(Received for publication, December 12, 1994; and in revised form, February 1, 1995)
From the
The high affinity immunoglobulin E (IgE) receptor is an
tetrameric complex. The truncated
extracellular segment (
t) of the heavily glycosylated
chain
is sufficient for high affinity binding of IgE. Here we have expressed
various
t mutants in eukaryotic and prokaryotic cells to analyze
the role of glycosylation in the folding, stability, and secretion of
t. All seven N-linked glycosylation sites in
t are
glycosylated and their mutations have an additive effect on the folding
and secretion of
t. Mutation of the seven N-glycosylation
sites (
1-7
t) induces misfolding and retention of
t in the endoplasmic reticulum. Similarly, tunicamycin treatment
reduces substantially the folding efficiency of wild-type
t. In
contrast, no difference in folding efficiency is detected between
wild-type
t and
1-7
t expressed in Escherichia
coli. In addition, maturation of N-linked
oligosaccharides and addition of O-linked carbohydrates are
not required for either the transport or the IgE-binding function of
t. Furthermore, complete enzymatic deglycosylation does not affect
the stability and the IgE-binding capacity of
t. Therefore,
glycosylation is not intrinsically necessary for proper folding of
t but is required for folding in the endoplasmic reticulum. Our
data are compatible with the concept that specific interactions between N-linked oligosaccharides and the folding machinery of the
endoplasmic reticulum are necessary for efficient folding of
t in
eukaryotic cells.
The high affinity IgE receptor (FcRI), which is required
for the initiation of IgE-mediated allergic reactions (Dombrowicz et al., 1993), is expressed constitutively on mast cells and
basophils (Metzger et al., 1986; Kinet, 1990). It is also
expressed on Langerhans cells (Bieber et al., 1992; Wang et al., 1992), on monocytes of allergic individuals (Maurer et al., 1994), and on eosinophils of some patients with
hypereosinophilia (Gounni et al., 1994). Fc
RI is a
multimeric complex consisting of an IgE-binding
chain, a
chain, and a dimer of disulfide-linked
chains which associate
through noncovalent interactions (Kinet, 1990; Ravetch and Kinet,
1991). The
chain is a type I integral membrane protein which is
homologous to Fc receptors for IgG (Fc
RII, Fc
RIII) (Hogarth et al., 1992). The extracellular domain of human
contains 181 amino acid residues, and is organized into two
immunoglobulin-like domains defined by two pairs of cysteine residues
forming two disulfide bridges (Kochan et al., 1988; Shimizu et al., 1988). Previous studies have shown that expression on
the plasma membrane of the extracellular domain of
reconstitutes
IgE binding (Blank et al., 1989; Hakimi et al.,
1990). Furthermore, truncation of the transmembrane and cytoplasmic
domains of human Fc
RI
results in a protein (
t)
efficiently secreted by CHO (
)cells and yet able to mediate
high-affinity binding of IgE (Blank et al., 1991). The same
t construct has also been expressed in a secreted and active form
in Escherichia coli (Robertson, 1993). Recently, the second Ig
domain has been shown to contain at least a portion of the receptor
binding site (Mallamaci et al., 1993; Robertson, 1993).
However, this second domain binds IgE only with low affinity.
Therefore, it is likely that the first domain is somehow also involved
in providing the receptor with a high-affinity binding site.
The
extracellular domain of the chain is heavily glycosylated. The
core protein of
has a theoretical molecular weight (M
) of 19,275, but the apparent M
estimated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) is 50,000 (Blank et al., 1991).
This increase in M
is primarily due to N-linked glycosylations, which represent 38-42% of the
total M
of the full
chain, and secondarily
to O-linked carbohydrates which represent 4% of the total M
(La Croix and Froese, 1993). Sequence analysis
reveals seven potential N-glycosylation sites in the
extracellular part of the human
chain (Kochan et al.,
1988; Shimizu et al., 1988). The mouse and rat Fc
RI
chains display 6 and 7 N-glycosylation sequons, respectively
(Kinet et al., 1987; Ra et al., 1989), but only one
sequon position is conserved among murine, rat, and human
chains.
