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Departments of Molecular Biology and Pharmacology and Obstetrics and Gynecology (M.M., I.B.) Washington University School of Medicine St Louis, Missouri 63110
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunit and ß-subunits that confer biological specificity.
LHß and CGß share more than 80% amino acid sequence identity;
however, in transfected Chinese hamster ovary (CHO) cells, LHß
assembles with the -subunit more slowly than does hCGß, and only a
fraction of the LHß synthesized is secreted, whereas CGß is
secreted efficiently. To understand why the assembly and secretion of
these related ß-subunits differ, we studied the folding of LHß in
CHO cells transfected with either the LHß gene alone, or in cells
cotransfected with the gene expressing the common -subunit, and
compared our findings to those previously seen for CG. We found that
the rate of conversion of the earliest detectable folding intermediate
of LH, pß1, to the second major folding form, pß2, did not differ
significantly from the pß1-to-pß2 conversion of CGß, suggesting
that variations between the intracellular fates of the two ß-subunits
cannot be explained by differences in the rates of their early folding
steps. Rather, we discovered that unlike CGß, where the folding to
pß2 results in an assembly-competent product, apparently greater than
90% of the LH pß2 recovered from LHß-transfected CHO cells was
assembly incompetent, accounting for inefficient LHß assembly with
the -subunit. Using the formation of disulfide (S-S) bonds as an
index, we observed that, in contrast to CGß, all 12 LHß cysteine
residues formed S-S linkages as soon as pß2 was detected. Attempts to
facilitate LH assembly with protein disulfide isomerase in
vitro using LH pß2 and excess urinary -subunit as substrate
were unsuccessful, although protein disulfide isomerase did facilitate
CG assembly in this assay. Moreover, unlike CGß, LHß homodimers
were recovered from transfected CHO cells. Taken together, these data
suggest that differences seen in the rate and extent of LH assembly and
secretion, as compared to those of CG, reflect conformational
differences between the folding intermediates of the respective
ß-subunits. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunit and
a distinct ß-subunit that confers biological specificity (1). Human
LHß of the anterior pituitary and placental hCGß are the most
closely related ß-subunits of this family, apparently having evolved
from the same ancestral gene (2). LH- and hCGß share more than 80%
sequence identity, including 12 conserved cysteine residues that form 6
intramolecular disulfide (S-S) bonds (1). The structural similarities
between LH and hCG account for the fact that they bind the same
receptor and elicit the same biological response (3). However, despite
these similarities, LHß and hCGß subunits exhibit dramatic
differences in their rates of secretion as monomer and assembly with
the common -subunit. hCGß is secreted and assembles with the
-subunit quantitatively, whereas the secretion and assembly of LHß
are inefficient. These intracellular characteristics of the subunits
are observed in transfected Chinese hamster ovary (CHO) cells (4, 5),
mouse C-127 mammary tumor cells (6), and somatotrope and
corticotrope-derived GH3 and AtT-20 cells (7, 8), respectively.
Previous studies have shown that the hCGß subunit undergoes multiple
maturation steps characterized by the formation of intramolecular S-S
bonds to attain an assembly-competent conformation (9, 10, 11). Thus,
variations in the rate and/or extent of folding of the LHß subunit
could be responsible for its inability to assemble and be secreted
efficiently.
The reported S-S bond pairing of the ovine LHß subunit between Cys
residues 34–88, 38–57, 9–90, 23–72, 93–100, and 26–110 (12) is
the same as that observed during the hCGß kinetic folding pathway
(10, 11, 13). However, the crystal structure of secreted hCGß (14, 15) reveals S-S bonds formed between Cys residues 38–90 and 9–57
rather than between Cys residues 38–57 and 9–90 and implies that a
S-S bond rearrangement occurs during the folding or processing of
hCGß. That the positions of the cysteine residues in hCGß and LHß
are conserved (1) and both ß-subunits assemble with a common
-subunit suggest that the folding steps leading to formation of
assembly-competent ß-subunits are similar and that disulfide bond
formation could be used as an index of LHß folding, as it has been
used for hCGß folding. We have previously shown that folding of
hCGß from an early detectable precursor, pß1, to an
assembly-competent intermediate, pß2, and assembly of hCG pß2 with
the common -subunit, can be monitored by hCGß S-S bond formation
(for recent reviews see Refs. 16, 17, 18). The pairing order of these six
hCGß S-S bonds is the same whether wild-type CGß folds in human
choriocarcinoma (JAR) cells (10), where the hCGß gene is eutopically
expressed, in transfected CHO cells (11), or in vitro using
purified hCGß subunit as substrate (13). Intracellular hCGß folding
occurs in the presence or absence of the -subunit (11).
