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Originally published In Press as doi:10.1074/jbc.M104979200 on August 30, 2001
J. Biol. Chem., Vol. 276, Issue 44, 40933-40939, November 2, 2001
Recovering Antibody Secretion Using a Hapten Ligand as a Chemical
Chaperone*
Gregory D.
Wiens §,
Thomas
O'Hare¶, and
Marvin B.
Rittenberg
From the Department of Molecular Microbiology and
Immunology and ¶ Department of Cell and Developmental Biology,
Oregon Health and Science University, Portland, Oregon 97201-3098
Received for publication, May 31, 2001, and in revised form, August 21, 2001
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ABSTRACT |
Engineered antibodies have come to the forefront
as research reagents and clinical therapeutics. However, reduced
stability or expression levels pose a major problem with many
engineered antibodies. As a model for understanding functional
consequences of variable region mutation, we have studied the assembly
and trafficking of anti-phenylphosphocholine antibodies. Previously, we
identified severe secretion defects because of mutations in the heavy
chain second complementarity determining region, which is involved in
antigen binding. Here we demonstrate that immunoglobulin secretion is
increased up to 27-fold by incubating stably transfected PCG1-1 cells
with cognate hapten p-nitrophenylphosphocholine. Secretion
was unaffected by nonbinding analogs. Radiotracer and metabolic
labeling experiments demonstrated specific cellular uptake of
p-nitrophenylphosphocholine and increased intracellular heavy and light chain assembly. Brefeldin A inhibited hapten-mediated immunoglobulin secretion but not assembly, indicating that assembly occurs early within the biosynthetic pathway. Recovery of secretion correlated with antigen binding capacity, suggesting that the rescue
mechanism involves stabilization of heavy and light chain variable
domains. This model system provides the first demonstration that
cognate ligands can increase intracellular assembly of functional anti-hapten antibody within mammalian cells and suggests that small
molecules of appropriate specificity and affinity acting as
chemical chaperones may find application for increasing or regulating
immunoglobulin expression.
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INTRODUCTION |
Engineered antibodies and variable region
(V-region)1 fragments are now
established as indispensable tools for scientific and clinical uses
(reviewed in Refs. 1 and 2). Optimization of V-region binding
properties is, in many cases, accompanied by impaired secretion from
eukaryotic cells (3-5). Previously, we demonstrated that 16 mutants in
the H chain framework region 2 and CDR2 region of the T15 and PCG1-1
anti-phenylphosphocholine antibodies have severe defects in secretion
(6, 7). The molecular basis of the secretion defect in several mutants
was traced to a single substitution (Ile51 to Arg or Lys)
in the H chain of the PCG1-1 antibody (8). Mutant H chains
co-immunoprecipitate with chaperones BiP and GRP94 and remain
endoglycosidase H-sensitive, consistent with retention in the
endoplasmic reticulum (ER). Mutant H chains rapidly assemble into
intracellular H2 intermediates but exhibit impaired L chain association and undergo intracellular degradation (8).
Recently, it has been demonstrated that defective maturation of mutant
proteins from the ER can be partially or fully corrected by incubation
with low molecular weight compounds termed chemical chaperones (9, 10).
Chemical chaperones can rescue both mutant secretory and transmembrane
proteins that are otherwise destined for degradation (10-15). High
concentrations of chemical chaperones, often in the millimolar range,
are generally required to reverse trafficking defects. The mechanism(s)
by which chemical chaperones function are not fully understood but are
thought to include stabilization of improperly folded proteins (10),
reduction of aggregation (13), and prevention of nonproductive
interactions with ER proteins (11). Chemical chaperones such as
glycerol and trimethylamine N-oxide appear to be nonspecific
as they are able to rescue diverse types of proteins (16), whereas
other chemical chaperones only function with a narrower set of proteins
(13, 17). Until now chemical chaperones have not been shown to restore
secretion of heteropolymers such as Ig.
Here we used anti-phenyphosphocholine antibodies as a model system to
determine whether a defect in Ig secretion could be corrected by the
use of the cognate hapten ligand acting as a chemical chaperone.