While a role for N-linked oligosaccharides in the folding
of glycoproteins is widely recognized, the mechanisms underlying this
function are still unclear. Current understanding does not allow
accurate predictions of the role of glycosylation for a particular
protein. For some glycoproteins, N-linked oligosaccharides are
necessary for their overall stability, while for others the presence of
the sugars is only needed during the folding process. Some proteins,
such as IgD, fold more efficiently without sugars, while the trimming
of the N-linked oligosaccharides is essential for others
(reviewed in Helenius(1994)). In the present study, we clarify the role
of glycosylation in the maturation of t. We show that the main
function of glycosylation is to facilitate the folding process of
t in the endoplasmic reticulum of eukaryotic cells.
Figure 1:
Mutations of N-linked
glycosylation sites. Panel A, schematic representation of
FcRI
t chain indicating the positions of the N-linked glycosylation sites within the polypeptide sequence.
The shaded bar represents the leader peptide, the open
bar, the mature polypeptide. The N-linked glycosylation
sites are numbered from 1 to 7 and their positions in the amino acid
sequence is indicated. Panel B, amino acid changes for each N-glycosylation site mutation.
In order to assess the usage of each individual sequon, COS-7 cells
were transfected with mutated or WT t constructs and metabolically
labeled with [
S]methionine-cysteine for 3 h. The
WT and mutated
t proteins, including both intracellular and
secreted forms, were then immunoprecipitated with the anti-
antibody, mAb 15-1, and resolved on SDS-PAGE. The intracellular
WT
t is composed of polypeptides with different degrees of
glycosylation. This generates a ladder of bands with the two at the
top, around 40 kDa, being most prominent (Fig. 2A). All
mutants of
t show a similar pattern but with a 4-kDa increase in
electrophoretic mobility. This systematic downward shift indicates that
all the potential N-glycosylation sequons of WT
t are
glycosylated (see also Fig. 9). The bands corresponding to the
secreted forms of WT and mutated
t are much broader, indicating an
heterogeneity most likely due to the Golgi processing of
t (Fig. 2B). These broad bands are not detected
intracellularly, probably because of a rapid secretion after Golgi
processing. The increase in electrophoretic mobility observed
intracellularly with the mutated forms of
t is also seen on
secreted proteins, except when glycosylation sites 1 and 5 are mutated.
This could result from an influence of the local environment of the
glycosylation site on the type of carbohydrate processing to which this
particular site is subjected.
Figure 2:
Expression of WT t and
t lacking
single N-glycosylation sites in COS-7 cells. COS-7 cells
transfected transiently with a plasmid encoding WT
t or
single-site N-glycosylation mutants were metabolically labeled
with [
S]methionine-cysteine for 3 h at 37
°C. The
t proteins were immunoprecipitated with mAb 15-1
from cell lysates (A) or cell supernatants (B) before
analysis by 14% SDS-PAGE in reducing conditions. The designation of the
transfected construct is indicated above each line. The
numbers correspond to the N-glycosylation sequons mutated. N, untransfected cells. The position of the M
standards is indicated on the left.
Figure 9:
Enzymatic deglycosylation of t
secreted by ldlD.Lec1. Secreted
t was purified on an agarose-bound
mAb 15-1 column (lane 1). The affinity purified product
was digested with Endo H (lane 2), followed by further
deglycosylation with N-glycanase (lane 3). Proteins
were resolved on SDS-PAGE under reducing conditions and stained with
Coomassie Blue. The position of the M
standards
are indicated on the left.
Figure 3:
Expression of WT t and mutant
1-7
t in E. coli. Periplasmic extracts from E. coli cells transformed with either WT
t (lane
2) or mutant
1-7
t (lane 4) DNA were
immunoprecipitated with mAb 15-1. Control immunoprecipitates
using an isotype matched antibody are in lanes 1 (WT
t)
and 3 (
1-7). The immunoprecipitates were subjected
to SDS-PAGE, and immunoblotted with antibody 997. The position of the M
standards is indicated on the left.
Figure 4:
Reactivity of WT t and
1-7
t produced in E. coli with mAb 15-1 and antibody
974. Panel A, periplasmic extracts from E. coli cells
transformed with either WT
t or mutant
1-7
t DNA
were immunoprecipitated with mAb 15-1, antibody 974, or control
antibodies. Panel B, the resulting supernatants were subjected
to a second immunoprecipitation with the same antibody, mAb 15-1
or 974. The supernatants depleted with mAb15-1 or 974 were then
immunoprecipitated with 974 or mAb15-1, respectively. All
immunoprecipitates were resolved on SDS-PAGE and immunoblotted with
antibody 997. The position of the M
standards is
indicated on the left.
