPH (215 kDa), as well as
intracellularly cleaved LPHm (160 kDa) can be detected (Naim et
al., 1987; Naim, 1992). The biopsy lysates were then treated with
DTT, alkylated with iodoacetamide, dialyzed to remove any residual
reagents, and finally immunoprecipitated with anti-LPH mAb (HBB 1/909)
or with anti-LPH pAb. Fig. 4(lane2)
demonstrates that mannose-rich and complex glycosylated precursor
pro-LPH as well as LPHm (160 kDa) (Naim et al., 1987; Naim,
1992) were immunoprecipitated with anti-LPH mAb (HBB 1/909) in the
absence of DTT treatment. By contrast, when the biopsy lysates were
treated with DTT prior to immunoprecipitation, no bands corresponding
to LPH molecules were detected (Fig. 4, lane3). Similar lysates were immunoprecipitated with anti-LPH
pAb. This antibody reacts with the denatured 160-kDa LPHm species on
Western blots (Fig. 4, lane1). Under native
conditions, anti-LPH pAb recognized only the mannose-rich species (Fig. 4, lane4). The reactivity of this
antibody with LPH forms increased substantially upon reduction of the
biopsy lysates. As shown in Fig. 4(lane5),
the intensity of the immunoprecipitated mannose-rich pro-LPH increased
by almost a 4-fold factor in comparison with that obtained under native
conditions (lane4). In addition, the 160-kDa LPHm
species was also immunoprecipitated with anti-LPH pAb, although to a
lesser extent than with anti-LPH mAb under native conditions (Fig. 4, lane2). Since anti-LPH pAb
recognizes the denatured form of LPH and since its binding to the
mannose-rich species increases upon reduction of the lysates with DTT,
we conclude that the mannose-rich form of pro-LPH recognized by
anti-LPH pAb under native conditions corresponds to an unfolded or
intermediate folded form of pro-LPH. On the other hand, anti-LPH mAb
(HBB 1/909) binds native LPH and fails to precipitate LPH that has been
reduced with DTT, strongly suggesting that the epitope recognized by
this antibody contains disulfide bonds. It should be mentioned that no
change in the electrophoretic mobilities of reduced and alkylated LPH
species (Fig. 4, lane5) relative to the
nonreduced species (lane2) was detected. This is
consistent with the notion that an electrophoretic assessment of
disulfide bonds in LPH (Fig. 1) is not possible.) as well as complex glycosylated (SI
) sucrase-isomaltase bands (see Naim et al.(1988)) were revealed in both the DTT-treated (lane7) and untreated (lane6) samples.
Moreover, the labeling intensity of these forms did not change. The
data show that the integrity of the solubilized intestinal proteins in
the biopsy did not change drastically as a consequence of reduction and
alkylation. Moreover, the epitope recognized by the monoclonal
sucrase-isomaltase antibody appears not to involve disulfide bonds, in
contrast to anti-LPH mAb. In essence, the data indicate that under
nondenaturing conditions, anti-LPH mAb recognizes an epitope in native
LPH in which disulfide bond(s) are essential.
Binding of Pro-LPH to BiP
In addition to enzymes
involved in protein folding, such as protein-disulfide isomerase, a
number of other ER resident proteins, known as molecular chaperones,
function in vivo as stabilizers of partially folded
intermediates of proteins. We wanted to determine whether partially
folded or unfolded intermediates of pro-LPH interact with BiP, a well
characterized molecular chaperone (Haas and Wabl, 1983). For this
purpose, pro-LPH was translated and translocated into the microsomal
membranes. The reaction was performed in the presence or absence of
GSSG. Furthermore, apyrase was added to the samples to deplete them of
ATP since hydrolysis of ATP bound to BiP may result in the release of
the associated protein (Munro and Pelham, 1986; Kassenbrock and Kelly,
1989; Flynn et al., 1989). As shown in Fig. 5,
immunoprecipitation of the membranes containing the translation
products with anti-BiP antibody revealed a glycosylated pro-LPH band
when the reaction was performed in the absence of GSSG (lane1), but not its presence (lane2).
Since GSSG promotes the formation of disulfide bonds and hence
facilitates the generation of a native pro-LPH conformation (see Fig. 3), we conclude that only unfolded or partially folded
pro-LPH structures bind BiP.
and Ala
(Mantei et al., 1988; Montgomery et al., 1991)
and therefore represents a potential trypsin cleavage site. In fact,
treatment of recombinant pro-LPH expressed in COS-1 cells with trypsin
generates a brush border-like species (Naim et al., 1991). It
is therefore possible that a correctly folded pro-LPH molecule should
be cleaved by trypsin to the mannose-rich analogue of LPHm, while
malfolded pro-LPH should reveal a different behavior toward trypsin.
For this purpose, pro-LPH species that bind or do not bind BiP were
translated in the absence or presence of GSSG, respectively,
immunoprecipitated with anti-LPH pAb or anti-LPH mAb, and finally
treated with trypsin. As expected, anti-LPH pAb precipitated the
nascent 200-kDa polypeptide as well as the glycosylated 215-kDa species (Fig. 6, lane1). On the other hand, anti-LPH
mAb reacted exclusively with the glycosylated species generated in the
presence of GSSG (lane3). Trypsin treatment of
pro-LPH translated in the absence of GSSG and immunoprecipitated with
the pAb resulted in a complete degradation of this species (Fig. 6, lane2). By contrast, pro-LPH
immunoprecipitated with anti-LPH mAb was cleaved into two polypeptides
in the presence of trypsin, a 135-kDa species and an
80-90-kDa polypeptide (Fig. 6, lane4). The variations in the reactivity with trypsin
corroborate the findings that GSSG assists the formation of a native
conformation of pro-LPH, while in its absence, a malfolded species that
binds to BiP is generated.
45% of total pro-LPH) that is cleaved off
during maturation of pro-LPH to LPHm (Mantei et al., 1988).
Ample evidence has accumulated to suggest that the native conformation
of proteins is promoted by the formation of disulfide bonds that
stabilize particular structures within the protein (Creighton, 1986,
1988). It is therefore important to determine whether disulfide bond
formation is an essential event in the folding process of pro-LPH.
While for many proteins the presence of disulfide bonds could be
manifested by shifts in the protein mobility on SDS-PAGE under reducing
conditions (Goldenberg and Creighton, 1984), this approach may not be
adequately sensitive when large proteins such as pro-LPH or LPHm are
studied. In fact, the initial analysis of the subunit structure of
pro-LPH and LPHm by SDS-PAGE under reducing or nonreducing conditions
did not provide any evidence of the presence of intramolecular
disulfide bridges since no major electrophoretic alterations of reduced versus nonreduced components could be discerned. This is even
more difficult to detect, if at all, if only a few cysteine residues
are implicated in disulfide bond formation. We therefore investigated a
possible role of disulfide bond formation in the folding of pro-LPH
using a rabbit reticulocyte lysate system supplemented with microsomal
membranes derived from dog pancreas. One of the advantages of this
system is that disulfide bond formation can be promoted and controlled
by the addition of the thiol oxidant GSSG. The promotion of a native
conformation in which disulfide bonds are implicated can then be
monitored by the binding capacity of pro-LPH to a conformation-specific
monoclonal antibody. However, this requires that the epitope recognized
by this monoclonal antibody contains disulfide bonds.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.