To determine whether differences in the folding pattern between the
hCG- and LH- ß-subunits could account for the intracellular behavior
of LHß, we studied LHß biosynthesis in CHO cells transfected with
the human LHß gene alone, or with the human glycoprotein hormone
-subunit. We report here that, although the early folding steps for
LHß and hCGß occur with similar kinetics in CHO cells, LH pß2, in
contrast to hCG pß2, had minimal ability to assemble with the common
-subunit. Greater that 90% of the LH pß2 synthesized was assembly
incompetent for at least 8 h after biosynthesis. Therefore, the
rate-limiting step in the attainment of LHß assembly competence does
not appear to be the pß1-to-pß2 conversion step, but
rather, the maturation of the LH pß2 folding intermediate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunit is markedly less efficient than that
of the hCG ß-subunit in transfected CHO cells (4, 5), mouse mammary
tumor C-127 cells (6), and somatotrope-derived GH-3 and AtT-20 (7, 8)
cells. These findings suggest that differences in the rate and extent
of formation of assembly-competent forms could explain the differences
in the intracellular fate of these two closely related ß-subunits. To
examine this issue, CHO cells transfected with only the LHß gene were
pulse labeled for 5 min with [35S]Cys and chased with
unlabeled medium (Fig. 1
). After a 5-min
pulse and a 0-min chase, two major forms of LHß were detected by
nonreducing SDS-PAGE (Fig. 1A). The more slowly migrating of these two
forms (Mr = 30,000) apparently represents the LHß
homodimer (LH ß/ß). There was no apparent precursor-product
relationship in the formation of LH ß/ß since large amounts of the
homodimer, representing equivalent percentages of total LHß seen at
later chase times, were recovered after chase periods of 0 min (Figs. 1, A and B). Also observed at 0 min of chase was an early form of LHß
that migrated with approximately half the apparent mol wt of LH
ß/ß, termed pß1 (Mr = 17,000). LH pß1 converted to
a second intermediate, pß2 (Mr = 24,000), with a
t1/2 of 7–8 min (Fig. 1A), demonstrating that
conversion of LHß folding intermediates pß1 to pß2 was analogous
to the conversion of hCG pß1 to pß2, which we have previously
demonstrated occurs with a t1/2 of 4–5 min (10, 11).
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-subunit would affect the
kinetics of formation of LHß folding intermediates, we examined cells
expressing both LHß and the -subunit. It should be noted (see Fig. 1
) that the intracellular -subunit migrates with a Mr of
19,000 in either nonreducing (panels B and C) or reducing gels (panels
E and F). On the other hand, nonreduced LH pß1 (panels A–C) and
reduced LHß (panels D–F) migrate more rapidly than the -subunit,
while LH pß2 (panels A–C) migrates more slowly than . At an
/ß-subunit ratio of either 0.4 (Fig. 1B) or 1.6 (Fig. 1C), the
t1/2 of conversion of LH pß1 to pß2 was not
altered. Conversion of LH pß1 to pß2 involves the formation of S-S
bonds since these forms collapse to a single band under reducing
conditions (Fig. 1D–F). These findings demonstrate that the rate of
conversion of LH pß1 to pß2 did not differ significantly from that
of hCGß, even in the presence of -subunit, and suggest that the
differences between the intracellular fates of the two ß-subunits
cannot be explained by differences in the rate of their early folding
events.
Role of pß2 in LH-ß Assembly
To ensure that the extent of heterodimer formation was not limited
by the amount of -subunit present in the following experiments, CHO
cells overexpressing the common -subunit relative to the LHß
subunit (i.e. at an -/ß-subunit ratio of 1.6) were
used. The extent of LH heterodimer formation was assessed by
immunoprecipitation with subunit-specific antisera. Precipitation of
the -subunit with ß-antiserum, or the precipitation of the
ß-subunit with -antiserum, indicates heterodimer formation.