Figure 5:
Demonstration of the specifities of mAb
15-1, 974, and IgE. COS-7 cells transiently expressing WT t
chain were metabolically labeled with
[
S]methionine-cysteine for 3 h at 37 °C.
Cell lysates were divided into 3 portions: four-sixths was
immunoprecipitated using IgE, one-sixth with mAb 15-1, and
one-sixth with 974. Three-fourths of the washed IgE precipitate was
then denatured and divided into 3 equal portions which were
immunoprecipitated with either IgE, or mAb 15-1 or 974.
Immunoprecipitates were analyzed by 12% SDS-PAGE under reducing
conditions. Lanes 1-3, immunoprecipitation in native
conditions; lanes 4-6, immunoprecipitation after
denaturation; lanes 1 and 4, immunoprecipitation
using IgE; lanes 2 and 5, using the mAb 15-1; lanes 3 and 6, using 974. The position of the M
standards is indicated on the left.
Figure 6:
Pulse-chase analysis of WT t, mutant
1-5
t, and mutant
1-7
t in transfected
COS-7 cells. COS-7 cells transfected transiently with plasmids encoding
WT-
t (A),
1-5
t (B), or
1-7
t (C) constructs were pulse-labeled with
[
S]methionine-cysteine for 20 min at 37 °C
and chased for 0, 2, or 6 h. The
t proteins were
immunoprecipitated either with mAb 15-1 (left) or
antibody 974 (right). Immunoprecipitates from cell extracts
were either treated with Endo H (+) or not(-), while
immunoprecipitates from supernatants (S) were untreated before
analysis on 14% SDS-PAGE under reducing conditions. Note that panels A and B correspond to an overnight exposure of
the gels while panel C corresponds to a 4-day exposure. The
position of the M
standards is indicated on the right.
The mutant with the first five sequons
inactivated, 1-5
t, shows an increased electrophoretic
mobility when compared with WT
t (Fig. 6B). The
amount of secreted
1-5
t which can be
immunoprecipitated with mAb 15-1 is much less than that of
secreted WT
t and corresponds well with the drop in IgE-binding
activity seen in the supernatant of the
1-5
t mutant
(see above). The amount of intracellular
1-5
t
immunoprecipitated by mAb 15-1 also decreases. In contrast, 974
immunoprecipitates more intracellular
1-5
t than WT
t (compare Fig. 6, B and A). A large
amount of
1-5
t remains stable and undegraded
intracellularly throughout the chase. Virtually no 974 reactive
1-5
t is secreted. Therefore, the mutation of sequons
1-5 affects the intracellular folding and hence the secretion of
1-5
t, but causes little change in its stability.
The effect of multiple mutations is even clearer when all the
sequons are mutated. Under these conditions, mAb 15-1 does not
precipitate any material around 22 kDa from intracellular or secreted
1-7
t (Fig. 6C). However, intracellular
1-7
t is clearly seen after 974 immunoprecipitation.
Thus, unlike in E. coli, mAb 15-1 and antibody 974
detect differences in reactivity between WT
t and
1-7
t expressed in eukaryotic cells. The amount of
t detected by
mAb 15-1 decreases with the number of mutations. In striking
contrast, the amount of
t detected by antibody 974 increases with
the number of mutations (compare panels A, B, and C in Fig. 6). Therefore, increasing the number of mutations
and thereby decreasing the glycosylation, induces misfolding of
t
in the pre-Golgi compartment. Taken together, these data indicate that
glycosylation of
t is necessary for its efficient folding in the
endoplasmic reticulum of eukaryotic cells.
Figure 7:
Comparison of WT t from cells grown
in the presence of tunicamycin with mutant
1-7
t. COS-7
cells transiently expressing WT
t grown without (lanes 1 and 2) or with tunicamycin (lanes 3 and 4) or expressing the mutant
1-7
t (lanes 5 and 6) were metabolically labeled with
[
S]methionine-cysteine for 3 h at 37 °C.
Cell lysates (lanes 1, 3, and 5) or supernatants (lanes 2, 4, and 6) were divided in two portions and
immunoprecipitated using the mAb 15-1 (A and B)
or 974 (C) before analysis on 12% SDS-PAGE under reducing
conditions. Note that Panel B is a longer exposure of the gel
in Panel A. The position of the M
standards is indicated on the left.