Incubation of stable transfectants with the cognate hapten,
p-nitrophenylphosphocholine (NPPC), led to rescue of
secretion in five of six low secreting PCG1-1 mutants. NPPC was
specifically taken up by cells and resulted in increased intracellular heavy and light chain assembly. In contrast, NPPC did not affect the
amount of secretion of wild type (WT) Ig nor did it nonspecifically induce the release of incompletely assembled Ig chains, indicating that
the interaction is highly specific. The capacity of
trafficking-impaired Ig to bind antigen correlated with ligand-mediated
rescue of secretion. These data indicate that the mechanism of rescue
involves the intracellular stabilization of
VH-VL pairing. Hapten ligands may find
application for regulating or increasing assembly and secretion of
anti-hapten Ig from hybridomas.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
The PCG1-1 heavy chain loss variant (PCG1-1
H ), and cell culture conditions were described previously
(7). For antibody purification, cells were cultured in HyQ-CCM1
serum-free medium (HyClone Laboratories, Logan, UT) for 4 days in the
presence of 5 mM p-nitrophenylphosphocholine
(NPPC). Antibody was purified from sterile filtered supernatants using
protein A-Sepharose.
Ig Secretion Assay--
Stable transfectants were plated in
triplicate at 1 × 106 cells ml1 of
tissue culture medium in a 12-well plate. After designated incubation
periods, culture supernatants and cell lysates were collected as
described (6).
Haptens--
Haptens NPPC and
p-aminophenylphosphocholine (APPC) were obtained from
Sigma-Aldrich and dissolved in tissue culture medium prior to
use. Phosphocholine (PC, Sigma-Aldrich) was dissolved in 0.1 M EDTA (pH 8.0) and diluted 1:10 in 0.1 M
sodium phosphate buffer (pH 8.0). The solution was adjusted to pH 8.0 with 1 M KOH, precipitated salt was removed by
centrifugation, and supernatant stored at  20 °C.
[3H]NPPC Synthesis--
All reagents were from
Sigma-Aldrich except [methyl-3H]choline
chloride (specific activity = 75.00 Ci mmol1), which
was from PerkinElmer Life Sciences. The procedure for the
synthesis of [3H]NPPC was based on a modification of the
method of Chesebro et al. (18) for the synthesis of
p-nitrophenyl-3-N,N,N-trimethylpropylammonium phosphate, as described by Moulton (19). Ethanol solutions of unlabeled
choline chloride (0.32 mmol) and
[methyl-3H]choline chloride (800 µCi) were
combined and evaporated to dryness (final specific activity = 2.5 mCi mmol1). In a separate tube,
p-nitrophenylphosphorodichloridate (0.35 mmol) and quinoline
(101 µl; 0.85 mmol) were dissolved in dry CH3CN (372 µl) and mixed with radiolabeled choline on ice. The tube containing
the reaction mixture was rotated end-over-end at 4 °C for 8 h.
Pyridine (300 µl) and H2O (50 µl) were then added, and
the solution was agitated at 25 °C for 30 min. Solvent was removed,
and the resulting oil was dissolved in H2O (1.0 ml), then
passed through a TMD-8 mixed bed ion exchange column (2 cm × 12 cm) with H2O as the elution solvent. The first 45 ml of
flow-through was lyophilized, yielding [3H]NPPC as a
white solid (0.11 mmol; 2.5 mCi mmol1; 34% isolated
yield). The product was analyzed by cellulose thin layer chromatography
(isopropanol/ammonium hydroxide/water, 7:2:1 (v/v/v);
RF = 0.56 for NPPC). Product obtained from a
side-by-side synthesis in which the radiolabeled choline chloride was
replaced by the unlabeled choline was examined by 1H NMR,
13C NMR, and 31P NMR. Each NMR spectrum was
identical to the corresponding spectrum of authentic NPPC. The final
[3H]NPPC was tested alongside authentic NPPC in an
inhibition ELISA and 24-h secretion assay and found to be
indistinguishable from the unlabeled hapten.
