From the
The role of asparagine (N)-linked oligosaccharide
chains in intracellular folding of the human chorionic gonadotropin
(hCG)-
Human chorionic gonadotropin (hCG)¹(¹)
is a
member of the glycoprotein hormone family and consists of two
noncovalently associated subunits (
hCG-
Depending on the protein, glycans may
contribute to protein conformation, stability, and binding to target
cells. A number of reports demonstrate that the N-linked
oligosaccharides and their processing are critical for proper
glycoprotein folding and assembly
(17) . Matzuk and Boime
(11) have demonstrated that the two N-linked
oligosaccharides of hCG-
Since proper
disulfide bond formation is a critical event in the folding and
maturation of functional hCG-
CHO
The
Since the C-terminal of hCG-
Taken together, these results indicate that a portion of
hCG-p
It should be noted that folding of the
Huth et al.(26) have suggested that hCG-
The two
N-linked (Asn
The data also
suggest that molecular chaperones are involved in the folding of
unglycosylated hCG-
Since intracellular folding of
unglycosylated hCG-
Some intracellular
degradation of unglycosylated
We have shown that the d
The
[
subunit was determined by examining the kinetics of folding
in Chinese hamster ovary (CHO) cells transfected with wild-type or
mutant hCG-
genes lacking one or both of the asparagine
glycosylation sites. The half-time for folding of p
1 into p
2,
the rate-determining step in
folding, was 7 min for wild-type
but 33 min for
lacking both N-linked glycans. The
p
1
p
2 half-time was 7.5 min in CHO cells expressing
the
subunit missing the Asn
-linked glycan and 10 min
for the
subunit missing the Asn
-linked glycan. The
inefficient folding of hCG-
lacking both N-linked glycans
correlated with the slow formation of the last three disulfide bonds
(i.e. disulfides 23-72, 93-100, and 26-110)
to form in the hCG-
-folding pathway. Unglycosylated hCG-
was
slowly secreted from CHO cells, and
subunit-folding intermediates
retained in cells for more than 5 h were degraded into a hCG-
core
fragment-like protein. However, coexpression of the hCG-
gene
enhanced folding and formation of disulfide bonds 23-72,
93-100, and 26-110 of hCG-
lacking N-linked
glycans. In addition, the molecular chaperones BiP, ERp72, and ERp94,
but not calnexin, were found in a complex with unglycosylated, unfolded
hCG-
and may be involved in the folding of this
form. These
data indicate that N-linked oligosaccharides assist hCG-
subunit folding by facilitating disulfide bond formation.
and
). It is synthesized
and secreted by nonmalignant and malignant trophoblast
cells
(1, 2) . The hCG-
subunit is a 145-amino acid
protein, in which 12 cysteine residues pair to form six intramolecular
disulfide bonds. The rate of disulfide bond formation in the
subunit is rate-limiting in formation of the
heterodimer
(3, 4) . The hCG-
-folding intermediates
have been characterized based on the order of formation of the six
disulfide bonds
(5, 6, 7) , and the
hCG-
-folding pathway has been defined as: p
1
p
2-free
p
2-combined
mature
-combined. The
conversion of p
1
p
2 is the rate-determining step in
this pathway
(3) . This folding pathway is identical in JAR
(human choriocarcinoma) cells
(5, 6) , in Chinese hamster
ovary (CHO) cells transfected with the wild-type hCG-
gene
(7, 20) , and in vitro(8) .
hCG-
is the only mammalian protein for which the intracellular
folding pathway has been defined
(8, 9) .
is
synthesized, folds, and assembles with the
subunit in the
endoplasmic reticulum (ER)
(2, 3, 4) . The forms
of the
subunit detected within the ER contain two high mannose
asparagine (Asn
and Asn
)-linked
oligosaccharide chains (3, 10, 11). During transport through the Golgi
apparatus, asparagine (N)-linked oligosaccharides are
processed to form complex oligosaccharides containing galactose and
sialic acid
(12) , and four O-linked oligosaccharides
are attached to the C-terminal extension of the hCG-
sequence
(13, 14, 15) . Although all the
information needed to determine the final conformation of a protein
exists in the polypeptide chain
(16) , addition of
N-linked oligosaccharides and intracellular factors such as
molecular chaperones significantly affect protein folding inside
cells
(17, 18) .
are critical for efficient assembly with
the
subunit and for secretion. In addition, we have demonstrated
that at least one N-linked oligosaccharide is required for
efficient hCG-
folding in vitro(19) . However, the
molecular mechanism by which the N-linked oligosaccharide
chain(s) affect protein folding remains unclear.
subunits
(3, 5, 6, 7, 20, 27) and altered glycosylation can cause alteration of folding
and disulfide bond formation
(21, 22) , we postulated
that N-linked oligosaccharides are involved in hCG-
folding by facilitating disulfide bond formation. To test this
hypothesis, we analyzed the kinetics of intracellular folding of
hCG-
in CHO cells transfected with either the wild-type hCG-
gene or hCG-
genes mutated at N-linked glycosylation
sites. We found that the two N-linked glycans increased
hCG-
subunit folding efficiency by facilitating formation of the
last three disulfide bonds (23-72, 93-100, and
26-110) to form. We also observed that inefficiently folded,
unglycosylated hCG-
was degraded to a
core fragment-like
protein. However, in the presence of the
subunit, unglycosylated
folded efficiently. In addition, the slow folding process of
unglycosylated hCG-
allowed us to detect molecular
chaperone-hCG-
complexes that may be involved in hCG-
folding.