Following pulse labeling, LH heterodimer precipitated with either -
or ß-antisera contains radiolabeled (newly synthesized) -subunit
during initial chase periods; however, very little radiolabeled LHß
associated with labeled -subunit is detected when the heterodimer is
precipitated with -antiserum (4, 5, 6). This is presumably due to the
presence of a stable, preexisting nonradiolabeled intracellular pool of
assembly-competent LHß subunit that accumulates because nascent LHß
requires time to become assembly competent (4, 5, 6).
To identify all forms of LH subunits present in our cells, we performed
Western blot analysis under nonreducing conditions of CHO cell lysates
expressing both the LH - and ß-subunits (Fig. 2). Panel A shows that when polyclonal
antiserum to the -subunit was used, two bands appeared: a band of
Mr = 19,000 was seen when intracellular lysates were probed
(-int; lane A1), while a heterogeneous band typical of secreted
-subunit (Mr = 22,000–26,000) was detected in the
medium (-sec; lane A2). When polyclonal antiserum to the LH
ß-subunit was used, multiple bands appeared (panel B). In lysates,
the most slowly migrating band (panel B, lane 1) is LH ß/ß
homodimer, while a single LHß band was detected as mature secreted
LHß (ß-sec) (panel B, lane 2). In addition to LH ß/ß, lysates
contained two bands that migrated at Mr = 22,000 and
Mr = 20,000 (Fig. 2B, lane 1, designated pß2-U (upper),
and pß2-L (lower), respectively]. Both of these bands are likely to
be pß2 forms, based on their apparent mol wts (pß1 migrates at
Mr = 17,000 (Fig. 1, A–C)).
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). The more predominant of the two
pß2 folding intermediates, pß2-U, migrated more slowly than the
lower band, pß2-L, and contained more than 90% of the radiolabeled
LH pß2. When CHO cells expressing both the LHß subunit and the
-subunit were pulse labeled and precipitated with LHß antiserum
(Fig. 3B), a small amount of pß2-L, was detected. The appearance of
LH pß2-L is clearer after a 60-min chase, when changes in the
migration rate of the -subunit, presumably due to processing of the
N-linked oligosaccharides, allows pß2-L to be more readily
distinguished from . At these later chase times, -subunit
secretion occurred, which reduced the amount of intracellular
-subunit detected. Figure 3C shows that the minor folding form,
pß2-L, associates with the -subunit. Here, CHO cells expressing
both the LHß subunit and the -subunit were labeled and
immunoprecipitated with antiserum against the -subunit. Under these
conditions the rapidly migrating LH pß2-L, but not LH pß2-U, was
detected at chase times of 1–4 h as -subunit processing proceeded,
indicating that pß2-L was not a spurious -subunit band. Further
verification that pß2-L was a form of LHß was obtained when we
immunoprecipitated CHO cell lysates of cells expressing only the
-subunit with -antisera and failed to detect its presence (Fig. 3D). Taken together, these data suggest that a form of LH pß2
representing less than 10% of the total pß2 in the cell was the only
form of LH pß2 competent to associate with the -subunit and
provide an explanation for why LHß assembly is inefficient.
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), we failed to detect any
peptides released from the S-S-linked core material of LH pß2 (Fig. 4A). The radioactivity eluting in fractions 4–6 (Fig. 4A) coincides
with the void volume of the column and does not contain LHß peptides
when rechromatographed and thus appears to be free
[35S]Cys or some other degradation product (Refs. 10, 11 and data not shown). This suggests that all of the LHß cysteine
residues were paired as soon as pß1-to-pß2 conversion was detected.
By contrast, in hCGß, peptides 96–104 (peaks 1a and b) and 105–114
(peak 2) are released from assembly-competent pß2 (Fig. 4B; and Refs.
10, 11, 13). The release of these two peptides indicates that S-S
bonds 93–100 and 26–110 remain unformed when hCG pß1-to-pß2
conversion occurs (10, 11, 13), which is consistent with the
observation that these two bonds form coincident with or shortly after
hCGß assembly with the -subunit (10). In the case of LHß,
however, it appears that all free thiols were converted to S-S bonds as
LHß folded from pß1 to pß2.