Metabolic Labeling, Immunoprecipitation, and
SDS-PAGE--
Transfected cells were cultured and labeled with 150 µCi of [35S]Express protein labeling mix for 20 min as
described (20) either in the presence or absence of NPPC.
Unincorporated label was removed by washing cells two times and
resuspending in complete tissue culture medium containing Met/Cys for
the indicated chase time periods. Ig was immunoprecipitated from
clarified cell lysates or supernatants by incubation with protein
A-Sepharose CL-4B (Amersham Pharmacia Biotech). For assembly
experiments the immunoprecipitates were washed with buffers as
described previously (20). All labeling experiments were quantified
using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and IP lab
gel software (version 1.5; Analytics, Vienna, VA). In some experiments,
cells were cultured in 5 µg ml1 brefeldin A (BFA;
Sigma) to block secretion. Cells were treated for 1 h with
brefeldin A prior to pulse labeling and during chase time points.
ELISA--
The concentration of Ig proteins in supernatants and
cell lysates was determined by sandwich ELISA as described previously (7). Briefly, to determine H+L in the supernatant or lysate, ELISA
plates were coated with rabbit anti-mouse IgG2b
(Zymed Laboratories Inc., San Francisco, CA), and the
amount of bound Ig from the lysate or supernatant was determined by a
secondary alkaline phosphatase-conjugated, goat anti-mouse  antibody
(Southern Biotechnology Associates, Birmingham, AL). All antisera were
used at a 1:1000 dilution. Standard curves were generated using
affinity-purified antibody from WT transfectant PCG1-1 ( 2b, ) or
hybridoma PCG2b-2 (2b, ). Antibody binding to PC-histone was
determined by a direct ELISA as described previously (7).
[3H]NPPC Uptake Assay--
Trypsinized cells were
washed twice and resusupended in tissue culture medium containing 20%
fetal bovine serum, then allowed to adhere to 12-well tissue culture
plates. For dose-response experiments, 1.0 × 106
cells were plated, whereas 0.8 × 106 cells were used
for the time-course and inhibition experiments. In all experiments,
after 12 h of incubation, nonadherent cells were removed with four
washes of 1 ml of serum-free CCM-1 medium (HyClone) and dilutions of
[3H]NPPC were added to cells in 0.5 ml of CCM-1 medium.
As a negative control, cells were fixed with 4% paraformaldehyde
solution in Dulbecco's phosphate-buffered saline (pH 7.4) for 15 min
prior to the addition of labeled hapten. For the dose-response and
inhibition experiments, cells were incubated for an additional 14 h at 37 °C prior to harvest. At harvest, free [3H]NPPC
was removed by washing cells four times with 1 ml of phosphate-buffered saline, and cells were solubilized in 0.5 ml of 0.5% SDS in 0.2 M NaOH solution as described (21). Lysates were transferred to vials, diluted in 10 ml of ScintiVerse (Fisher Scientific, Fair
Lawn, NJ), and the radioactivity associated with the cells was
quantified by liquid scintillation counting (Beckman model LS 3801).
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RESULTS |
NPPC Rescues Ig Secretion from Ile51  Arg Stable Low
Secretion Transfectants--
Previously we proposed that
VH mutations may impair Ig assembly and/or destabilize the
H-L complex (20). A single mutation at position 51 appears sufficient
to prevent stable H-L complex formation; we predicted that compounds
stabilizing H-L interaction should increase secretion of this antibody.
Because ligand binding can stabilize recombinant
VH-VL pairing in vitro (22, 23), we
tested whether addition of the hapten NPPC (5 mM) to
cultured cells would increase the secretion of Ig into the surrounding medium. Incubation of PCG1-1 Ile51 Arg transfectant
cells in NPPC significantly increased the amount of Ig in culture
supernatant as determined by ELISA (15-fold ± 3 (mean ± S.E.); n = 7 independent experiments, Table
I). Similar results were obtained with
three independently created PCG1-1 Ile51 Arg
transfectants, indicating this is not a transfectant-specific effect
(Data not shown). The addition of NPPC also augmented secretion of
another Ile51 single-site secretion mutant, PCG1-1
Ile51 Lys (9-fold ± 3; n = 4 independent experiments). The increase of Ig in the supernatant was
dose-dependent, and the effect was specific to NPPC (Fig.