Cell Culture
CHO cells transfected with the
wild-type or mutated hCG- genes alone, or co-transfected with the
glycoprotein hormone
gene
(11) , were maintained in
Ham's F-12 medium supplemented with 5% fetal bovine serum,
penicillin (100 units/ml), streptomycin (100 µg/ml), and glutamine
(2 mM) (23). The mutated hCG-
genes contained mutations
Asn
Gln at the Asn
and/or Asn
codons
of the two N-linked glycosylation consensus
sequences
(11) . The terminology used here is: CHO
WT
or CHO
WT for CHO cells transfected with wild-type hCG-
genes
with or without co-transfection of the
gene, respectively; CHO
Asn(1+2) and CHO
Asn(1+2) for CHO cells
transfected with hCG-
genes mutated at both N-linked
glycosylation sites with or without co-transfection of the
gene,
respectively; CHO
Asn1 or CHO
Asn2 for CHO cells transfected
with hCG-
genes mutated at the Asn
or Asn
glycosylation sites, respectively.
Biosynthetic Labeling
CHO cells were metabolically
labeled as described previously
(20) . Briefly, 100-mm Petri
dishes of 90% confluent CHO cells were starved in cysteine- or
leucine-free and serum-free Dulbecco's modified Eagle's
medium for 30 min. These cells were then pulse-labeled for 5 min with
300-400 µCi/ml L-[S]cysteine
(1100 Ci/mmol; DuPont NEN) or
L-[4,5-³H]-leucine (60 Ci/mmol; DuPont
NEN) in serum-free Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) lacking cysteine or leucine, washed with
phosphate-buffered saline (pH 7.4), and chased for the times indicated
in the text. Then, cells were rinsed with cold phosphate-buffered
saline and lysed in 5 ml of phosphate-buffered saline (pH 8) containing
detergents (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium
dodecyl sulfate), protease inhibitors (20 mM EDTA and 2
mM phenylmethanesulfonyl fluoride), and 50 mM
iodoacetic acid to alkylate-free sulfhydryl groups of folding
intermediates to prevent further disulfide bond formation or
rearrangement.
Immunoprecipitation of Cell Lysates
The cell
lysates were immunoprecipitated with an anti-hCG- polyclonal
antibody (1:1000), which recognizes all forms of the hCG-
subunit,
for 16 h at 4 °C, and the immune complexes were precipitated with
protein A-Sepharose (Sigma) as described previously
(3) .
Separation of hCG-
The protein
A-Sepharose-hCG--folding Intermediates by
SDS-Polyacrylamide Gel Electrophoresis (PAGE)
immune complexes were eluted with 2
concentrated SDS-PAGE sample buffer (125 mM Tris-HCl, 2% SDS,
20% glycerol, and 40 µg/ml bromphenol blue) and analyzed by
5-20% gradient, nonreducing SDS-PAGE
(24) as described
previously
(20) .
Determination of Kinetics of hCG-
[ in Vivo
Folding
S]Cysteine-labeled hCG-
folding intermediates (p
1 and p
2) were visualized by exposing
to x-ray film. The integrated optical density (IOD) values of each band
on autoradiograms were quantitated and analyzed using a
BioImage
110S image analyzer and Whole Band
software (Millipore). These bands, representing the
hCG-
-folding intermediates (p
1 or p
2), were measured as
the product of the band area (mm²) and optical density (OD)
as calibrated with a Kodak standard 21-step gray-scale wedge (Eastman
Kodak Co.). To evaluate folding efficiency of hCG-
, we compared
the IOD of folded p
2 with that of p
1. For instance, if the
values of IOD of p
2 and p
1 were 0.80 and 0.20, respectively,
this indicated that 80% (0.8/(0.8+0.2)) of p
1 converted to
p
2, and 20% of p
1 remained unfolded. Therefore, the folding
efficiency at this time point was 80%. Each time point served as its
own control in that the ratio of p
2/(p
1 + p
2) was
obtained for each gel lane. The t reported here for folding of
p
1 to p
2 in CHO cells transfected with the wild-type
gene was slightly longer than that previously reported (7 versus 5 min)
(20) because a longer pulse time (5 versus 3 min) was used here to incorporate sufficient counts/min for
tryptic peptide mapping.
Purification of hCG-
The protein A-Sepharose beads containing hCG--folding
Intermediates
immune complexes were eluted with 6 M guanidine hydrochloride
(pH 3) (Sequanal grade; Pierce Chemical Co.) for 16 h at room
temperature. Eluates were purified using Vydac 300-Å C
reversed-phase high performance liquid chromatography (HPLC) with
elution by an acetonitrile gradient as described previously
(6) .
The fractions containing hCG-p
1 or -p
2 subunits were
collected and concentrated by Speed-Vac
concentrator.
Tryptic Digestion and Separation of Tryptic Peptides on
HPLC
Purified [S]cysteine-labeled
hCG-
-folding intermediates were digested with diphenylcarbamyl
chloride-treated trypsin. Trypsin-digested samples were loaded onto a
Brownlee 10-µm C
RP-300 reversed-phase column
(Brownlee/ABI) eluted with an acetonitrile gradient as described
previously
(6, 7) . The profile of peaks generated
indicated which hCG-
disulfide bonds had not formed.
Amino Acid
Sequencing
[S]Cysteine- or
[³H]leucine-labeled peptides were concentrated to
less than 50 µl using a Speed-Vac
concentrator.
Each sample was loaded onto a polybrene-coated, trifluoroacetic
acid-treated cartridge filter (Applied Biosystems) and sequenced using
a pulsed liquid protein sequencer (Applied Biosystems model 477A). The
phenylthiohydantoins obtained from each cycle of the Edman degradation
were collected and analyzed by liquid scintillation counting to
determine the positions of radiolabeled residues in each peptide.