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-subunit (13) because this is the
last S-S bond to form during hCGß folding (10, 11, 13) and forms a
"seatbelt" (14) that stabilizes native hCG following heterodimer
assembly; preformation of the 26–100 bond inhibits CGß assembly with
the -subunit (13). We, therefore, examined whether the 26–110 S-S
bond of LHß had already formed, preventing LH assembly. To do this,
we used an in vitro assembly assay (13). We have previously
demonstrated that protein disulfide isomerase is capable of reducing
the 26–110 and 93–100 S-S bonds of hCGß, thereby enhancing its
in vitro assembly with the -subunit under appropriate
redox conditions (13). Similarly, we determined whether protein
disulfide isomerase was capable of enhancing LH assembly. Using
HPLC-purified pß2 (see Materials and Methods) derived from
CHO cells expressing either the LHß or CGß subunits, we examined
the ability of the respective pß2 subunits to assemble with a large
molar excess of purified urinary -subunit in vitro (Fig. 5). Under conditions where hCG pß2
efficiently assembled with the -subunit (Fig. 5A; also Ref. 13), no
heterodimer containing LH pß2 was detected (Fig. 5B). This result
supports the hypothesis that there is some structural constraint other
than reduction of the 26–110 S-S bond in LH pß2 that must be
overcome before the LHß subunit can form heterodimer. Conceivably,
these constraints might make the LHß 26–110 S-S bond insusceptible
to protein disulfide isomerase.
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, A and
B, and Fig. 3, A and B). There appeared to be no precursor-product
relationship between LH ß/ß and other LHß folding intermediates
as large amounts of the homodimer representing equivalent percentages
of total LHß seen at later chase times were recovered following chase
periods of 0 min (Fig. 1, A and B). These forms persisted within the
cell for chase periods of up to 8 h (Fig. 3, A and B). Moreover,
since LH ß/ß comigrated with LHß monomer (Mr =
17,000) under reducing conditions (Fig. 1, D and E), it appears that LH
ß/ß is formed by intermolecular S-S bonds. For these reasons, and
because the tryptic digestion of LH ß/ß produced a peptide map
similar to those seen when intermolecular S-S bond containing
homodimers and multimers of hCG-ß were analyzed (Ref. 19 and data not
shown), we have concluded that this material represents a homodimeric
form of the LHß subunit.
High levels of LH ß/ß were detected in LHß transfected CHO cells
either lacking (Figs. 1A and 3A) or underexpressing (Fig. 1B) the
-subunit relative to LHß (/ß subunit ratio = 0.4).
However, when the -subunit was overexpressed (/ß subunit
ratio = 1.6), very little LH ß/ß was seen, especially at early
chase times (Figs. 1C and 3B). Thus, it appears that the -subunit is
affecting the kinetics of formation and the amount of LH ß/ß
formed. LH ß/ß was not detected in the media following chase
periods of up to 8 h (Figs. 3, A and B), demonstrating that, like
unassembled LHß subunits (Fig. 3A), secretion of LH ß/ß was
inefficient.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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hCGß folds via the following kinetic pathway (19):
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By contrast, conversion of LH pß1 to pß2 did not produce an
assembly-competent subunit. Rather, for LHß, the folding and assembly
steps appeared to require additional conformational changes, possibly
involving S-S bond rearrangement before becoming assembly competent. In
contrast to hCGß, all 12 LHß cysteine residues appeared to be
involved in S-S linkages as soon as pß1-to-pß2 conversion was
detected. This conclusion was based on the observation that no peptides
were released from LHß S-S linked core protein following tryptic
digestion (Fig. 4A). While we cannot rule out the possibility that
tryptic cleavage sites were buried due to collapse of the hydrophobic
domains of the LHß subunit, this seems unlikely because the purified
form of LHß subjected to trypsin had been highly denatured by
exposure to SDS and 6 M guanidinium hydrochloride during
the extraction and purification procedures before trypsin
treatment.
It is not clear why LHß folds and assembles differently from hCGß.