The t-containing pEE14 vector was transfected into the
wild type CHO (CHOK1) and the two mutant Lec3.2.8.1 and ldlD.Lec1 cell
lines. The
t secreted from stable clones was metabolically labeled
with [
S]methionine-cysteine for 16 h,
precipitated with IgE, and analyzed by SDS-PAGE (Fig. 8). As
expected, the fully glycosylated
t secreted by CHOK1 migrates as a
broad heterogenous band around 50 kDa. The electrophoretic mobility of
t secreted by the mutant ldlD.Lec1 and Lec3.2.8.1 cell lines shows
a 10-15 kDa reduction. In the three cell lines, secreted
t
can be precipitated with IgE and is therefore properly folded.
Furthermore, after 16 h of metabolic labeling,
t is detected
almost exclusively in the cell supernatant with virtually no
intracellular
t remaining (data not shown). This indicates an
efficient secretion of
t. Therefore the processing of high
mannose-type to complex-type oligosaccharides and the addition of O-linked oligosacharides are not required for transport and
secretion of efficiently folded
t.
Figure 8:
Secretion of t by stable CHO
transfectants. CHOK1, ldlD.Lec1 (Lec1), and Lec3.2.8.1. (Lec3), either untransfected (lanes 1, 3, and 5) or stably expressing
t (lanes 2, 4, and 6), were metabolically labeled with
[
S]methionine-cysteine for 16 h at 37 °C.
The
t proteins were precipitated from the cell supernatants with
IgE before analysis on 11% SDS-PAGE under reducing conditions. The
position of the M
standards is indicated on the left.
The
IgE-binding activity of glycosylated and deglycosylated t was
compared quantitatively. No difference was detected (data not shown).
Therefore once folded properly,
t remained active and was still
capable of binding IgE.
Analyzing the role of carbohydrates in the folding and stability of glycoproteins is an area of intensive investigation. At present, our understanding of carbohydrate function is still fragmentary. Recent works indicate that glycoprotein glycans may play multiple roles and that different proteins have different requirements for carbohydrates. Glycoprotein glycans have been shown to be involved in several functions which include regulation of intracellular trafficking, modulation of enzyme and hormone activities, and participation in cell-cell interactions (Lis and Sharon, 1993). It has been proposed that N-linked oligosaccharides could play several roles during the conformational maturation of glycoproteins (Helenius, 1994). Although for many glycoproteins it has been reported that N-linked oligosaccharides are needed for folding, some, such as IgM or major histocompatibility complex molecules fold efficiently without any. Carbohydrate trimming may also be essential for the folding of many proteins (reviewed in Helenius(1994)).
Our
first question was whether the core N-glycosylation which
occurs in the pre-Golgi compartment has any relevance to the folding of
t. We first generated a series of single glycosylation site
mutants of
t (Fig. 1). Transient expression of the single
site mutants in COS-7 cells shows that all 7 potential N-glycosylation sites of the
t chain are glycosylated in
the mature native molecule (Fig. 2). The individual elimination
of any one site has no significant effect on the secretion of active
t (data not shown).
Once we had established that all 7
glycosylation sites were modified in vivo, and that no one
site was particularly critical for the folding process, we constructed
a panel of multiple mutants to test the effect of the cumulative loss
of core glycosylation on the intracellular transport of t. As
illustrated in Table 1, there is a strong cumulative effect of
the number of mutated glycosylation sites on the amount of active
t secreted, raising the question of the cause of the decreased
secretion. Possible mechanisms include a primary effect of the amino
acid substitutions, misfolding due to the loss of N-glycosylation, and either increased degradation or
intracellular retention of unglycosylated
t. While single
replacements of serine or threonine with alanine rarely induce
conformational changes (Argos, 1987), the effect of multiple
substitutions could have resulted in the misfolding of
t.
To
determine whether the multiple substitutions in the primary sequence
could affect the folding, we expressed WT t and mutant
1-7
t in E. coli. As shown in Fig. 3,
the mutation of the seven N-linked glycosylation sites does
not influence the production and the folding of
t. This was also
confirmed with IgE-binding activity data where no significant
difference was noticed between the WT
t and the mutant
1-7
t. The above data demonstrate that the amino acid
substitution per se does not interfere with the folding of
t.