Western Blot Analysis
To detect the presence of
proteins that co-immunoprecipitated with unglycosylated -folding
intermediates, cell lysates derived from six 100-mm Petri dishes CHO
Asn(1+2) cells were immunoprecipitated with anti-hCG-
,
and the hCG-
subunits were eluted from protein A-Sepharose beads
with 6 M guanidine (pH 3.0) for 16 h with rotation at room
temperature and purified by C
reversed-phase HPLC (as
described above). The 80-95-min and 100-115-min fractions
(termed C1 and C2, respectively) were high molecular weight complexes
containing unglycosylated hCG-
. These complexes were separated by
SDS-PAGE under reducing conditions
(24) , and the separated
proteins were transferred to Immobilon poly(vinylidne fluoride)
transfer membranes (Millipore) in a Trans-Blot
cell
(Bio-Rad) at 480 mA for 1-2 h at 4 °C. The membranes were
immunoblotted with either rat anti-BiP (1:500), rabbit anti-ERp72
(1:100), rabbit anti-ERp94 (1:100), or rabbit anti-calnexin (1:2000)
overnight at 4 °C with gentle shaking. Membranes were washed
several times with buffer containing 20 mM Tris, 150
mM NaCl, 1% non-fat milk, and 0.2% Tween (pH 7.4) and
incubated with anti-rat or -rabbit IgG peroxidase conjugate (1:1000,
Sigma) for 30 min at 4 °C. Enhanced chemiluminescence (ECL Western
blotting kit, Amersham Corp.) was used to identify the blotted
proteins. Rat anti-BiP polyclonal antibody was provided by Dr. David
Bole (University of Michigan, Ann Arbor, MI). Rabbit anti-ERp72
(against the 16 C-terminal amino acids of murine ERp72) and rabbit
anti-ERp94 (against the 16 C-terminal amino acids of murine ERp94) were
provided by Dr. Michael Green (St. Louis University Medical Center, St.
Louis, MO). The rabbit anti-calnexin (against the C-terminal 19 amino
acids of canine calnexin) was provided by Dr. Ari Helenius (Yale
University, New Haven, CT). Rat and rabbit nonimmune sera (Sigma) were
used as controls for specificity.
RESULTS
Kinetics of Intracellular Folding of hCG-
To determine what role N-linked
oligosaccharide chains play in intracellular hCG-
Glycosylation Mutants
folding, CHO
cells transfected with wild-type hCG-
gene (
WT) or mutated
hCG-
genes (
Asn1,
Asn2, or
Asn(1+2)) were
pulse-labeled for 5 min with [
S]cysteine and
chased for the times indicated in Fig. 1. The
[
S]cysteine-labeled hCG-
-folding
intermediates (p
1 or p
2) were immunoprecipitated with
polyclonal anti-hCG-
and analyzed by nonreducing SDS-PAGE
(Fig. 1, A and B). With increasing chase time,
p
1 synthesized in CHO cells transfected with wild-type or mutated
genes folded into their corresponding p
2 forms. To evaluate
the folding efficiency, the IOD of each band on the autoradiograms was
quantitated. The extent of p
1 conversion to p
2 was calculated
(p
2/(p
1 + p
2)) at each chase time and plotted in
Fig. 1C. Based on the calculated linear initial folding
rate, the half-times for the conversion of p
1 to p
2 were 7,
7.5, and 10 min for CHO
WT, CHO
Asn1, and CHO
Asn2,
respectively. These data indicate that hCG-
lacking the
Asn
-linked oligosaccharide had marginally decreased
folding efficiency inside cells, compared to wild-type hCG-
and
hCG-
lacking the Asn
-linked oligosaccharide. However,
the t of conversion of unglycosylated p
1 to p
2 was
33 min, demonstrating that at least one N-linked glycan is
required for efficient hCG-
folding in CHO cells.
Figure 1:
Kinetics of intracellular folding of
wild-type and glycosylation mutants of hCG-. CHO cells transfected
with wild-type or glycosylation mutants of hCG-
genes were
pulse-labeled with [
S]cysteine for 5 min and
chased for the indicated times. hCG-
subunits with different
numbers of N-linked oligosaccharides were immunoprecipitated
and analyzed by 5-20% gradient SDS-PAGE under nonreducing
conditions (see ``Experimental Procedures''). Left
lanes, Panels A and B:
C-labeled
molecular weight markers (Sigma): carbonic anhydrase (M
= 29,000), and
-lactalbumin (M
= 14,000). Panel A, CHO-
WT, CHO
cells transfected with wild-type hCG-
genes and CHO
Asn(1+2), CHO cells transfected with hCG-
genes
mutated at both N-linked (Asn
and
Asn
) glycosylation sites; Panel B, CHO
Asn1, CHO cells transfected with hCG-
genes mutated at
the Asn
-linked glycosylation site and CHO
Asn2, CHO cells transfected with hCG-
genes mutated at
the Asn
-linked glycosylation site. Arrows indicate the respective positions of p
1 (p
1
lacking both N-linked glycans), p
1 (p
1
lacking either N-linked glycan), p
2 (p
2
lacking both N-linked glycans), and p
2 (p
2
lacking either N-linked glycan). Panel C, the
intensities of each band on A and B were quantitated
with a BioImage
analyzer as described under
``Experimental Procedures.'' The folding efficiencies were
calculated by dividing the integrated intensities of p
2 by that of
the integrated intensities of p
2 plus p
1 for each lane (see
``Experimental Procedures'').