While the two molecules share extensive homology, they have very
different C-terminal amino acid sequences (1). hCGß has a 31-amino
acid hydrophilic C-terminal peptide that contains four
O-linked glycans. Unlike hCGß, LHß possesses a
seven-amino acid hydrophobic C terminus. The hydrophobicity conferred
by the LHß C-terminal heptapeptide, together with hydrophobic amino
acid residues localized at the amino terminus of the molecule, appear
capable of serving as nucleation sites for LHß aggregation soon after
the nascent polypeptide is synthesized (4, 5). This is consistent with
previous studies showing that an interaction of the hydrophobic LHß C
terminus with other LHß residues is critical in delayed secretion and
assembly of the LHß subunit (5). This hypothesis is also supported by
the detection of a homodimeric species of LHß, LH ß/ß. There is
apparently no precursor-product relationship between the LH ß/ß
form and the assembly-competent subunit since it was detected following
a 0-min chase. LH ß/ß was detected intracellularly in all CHO cell
clones, regardless of the expression level of LHß (data not shown)
following chase periods of up to 8 h. Because of this
intracellular stability, LH ß/ß may represent a dead-end
nonproductive product or an assembly-incompetent storage form of LHß,
a fraction of which can be rescued when sufficient amounts of
-subunit are present to drive assembly.
By decreasing LH ß/ß formation (aggregation), and facilitating
folding and assembly, the -subunit, when overexpressed in CHO cells,
appears to be acting in a chaperone-like manner, presumably by binding
to LHß subunits before they bind each other. Since LHß is not
assembly competent following 0 min of chase, the -subunit seems to
bind assembly-incompetent LHß at one epitope accessible in
assembly-incompetent LHß and at a different epitope, found only in
assembly-competent LHß, during heterodimer formation. This mechanism
explains why LHß need not be assembly competent to bind . It is
likely that the endoplasmic reticulum chaperones play a role in
facilitating the folding and assembly of LHß. We previously
identified hCGß-chaperone complexes that facilitate folding and
assembly of CG in transfected CHO cells (22). The endoplasmic reticulum
chaperones, BiP, ERp72, GRp94 (22), calreticulin, and calnexin (E.
Bedows, unpublished), associate with hCGß as pß1 folds into
assembly-competent pß2. Because of the large degree of amino acid
identity between CGß and LHß subunits, and the hydrophobic nature
of the LHß C terminus, molecular chaperone intervention likely
determines how and when LHß folding intermediates proceed along their
kinetic folding pathway.
The intracellular behavior of these two gonadotropin ß-subunits may
reflect their respective biological roles. Secretion of hCG from the
placenta is primarily constitutive to maintain the corpus luteum,
whereas secretion of LH from the pituitary is pulsatile and regulated
by LHRH levels (for a review see Ref. 23). Since unassembled LHß is
not secreted efficiently, the ability of the pituitary to build up
stores of free LHß may assist secretion of large quantities of the
hormone from the anterior pituitary during the LH surge before
ovulation (4). There remain several unanswered questions about the
mechanisms for the selective retention of LHß in the endoplasmic
reticulum. Cellular factors including chaperone association (24, 25, 26)
and the presence of intracellular retention signals (27) may influence
how quickly secretory proteins such as LH are allowed to exit the
endoplasmic reticulum. It will be important to explore how these
factors differentially control the folding, assembly, and secretion of
the glycoprotein hormone ß-subunits with their common
-subunit.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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gene, were grown in F-12 medium supplemented with 5% FBS,
the neomycin analog G-418 (GIBCO, Grand Island, NY), and antibiotics
(19, 28).
Metabolic Labeling of Cells with Radioactive Substrates
CHO cells grown to 90–95% confluency in 100-mm plastic dishes
were pulse labeled for the times indicated in the text with
L-[35S]cysteine (
1100 Ci/mmol; Du Pont-New
England Nuclear, Boston, MA), at a concentration of 200–300 µCi/ml,
in serum-free medium lacking cysteine (19). All pulse incubations were
carried out as described previously (19), and the cells were incubated
for the chase times indicated in the text. Cells were harvested by
rinsing with cold PBS and immediately lysed in 5 ml PBS containing
detergents (1.0% Triton X-100, 0.5% sodium deoxycholate, and 0.1%
SDS); protease inhibitors (20 mM EDTA and 2 mM
phenylmethanesulfonyl fluoride); and 50 mM iodoacetic acid
(pH 8.0), to trap the free sulfhydryl groups of the ß folding
intermediates. Cell lysates were incubated 20–30 min at 22 C in the
dark, followed by disruption through a 22-ga needle (three times),
centrifuged for 1 h at 100,000 x g, and
immunoprecipitated (see below) or frozen at -70 C for further
use.