To compare the fate of the various forms of t, WT
t,
1-5
t, and
1-7
t, we used a
monoclonal antibody that precipitates only efficiently folded
t
molecules (mAb 15-1) and an anti-peptide antibody that
precipitates unfolded
t molecules (974) ( Fig. 4and Fig. 5). In transfected COS-7 cells, immediately after
biosynthetic labeling, a mixture of properly folded and misfolded
t is detected intracellularly in all 3 cases. The ratio of
misfolded over properly folded
t increases with the extent of loss
of glycosylation (Fig. 6). The Endo H sensitivity of misfolded
WT and
1-5
t indicates retention in a pre-Golgi
compartment. The low levels of secretion of
1-5 and
1-7
t are most likely due to retention of misfolded
t.
The above data seem to contradict our previously published
work on tunicamycin-treated cells where glycosylation was demonstrated
not to be required for the secretion of active t (Blank et
al., 1991). However, in this previous study no attempt was made to
determine the efficiency of secretion. To clarify this apparent
contradiction we used the antibodies mAb 15-1 and 947 to compare
t produced by the
1-7 multiple mutant to that produced
by WT in cells treated with tunicamycin. Tunicamycin treatment of COS-7
cells transiently transfected with WT
t results in misfolding and
intracellular trapping of approximately 95% of the unglycosylated
proteins, nearly identical to the results with the
1-7 (see Fig. 7, A and C). Despite the virtually
complete absence of glycosylation in both cases, small amounts of
efficiently folded
t are formed and secreted (see Fig. 7B). This confirms the previous work demonstrating
that glycosylation is not absolutely required for secretion (Blank et al., 1991), but indicates that the loss of N-glycosylation results in misfolding of
t and a
substantial decrease in secretion of
t. Taken together, these
results demonstrate that core N-glycosylation facilitates
correct folding, with the individual contributions of the carbohydrate
chains being additive in promoting folding and, as a consequence,
transport of
t through the secretory pathway.
Our next question
was whether the maturation of core N- or O-glycosylation which occurs in the Golgi compartment is
involved in the intracellular transport and in the folding of t.
We chose to address this question by stably expressing
t in two
glycosylation deficient cell lines, Lec3.2.8.1 and ldlD.Lec1. Both cell
lines produce
t which binds IgE (Fig. 8) and mAb 15-1
(data not shown). In addition, equilibrium labeling shows that the
t produced is completely secreted with essentially no retention in
either the Golgi or the endoplasmic reticulum. Retention would be
expected if the missing saccharide residues were necessary for proper
folding. This result is identical to that obtained after transfection
of
t into CHOK1 cells, the parental cell line for both Lec3.2.8.1
and ldlD.Lec1. These results demonstrate that the Golgi compartment
mediated maturation of N-glycosylation and the addition of O-glycosylation are dispensable for the proper folding and
efficient intracellular transport of
t.
Our data indicate that
t belongs to a group of molecules such as human transferrin
receptor, human glucuronidase, and influenza A virus hemagglutinin
which are also retained in the endoplasmic reticulum as a consequence
of a loss of oligosaccharides (Helenius, 1994). However, once folded,
these molecules remain stable. Therefore, the oligosaccharides seem to
be necessary only in the endoplasmic reticulum and pre-Golgi
compartment. These striking features have been the basis for the theory
of specific interactions between the carbohydrate moieties of
glycoprotein and the folding machinery. These interactions would be
necessary for efficient folding and would control the retention of the
non-folded or partially folded forms in the endoplasmic reticulum. Our
results are consistent with this concept.
Interactions of proteins
with molecular chaperones such as BiP have been well documented.
However, these chaperones interact directly with the polypeptide moiety
(reviewed in Hartl et al.(1994)). Interactions with chaperones
that are dependent on the presence of carbohydrates have been
investigated only recently. One example is the folding of vesicular
stomatitis virus G protein which interacts sequentially with BiP and
calnexin (Hammond and Helenius, 1994). The latter interaction is
necessary for proper folding and is prevented by removal of the sugars
or by inhibition of the endoplasmic reticulum glucosidases. It will be
interesting to investigate whether t could also interact with
calnexin or a similar, carbohydrate-dependent, chaperone.