, p
1 with both
Asn-linked glycans;
, p
1 lacking Asn
-linked
glycan;
, p
1 lacking Asn
-linked glycan;
,
p
1 lacking both Asn-linked glycan.
N-Linked Oligosaccharides Facilitate Formation of
Disulfide Bonds in Later Steps of the hCG-
Since the evidence shown above demonstrated that
N-linked oligosaccharides were involved in hCG--folding
Pathway
subunit
folding, we examined whether this effect was due to changes in
disulfide bond formation. To test this hypothesis, a strategy of
identifying the disulfide bonds formed in each folding intermediate was
undertaken
(6) . Fig. 2illustrates the hCG-
tryptic
peptides arranged according to the disulfide bond assignments of Mise
and Bahl
(25) . We employed this strategy because our
intracellular kinetic folding data
(8, 26) and data from
analysis of the intracellular folding of
mutated at each of the
six disulfide bonds
(7, 27) strongly suggest that
disulfide bonds 38-57 and 9-90 form early in the folding
pathway. Since the crystal structure of mature, native hCG-
indicates that the disulfide bonds 9-57 and 38-90 are
present
(28, 29) , it is likely that there are disulfide
bond rearrangements in the hCG-
-folding pathway.
Figure 2:
hCG- disulfide-linked tryptic
peptides. Shown are the disulfide bond linked peptides generated
following trypsin digestion of hCG-
using the disulfide bond
assignments by Mise and Bahl (25) and confirmed by intracellular and
in vitro folding kinetics (7, 26). Seven tryptic peptides are
linked by six disulfide bonds in mature hCG-
. C
reversed-phase HPLC peaks of tryptic peptides (Fig. 10) that have
been previously identified (6, 7) were renumbered and indicated
above for nine of the peptides. Cystine or leucine residues
are shown in bold. Peptides or fragments of peptides missing
in the proteolytic degradation product of unglycosylated
(d
) are
shadowed.
The extent of
formation of each disulfide bond was evaluated by quantitating the
amount of hCG- tryptic peptides released from the remaining
disulfide-linked hCG-
polypeptides
(6) . For instance, when
hCG-
is trypsinized under nonreducing conditions, no peptides
containing free thiols would be released if all the disulfide bonds are
formed. However, if, for example, the disulfide bond between Cys
and Cys
was unformed, a peptide
containing Cys
with a free
thiol would be released from the remainder of the disulfide-linked
polypeptides. Thus, the percent of the released
peptide represents the extent of unformed disulfide bond between
Cys
and Cys
.
WT and CHO
Asn(1+2) mutant cells were pulse-labeled for 5 min with
[
S]cysteine and chased for the times shown in
Fig. 3
. Unformed thiols were trapped by alkylation with
iodoacetate at the time of cell lysis (thereby generating a
carboxymethyl derivative of nondisulfide-linked Cys residues). The
p
1- and p
2-folding intermediates were purified by
immunoprecipitation and C
reversed-phase HPLC. The purified
intermediates were digested with trypsin and the peptides analyzed by
C
reversed-phase HPLC to quantitate the amount of cysteine
residues 9, 57, 72, 88, 100, and 110 that were not disulfide linked in
each folding intermediate
(6, 7) . The percent of
unformed disulfide bonds involving each of the six disulfide bonded
cysteines of
was plotted according to the order in which they
form (Fig. 3, A
F). Compared to CHO
WT, disulfide bonds 23-72, 93-100, and 26-110
were predominately unformed in p
1 lacking both N-linked
oligosaccharides (Fig. 3, D-F). The lag in
conversion of p
1 to p
2 for the
Asn(1+2) mutant may
be due to less efficient formation of the 23-72 bond since
conversion of p
1 to p
2 requires this
bond
(6, 7) . Furthermore, 70% of disulfide bond
93-100 and 95% of disulfide bond 26-110 remained unformed
in unglycosylated p
2 even after a 120 min chase (Fig. 3,
E and F). These results suggest that
N-linked oligosaccharides facilitate formation of the three
late-forming disulfide
bonds
(23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 93, 94, 95, 96, 97, 98, 99, 100, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110) .
The data also suggest that the p
2
formed by the
folding of unglycosylated p
1
contained unstable
93-100 and 26-110 disulfide bonds because they were less
completely formed in p
2
than in p
1
(Fig. 3, E and F). This was unexpected
and was the opposite of wild-type p
2, which has more complete
formation of these bonds than wild-type p
1 (Fig. 3, E and F).
Figure 3:
Kinetics of disulfide bond formation in
wild-type hCG- and in
glycosylation mutants. CHO
WT and
CHO
Asn(1+2) cells were pulse-labeled with
[
S]cysteine for 5 min and chased for the
indicated times. hCG-
subunits were immunoprecipitated with
anti-hCG-
and protein A-Sepharose and purified on C
reversed-phase HPLC. The purified
[
S]cysteine-labeled hCG-p
1 and -p
2
were digested with trypsin and analyzed by C
reversed-phase and ion-exchange HPLC (see ``Experimental
Procedures''). The percent of unformed bonds for each of the six
disulfide bonds of
were calculated as described in the text, and
data are shown for
WT p
1 (
),
WT p
2 (
),
Asn(1+2) p
1 (
), and
Asn(1+2) p
2
(
). The disulfide bonds indicated at the top of each
panel are arranged in the order in which they form (A-F)
(6, 7).