Immunoprecipitation of Cell Lysates and Culture Media
The immunoreactive forms of LHß or hCGß were
immunoprecipitated with a rabbit (4) or goat (19) polyclonal antiserum
that recognizes the folding intermediates of both ß-subunits. Because
of the sequence identity between them, efficient precipitation of both
subunits was observed. All immunoprecipitations were carried out for
16 h at 4 C with rotation in the dark. Immune complexes were
precipitated with Protein A-Sepharose (Sigma Chemical Co., St. Louis,
MO) and prepared for SDS-PAGE or reversed-phase HPLC as described
below.
SDS-PAGE, Fluorography, and Western Blot Analysis
Radiolabeled LH or CG forms that adsorbed to Protein A-Sepharose
beads were eluted with 2 x concentrated SDS gel sample buffer
(125 mM Tris-HCl, pH 6.8, containing 2% SDS, 20%
glycerol, and 40 µg/ml bromophenol blue). Samples run under reducing
conditions were boiled for 4 min in sample buffer containing 2%
ß-mercaptoethanol, while samples run under nonreducing conditions
were boiled for 4 min in sample buffer lacking ß-mercaptoethanol. The
washed samples, including the Protein A-Sepharose beads, were applied
to polyacrylamide gradient slab gels (5–20%) that were run by the
method of Laemmli (29). Gels were rinsed in water, dried in
vacuo on filter paper, and exposed to x-ray film. Fluorographs
were photographed with a Kodak CCD (charged caption device) camera
(BioImage 110S System, Genomic Solutions Inc., Ann Arbor, MI).
Quantitation of gel images was obtained by transferring photographed
fluorograph images to a Sun SPARCstation 1+ computer and analyzed using
BioImage Whole Band software and printed on a Seiko Instruments USA
Inc. (San Jose, CA) CH-5504 color printer.
Western blot analysis was performed using aliquots (50 µl) of media
or cell lysates from CHO cells expressing LHß and the common
-subunit following a 24-h incubation in conditioned media lacking
serum and were resolved by SDS-PAGE on a 5–20% gradient gel under
nonreducing conditions. Gels were transferred to nitrocellulose
membranes and probed with either -antiserum or hCGß antiserum.
Proteins were detected by the Tropix (Bedford, MA) chemiluminescent
detection system.
Purification of ß-Subunit Folding Intermediates and HPLC
Analysis
The hCGß and LHß folding intermediates pß1 and pß2 were
purified by a two-step process (immunoprecipitation followed by
C4 reversed-phase HPLC) as described by Huth et
al. (10). Briefly, pß1 and pß2 were immunoprecipitated from
cell lysates with polyclonal antisera, and immunocomplexes were
precipitated with Protein A-Sepharose beads. To dissociate precipitated
immunocomplexes, pellets were treated with 6 M
guanidine-HCl (pH 3) (Pierce, Rockford, IL; sequencing grade) for
16 h at room temperature with 100 µg of myoglobin (Sigma) as
carrier. Following low-speed centrifugation to remove Protein-A
Sepharose beads, the guanidine eluates were injected onto a Vydac 300
Å C4 reversed-phase column (Hesparia Separations Group,
Hesparia, CA) equilibrated with 0.1% trifluoroacetic acid (TFA) and
eluted using an acetonitrile gradient as previously described (10).
Fractions containing LHß or hCGß forms were concentrated by vacuum
centrifugation and pooled for tryptic analysis.
Tryptic Digestions and HPLC Purification of Tryptic Peptides
Nonreduced LHß or hCGß forms were digested for 16 h at
37 C in silanized polypropylene tubes containing 100 µg myoglobin,
0.03% diphenylcarbamyl chloride-treated Trypsin (Sigma), 5
mM CaCl2, and 100 mM Tris-HCl, pH
8. The digestion was continued with the addition of two sequential
aliquots of 50 µg trypsin (0.06% final concentration) for 2 h.