The
Previous studies in our laboratory showed that
completion of the last disulfide bond (26-110) occurs after
assembly with the Subunit Stimulates Folding and Disulfide Bond
Formation of
Subunit Lacking N-Linked
Oligosaccharides
subunit
(26) and that the
subunit
increases the rate and extent of hCG heterodimer assembly (20). This is
consistent with the crystallographic structure which reveals that
formation of the disulfide bond 26-110 of
forms a seat belt
around the
subunit after heterodimer assembly occurs (28). These
data imply that the
subunit assists hCG-
folding by
facilitating disulfide bond formation. To examine whether the
subunit can increase folding of the unglycosylated
subunit, CHO
cells containing the
Asn(1+2) mutant gene were co-transfected
with the
gene.
Asn(1+2)- and
Asn(1+2)-expressing cells were pulse-labeled for 5 min with
[
S]cysteine and chased as described in
Fig. 4
. The percent of each of the six disulfide bonds that was
not formed was calculated as described above and plotted in the order
in which they form (Fig. 4, A
F). The
rates of disulfide bond formation involving cysteines 9, 34, 38, 57,
88, and 90 were similar in the either presence or absence of the
subunit (Fig. 4, A-C). However, the slow formation
of disulfide bonds 23-72, 93-100, and 26-110 in
-lacking N-linked oligosaccharides was accelerated in the
presence of the
subunit, suggesting that
subunit
facilitates formation of disulfide bonds that are completed later in
the hCG-
-folding pathway.
Figure 4:
Kinetics of disulfide bond formation in
glycosylation mutants in the presence or absence of
subunit. CHO cells transfected with N-linked glycosylation
mutants with or without co-transfection with wild-type glycoprotein
hormone
gene (CHO
Asn(1+2) or CHO
Asn(1+2), respectively) were pulse-labeled with
[
S]cysteine for 5 min and chased for the
indicated times. hCG-
subunits were prepared as described in Fig.
3. Shown are
Asn(1+2) p
1 (
),
Asn(1+2)
p
2 (
),
Asn(1+2) p
1 (
),
Asn(1+2) p
2 (
).
A Potential Role for Chaperones in Folding of
Glycosylation Mutants of hCG-
When CHO cells containing
WT and
Asn(1+2) mutated genes were pulse-labeled for 5
min with [
S]cysteine and chased for 5 min to 5
h, the hCG-
-folding intermediates purified on C
reversed-phase HPLC showed different profiles (Fig. 5).
With increasing chase time, wild-type p
1 (Fig. 5,
A-C) converted more efficiently into p
2 than the
unglycosylated p
1 (Fig. 5, D-F), and two
additional peaks appeared at 90 min (C1) and 110 min
(C2) in the HPLC profile of the unglycosylated
(Fig. 5, D-F). The C1 and C2 peaks diminished with
increasing chase times. When the C1 and C2 peaks were collected and
analyzed by SDS-PAGE (Fig. 6), it was observed that
hCG-p
1
(lacking two N-linked glycans) was
complexed with a variety of high molecular weight proteins.
Figure 5:
Purification of
[S]cysteine labeled hCG-
folding
intermediates and protein complexes from CHO
WT and CHO
Asn(1+2) cell lysates. CHO
WT and CHO
Asn(1+2)
cells were pulse-labeled with [
S]cysteine for 5
min and chased for the indicated times. hCG-
subunits were
immunoprecipitated with anti-hCG-
and protein A-Sepharose. Then,
the [
S]cysteine-labeled hCG-
subunits were
eluted with guanidine and analyzed by C4 reversed-phase HPLC as
described under ``Experimental Procedures.'' The cell types
and chase times are indicated at the top of each panel.
WT-0,
WT-5, or
WT-15, CHO
WT cells chased for 0, 5, or 15 min, respectively;
Asn(1+2)-15,
Asn(1+2)-60, or
Asn(1+2)-300, CHO
(1+2) cells chased for
15, 60, or 300 min, respectively; p
1, p
1 with both
N-linked glycans; p
2, p
2 with both
N-linked glycans; p
1, p
1 lacking both
N-linked glycans; p
2, p
2 lacking both
N-linked glycans; C1 and C2, protein
complexes containing hCG-p
1
and chaperone-like
proteins (see Fig. 6).
Figure 6:
Identification of protein complexes on
reducing SDS-PAGE. The [S]cysteine-labeled
p
1, p
1
C1, and C2 peaks (Fig. 5) were analyzed
by 5-20% reducing SDS-PAGE. Lane 1,
C-labeled molecular weight markers (Sigma): bovine serum
albumin (M
= 66,000), chicken egg albumin
(M
= 45,000), carbonic anhydrase
(M
= 29,000), and
-lactalbumin
(M
= 14,000); lane 2,
hCG-p
1 (p
1 with both N-linked glycans) and
hCG-p
1 (p
1 lacking either N-linked glycan);
lane 3, hCG-p
1 (p
1 lacking both
N-linked glycans); lane 4, protein complex 1 (C1);
lane 5, protein complex 2 (C2). P1-P7 represent
chaperone-like proteins that were co-immunoprecipitated as part of
hCG-
1
complex.
To
identify these proteins, the C1 and C2 peak fractions were analyzed by
5-20% gradient SDS-PAGE under reducing conditions. The separated
proteins were transferred to nitrocellulose membranes and subjected to
Western blot analysis using antibodies against BiP
(Fig. 7A, lanes 3 and 4), ERp72
(Fig. 7B, lanes 3 and 4), and ERp94
(Fig. 7B, lanes 5 and 6). To rule out
nonspecific blotting, nonimmune rat serum (Fig. 7A,
lanes 1 and 2) and rabbit serum
(Fig. 7B, lanes 1 and 2) were used as
controls. The data in Fig. 7suggested that C1 and C2 are protein
complexes of unglycosylated hCG-p1 with molecular chaperones such
as BiP, ERp72, and ERp94. Western blot analysis of the C1 and C2
protein complexes indicated that they did not contain calnexin (data
not shown), which might be expected since unglycosylated hCG-p
1
lacks calnexin binding sites (glucose residues)
(17) .