Tryptic digests of ß-subunits were injected onto a Brownlee 300 Å
C8 reversed-phase column (Applied Biosystems, Foster City,
CA) equilibrated with 0.1% TFA (10, 11). The column was eluted
isocratically for 3 min with 0.1% TFA followed by a 0.32%/min
acetonitrile gradient in 0.1% TFA for 100 min. The column was washed
with 80% acetonitrile, 0.1% TFA for 5 min and then reequilibrated in
0.1% TFA. The flow rate was 1.0 ml/min. One-minute fractions were
collected in silanized polypropylene tubes. Tubes into which S-S-linked
peptides eluted contained 5 µg myoglobin as carrier. Samples were
concentrated by vacuum centrifugation and stored at -20 C.
Identification of Peptides Following Tryptic Digestion
Fully folded hCGß contains 6 disulfide bonds and 13 Arg and
Lys residues that are arranged such that all of the Cys-containing
tryptic peptides remain attached to each other as a result of their
covalent disulfide bridges (9, 10). If, however, particular disulfide
bonds are not formed in a given hCGß folding intermediate, specific
Cys-containing tryptic peptides are released from the S-S-linked CGß
core. For example, if the 26–110 bond is unformed, then CGß peptide
105–114 (containing Cys-110) would be released. The pattern of
tryptic-released peptides, distinguished from the disulfide-linked
peptides by HPLC, reveals incomplete bond formation (10). By lysing
cells in the presence of the alkylating agent iodoacetate, the Cys
residues of the unformed hCGß S-S bonds are trapped. The alkylated
folding intermediates are then resolved by C8
reversed-phase HPLC (10). Identification of HPLC peptide peaks was made
by comparing elution times of peaks generated from wild-type CGß
tryptic digests (10, 11) that had been verified by microsequencing.
Amino acid sequence analysis revealed whether the Cys-containing
peptides had been alkylated (indicating that the S-S bond had not been
formed in the intact molecule).
In Vitro Assembly of LH- and CG and -ß
Subunits
In vitro assembly reactions were performed by a
modification of the procedure described by Huth et al. (13).
To generate the pß2 used in the experiment shown in Fig. 5, CHO cells
expressing either CGß or LHß were pulse labeled for 5 min with
[35S]Cys and chased with unlabeled medium for 20 min for
CGß or 30 min for LHß, followed by lysis in PBS containing EDTA,
phenylmethanesulfonyl fluoride, and the detergent mixture described
above, but lacking alkylating agent. Respective CG and LH pß2
subunits were purified by immunoprecipitation followed by
reversed-phase HPLC. HPLC-derived fractions containing radiolabeled
pß2 substrate to be used in CG or LH assembly reactions were
concentrated under vacuum. Each reaction contained a final
concentration of 1 µM urinary hCG, 150,000 cpm (2
ng) [35S]cysteine-labeled pß2, 1.7 mM
reduced glutathione, 0.27 mM oxidized glutathione, 1
mM EDTA, and 20 mM sodium phosphate (pH 7.8); a
final concentration of 17.5 µM bovine liver protein
disulfide isomerase (Takara Biochemical Inc., Berkeley, CA) was also
included in the assay. The reactions were incubated at 37 C, and
aliquots were withdrawn at the times indicated and terminated with a
solution of 900 mM iodoacetate containing 450
mM Tris-HCl (pH 8.7). Samples were mixed with ice-cold
nonreducing electrophoresis buffer and analyzed by SDS-PAGE at
4 C.
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FOOTNOTES |
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This work was supported in part by NIH Grants HD-23398 and CA-32949, by American Cancer Society Institutional Grant IRG-165G and NCI Cancer Center Support Grant P30CA3627.
1 Current address: University of Rochester Medical Center, Department
of Biochemistry, Box 607, 601 Elmwood, Rochester, New York 14620. 
2 Current address: Corporate Office of Science and Technology,
Johnson & Johnson, 410 George Street, New Brunswick, New Jersey 08901.
Received for publication April 8, 1998. Revision received June 10, 1998. Accepted for publication June 21, 1998.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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ß dimer assembly competent form.
J Biol Chem 265:312–317ß dimer by protein disulfide
isomerase. J Biol Chem 268:16472–16482This article has been cited by other articles:
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