Figure 7:
Identification of chaperone-like proteins
by Western blotting analysis. Cell lysates from six 100-mm Petri dishes
of CHO Asn(1+2) cells were immunoprecipitated with
anti-hCG-
and protein A-Sepharose. Then, the
nonradioactive-labeled hCG-
subunits were eluted with guanidine
and purified by C
reversed-phase HPLC. The C1
(80-95th min) and C2 (100-115th min) fractions were
collected and analyzed by 5-20% gradient SDS-PAGE under reducing
conditions. The protein bands were transferred from acrylamide gels to
nitrocellulose membranes and immunoblotted with specific antibodies
indicated below or preimmune IgG (rat or rabbit). Secondary
peroxidase-conjugated antibodies and enhanced chemiluminescence
reagents were used to detect the chaperone proteins (see
``Experimental Procedures''). The molecular weight was
determined based on prestained molecular weight markers (Bio-Rad):
bovine serum albumin (M
= 97,200),
ovalbumin (M
= 50,000), carbonic anhydrase
(M
= 35,100), soybean trypsin inhibitor
(M
= 29,700), lysozyme (M
= 21,900). Panel A, lanes 1 (C1) and 2 (C2) were immunoblotted with
preimmune rat IgG; lanes 3 (C1) and 4 (C2) were immunoblotted with rat anti-BiP. Panel
B, lanes 1 (C1) and 2 (C2)
were immunoblotted with preimmune rabbit IgG; lanes 3 (C1) and 4 (C2) were immunoblotted with
rabbit anti-ERp72; lanes 5 (C1) and 6 (C2) were immunoblotted with rabbit
anti-ERp94.
Production of
In
the absence of the Core Fragment-like Protein
subunit, unglycosylated hCG-
is still
secreted as free
subunit with t of 5 h (t of
wild-type hCG-
= 2.5 h)
(11) . However, our results
indicated that unglycosylated p
2 subunits contain more unformed
93-100 and 26-110 disulfide bonds than the wild-type
p
2 (Fig. 3, E and F). Even after a 24-h
chase, 60% of the 93-100 and 95% of 26-110 disulfide bonds
still were not formed in the
subunit that remained inside the
cells (data not shown). However, with chase times longer than 5 h, a
new peak (d
) appeared on the HPLC profiles
(Fig. 8, C-F). In order to identify this new peak,
CHO
Asn(1+2) cells were pulse-labeled with
[
S]cysteine and chased for 15-24 h. The
cell lysates were immunoprecipitated with anti-hCG-
and purified
on C
reversed-phase HPLC. The d
fractions were collected and analyzed by SDS-PAGE under either
reducing (Fig. 9A) or nonreducing
(Fig. 9B) conditions. The results indicated that
d
migrated faster than unglycosylated p
1
(p
1
) (compare to Fig. 1) and was composed of
two disulfide-linked polypeptides. To further identify this apparent
subunit degradation product, d
was reduced and
digested with trypsin and tryptic peptides analyzed on C
reversed-phase HPLC. In comparison with the tryptic peptide map
of unglycosylated p
2 (p
2
)
(Fig. 10A), four peaks (1, 5,
8, and 12) were missing (Fig. 10B) and
two new peaks (3 and 9) were generated. Previous
studies
(6) had identified peaks 1, 5,
8, and 12 as peptides
,
,
, and
, respectively (). Since
d
lacked these four peaks, the data suggested that
the
,
,
, and
peptides
were missing or at least not intact in d
. Moreover,
amino acid sequence data indicated that new peaks 3 and 9 were peptides that contained [
S]cysteine at
positions 3 (Fig. 10E) or 6 and 8
(Fig. 10F), respectively. Since peptides
and
were
missing and since Gln
and Leu
are putative
proteolytic cleavage sites in hCG-
(30) , the most likely
explanation is that peaks 3 and 9 represent peptides
and
. This is
consistent with the finding that peak 3 contained
[
S]cysteine at cycle 3 and peak 9 contained
[
S]cysteine at cycles 6 and 8 (see
Fig. 2
). Thus, we concluded that peaks 3 and 9 represent peptides
generated by proteolytic degradation of p
2
.
Figure 8:
Identification of
[S]cysteine-labeled hCG-
proteolytic
products from CHO
Asn(1+2) cell lysates. CHO
Asn(1+2) cells were pulse-labeled with
[
S]cysteine for 5 min and chased for periods of
0-24 h. hCG-
subunits were immunoprecipitated and analyzed
by C
reversed-phase HPLC as described above. Panels
A-F represent the HPLC profiles of the
[
S]cysteine-labeled CHO
Asn(1+2) cell
lysates chased for 0, 1, 5, 10, 15, and 24 h, respectively.
p
1, p
1 lacking both N-linked glycans;
p
2, p
2 lacking both N-linked glycans;
C1 and C2, protein complexes containing
hCG-p
1
and chaperone-like proteins; d
,
proteolytic form(s) of the p
2
;
, mature
hCG-
with four O-linked
glycans.
Figure 9:
Identification of the proteolytic
hCG- substrate (d
) on SDS-PAGE.
The [
S]cysteine-labeled d
peak fractions (Fig. 8F) were analyzed by either
nonreducing (Panel A, lane 2) or reducing (Panel
B, lane 2) SDS-PAGE. Panels A and B,
lanes 1, [
C]-labeled molecular weight
markers. d
represents proteolytic form(s) of the
p
2
. d
f1 and
d
f2 represent the disulfide bond-linked
fragments of d
.
Figure 10:
Separation and identification of tryptic
peptides of reduced [S]cysteine-labeled
hCG-p
2
or -d
.
[
S]Cysteine-labeled hCG-p
2
and -d
were reduced and digested with trypsin.
The tryptic peptides were separated on C
reversed-phase
HPLC (see ``Experimental Procedures''). Panel A,
tryptic peptide HPLC profile of reduced
[
S]cysteine-labeled hCG-p
2
.
Every peak that contains a known specific peptide (6, 7) is numbered in
the order that it appears (Table I). Panel B, tryptic peptide
HPLC profile of reduced [
S]cysteine-labeled
hCG-d
The peaks 3 and 9 were
identified by amino acid sequencing (Panels E and F).
Panel C, ion-exchange HPLC analysis of peak 4 generated in Panel A showing that peak 4 contains two
co-eluted pepetides (
and
). Panel D, ion-exchange HPLC
analysis of peak 4 generated in Panel B showing that peak 4
contains only
peptide. Panel E,
amino acid sequencing of peak 3 generated in Panel B showing
that the third amino acid from the N-terminal end peptide is
[
S]cysteine-labeled cysteine. Panel F,
amino acid sequencing of peak 9 generated in Panel B showing
that the sixth and eighth amino acids from the N-terminal of the
peptide are [
S]cysteine-labeled
cysteines.
To
separate peptides (4a) and
(4b) that co-migrate on C
reversed-phase HPLC
(6, 7) , the peaks 4 of Fig. 10, A and B, were separated on
ion-exchange HPLC. As can be seen in Fig. 10C, peak 4
from the p
2
contained both 4a and 4b peaks,
indicating that both peptides
and
were present. However, only peak 4a was
present in the d
(Fig. 10D), indicating
was missing in d
.
contains leucine but not cysteine
residues, CHO
Asn(1+2) cells were labeled with
[³H]leucine to see if the C-terminal of
was
present in d
.
[³H]leucine-labeled hCG-p
2
and
-d
were digested with trypsin and analyzed as above.
Peaks 2 (
), 5
(
), 6 (
), 8
(
), 10 (
), and
12 (
) found in p
2
were
not found in d
(data not shown). The missing 2, 6,
and 10 peaks suggested that d
does not contain
and
. The
lack of peak 12 and peak 4b further support the conclusion that
d
did not contain intact
and
. The absence of peak 8 and the
presence of peak 9 indicated, as above, that
was clipped into
and
. Due to the absence of cysteine and leucine
residues in
, the
[
S]cysteine- and
[³H]leucine-labeled hCG-
would not detect
whether
was present in
d
. Since, however,
and
peptides were missing, it is likely that
was missing in d
as
well.
2
is degraded proteolytically into
d
lacking
and
. This strongly suggests that
d
is a
corelike fragment containing only
peptides
and
(30) .
DISCUSSION
The data reported here indicate that the t of
folding of hCG-p1 lacking both Asn
- and
Asn
-linked glycans into p
2 is significantly longer
(t = 33 min) than that of wild-type p
1 (t =7 min). However, most of the mutant p
1 can slowly
fold into the corresponding p
2 as seen after a 90 min chase
(Fig. 1C). We have previously reported that when
wild-type or glycosylation mutants of hCG-
are folded in
vitro, 60% of wild-type p
1 (two N-linked glycans),
60% of p
1 lacking the Asn
-linked glycan, 40% of
p
1 lacking the Asn
-linked glycan, and 10% of
hCG-p
1 lacking both Asn
- and Asn
-linked
glycans, were able to fold into the corresponding p
2
(19) .
The discrepancy in the extent of folding of the various
subunit
types in vitro and in vivo may be due to more optimal
conditions such as a favorable redox potential and the presence of
molecular chaperones that increase hCG-
-folding efficiency inside
cells.
subunit and its
assembly with the
subunit are completed within the ER while the
subunit contains high mannose type N-linked
oligosaccharides. Processing of the N-linked glycans to
complex oligosaccharides and addition of O-linked
oligosaccharides occur in the Golgi after
assembly and
translocation from the ER. Also, unglycosylated
secreted from CHO
cells transfected with
genes mutated in the Asn codons of the
N-linked glycan consensus sequences does become
O-glycosylated (data not shown). All of the disulfide bonds
are formed in
when it is secreted as
dimer from CHO
cells cotransfected with the Asn mutant
and
genes. This was
demonstrated by the absence of trypsin-releasable peptides digested
under nonreducing conditions by the methods previously
described
(6, 8) .
does not fold by a simple
sequential pathway and that folding occurs independently in different
domains of the molecule. The disulfide bonds of the putative domain(s)
involving amino acids 1-90 of hCG-
form in a discrete order.
However, the later forming disulfide bonds 93-100 and
26-110 begin to form before the complete formation of the early
forming disulfide bonds that stabilize the amino acid 1-90
domain(s) and continue to form after complete formation of the early
forming disulfide bonds. We have also shown that completion of the
26-110 disulfide bond of
does not occur until after
assembly
(26) , and indeed, if it is preformed by
complete oxidation in vitro,
will not assemble with
(8) . This is consistent with the crystal structure of the
hCG-
dimer in that the C terminus of
forms a seatbelt
around
(28) . This could only occur if the 26-110
disulfide bond is formed after
assembly.
and Asn
)
oligosaccharides may protect critical folding domains of the
molecule until proper folding occurs. This may account for the
deficiency of formation of disulfide bonds 93-100 and
26-110 in the absence of both N-linked oligosaccharides.
The crystal structure of hydrofluoric acid-treated
hCG-
(28, 29) suggests that in an early folding
intermediate the N-linked glycans may be located between two
important folding loops that are held together by disulfide bond
23-72. The N-linked glycans may protect important
hydrophobic residues in this region (such as Leu
,
Val
, Ile
, and Val
) and assist
disulfide 23-72 formation, which occurs before completion of
disulfide bonds 93-100 and 26-110. Thus, the instability of
93-100 and 26-110 disulfide bonds may result from less
efficient formation of disulfide bond 23-72.
. At least two classes of proteins, widely
distributed in prokaryotes and eukaryotes, are involved in polypeptide
folding
(18) . These proteins include: 1) enzymes such as
peptidylprolyl cis/trans-isomerase and protein disulfide
isomerase that catalyze specific rate-limiting isomerization steps in
protein folding; 2) binding proteins (molecular chaperones) that
stabilize unfolded or partially folded structures and prevent the
formation of inappropriate intra- or interchain interactions. The
molecular chaperones, composed of two major families (stress-70 and
stress-90) proteins in eukaryotic cells, are induced under a variety of
stress conditions and function in the stabilization, translocation, and
degradation of partially folded intermediates during polypeptide
folding and assembly. ERp72 has been identified as an ER protein
containing protein disulfide isomerase homology units
(31) . BiP
is a member of the stress-70 chaperone family in the ER of eukaryotic
cells. It has been proposed that BiP associates transiently with
unfolded or misfolded proteins to modulate protein folding and
translocation of folding intermediates across the ER
membrane
(18) . ERp94, a member of stress-90 chaperones, appears
to function with BiP to assist protein folding in the ER
lumen
(32) . In addition, calnexin (also called p88, IP90), an ER
trans-membrane protein, also plays a chaperone-like role by binding
with glucose-containing N-linked glycans of newly synthesized
glycoproteins
(33) .
is inefficient but does occur slowly,
accumulation of unfolded or misfolded hCG-
might stimulate the
formation of slowly dissociated unglycosylated
-chaperone
complexes. The C1 and C2 peaks observed in Fig. 5contain at
least seven proteins with a variety of molecular weights, some of which
have been identified by Western blots as the ER chaperones BiP, ERp72,
and ERp94. Both the C1 and C2 peaks disappear with increasing chase
time up to 5 h, during which time a greater amount of folded p
2
appears (Fig. 5, D-F) and some of the
d
degradation product is evident as well. This
strongly suggests that the
complexes contained in the C1 and C2
peaks are not dead-end folding complexes targeting
for
degradation, but rather are chaperone-
complexes that assist
subunit folding. Therefore, the slower folding rate of unglycosylated
may have enabled us to detect such complexes that are transiently
formed in the folding pathway of wild-type
. This hypothesis is
currently being tested in our laboratory.
, however, eventually occurs in CHO
cells ( Fig. 8and Fig. 9). This appears to be derived from
p
2
forms that are unstable without N-linked
glycans since that is the form of
mainly present after a 5-h
chase when the degradation product d
begins to
appear (Fig. 8). There is some precedent for this based on
observations with other glycoproteins. Hoe and Hunt (34) reported that
human transferrin receptor lacking N-linked oligosaccharide is
unable to form intermonomer disulfide bridges and undergoes
site-specific proteolysis after a long period of time in the ER lumen.
It was also shown that unglycosylated subunits of thyroid-stimulating
hormone are 50-65% degraded intracellularly
(35) .
Furthermore, unglycosylated vesicular stomatitis virus G protein forms
non-native intramolecular disulfide bonds and is degraded
intracellularly
(22) .
degradation product of unglycosylated
is a
core-like
fragment. The
core fragment, which contains the
and the
portions
of
subunit linked by disulfide bonds, is a major immunoreactive
component in the urine of pregnant women
(30, 36) . This
fragment has been considered as a partially degraded
subunit of
hCG produced in the kidney
(37) . The C-terminal region of
is susceptible to degradation and is cleaved free from a
disulfide-bridged core product. Also, the region between residues 40
and 50 is highly susceptible to proteolysis
(13, 30) . It
seems that the N-linked glycans shield the protease-cleavage
sites in hCG-
or that the conformation of misfolded or unfolded
unglycosylated hCG-
is more accessible to endopeptidases.
Table:
Trypsin-released peptides from reduced
hCG-p2
and
core fragment-like protein
S]cysteine- or
[³H]leucine-labeled hCG-p
2
and
core fragment-like protein were reduced and digested with
trypsin. The tryptic peptides were separated on HPLC (see
``Experimental Procedures''). The peaks were numbered in the
order in which they appear in the HPLC profiles (Fig. 10, A and B). The peaks contained the
peptides identified
previously (6, 7) or in Fig. 10, E and F. It should
be noted that the incomplete digestion at tryptic site between
Arg
and Leu
results in the appearance of
(peak 6) and that the proteolytic clip of
the Leu
-Ser
peptide bond produces peptides
and
(peak 5)
that are derived from
(peak 8) (6, 7).
WT, wild type hCG-
gene product;
Asn1,
hCG-
lacking Asn
;
Asn2, hCG-
lacking
Asn
;
Asn(1+2), hCG-
lacking both Asn
and Asn
; CHO, Chinese hamster ovary; ER, endoplasmic
reticulum; PAGE, polyacrylamide gel electrophoresis; HPLC, high
performance liquid chromatography; IOD, integrated optical density.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.