1A). Addition of the
structurally related hapten PC, which does not bind to PCG1-1 WT
antibody, did not increase the amount of Ig in culture supernatants.
Similarly, the addition of 5 mM choline or 0.05 mM p-nitrophenol had no effect on secretion
(data not shown). The addition of 5 mM APPC resulted in a
modest, 2.4-fold increase in secretion. APPC binds with ~12-fold lower affinity as compared with NPPC (24), suggesting that binding affinity may correlate with efficiency of rescue. Increased supernatant Ig could not be attributed to toxicity as cell viability was greater than 95% in medium alone or medium with NPPC (Fig. 1B). To
examine whether the hapten nonspecifically increased Ig chain
secretion, the secretion of PCG1-1 L chains was measured as a control
as it is produced in excess over transfected H chains (7). NPPC addition did not increase free PCG1-1 L chain secretion (Fig. 1C). Furthermore, NPPC did not increase secretion of
H2L2 from WT PCG1-1 or T15 cells (data not
shown).
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Table I
Immunoglobulin concentration in culture supernatants or cell lysates
after incubation in the presence or absence of 5 mM NPPC
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Fig. 1.
A, influence of several haptens on Ig
secretion by the PCG1-1 VH Ile51 Arg cell
line. Incubation with NPPC but not PC or APPC resulted in a significant
increase in Ig in the supernatant as measured by ELISA. Cells were
incubated for 24 h in tissue culture medium with or without
hapten. Data are expressed as an average ± S.E. of triplicate
cultures. Statistically significant increase versus control
(*, p < 0.05 one-way analysis of variance with
Student-Newman-Keuls post hoc test). Results are
representative of three experiments. B, cell viability as
determined by trypan blue exclusion staining was not altered by hapten
addition. C, light chain secretion as determined by ELISA.
D, secreted WT and mutant Ig display similar binding
activity to PC-histone. Cells were incubated with NPPC for 4 days and
antibody purified using protein A-Sepharose. Purified antibody was
extensively dialyzed in phosphate-buffered saline to remove any bound
hapten, and Ig binding to PC-histone was determined by ELISA.
Open square,WT Ig; open
diamond, Ile51 Arg Ig.
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Although NPPC did not affect the amount of secretion of WT Ig, the
presence of this compound might nonspecifically induce the release of
intracellularly retained, incompletely assembled Ig chains. This
possibility was tested in two ways. First, we incubated NPPC with
stable transfectant cell lines that only express either the T15 H chain
or the T15 L chain (V22 gene). We have previously demonstrated that
both the T15 H chain and the T15 L chain, when expressed alone, are
retained in the ER and that the T15 L chain is degraded via a
proteasome-dependent pathway (25). The addition of 5 mM NPPC did not increase the amount of either T15 H chain
or T15 L chain in supernatants, as measured by ELISA (Table I). These
data indicate that NPPC does not nonspecifically disrupt the retention
of unassembled Ig H or L chains in transfected cells. The presence of
NPPC also did not alter the steady state balance between synthesis and
degradation of the nonsecreted T15 H and T15 L chain (data not shown)
or the intracellular amount of the mutant PCG1-1 H chain (Table I).
Second, we examined the binding activity of Ig secreted in the presence
of hapten (Fig. 1D). If NPPC increased secretion of
partially assembled or nonfunctional antibody, then antigen binding
activity would be reduced. However, protein A-purified WT and mutant
antibody exhibited similar binding activity for PC protein, indicating
that functional antibody was secreted into supernatant. Thus, these
data indicate that NPPC increases the amount of mutant Ig in the
supernatant and this secreted Ig is stable with respect to antigen binding.
[3H]NPPC Is Specifically Taken up by
Cells--
Because NPPC would not be expected to enter cells
passively, we tested its capacity to be taken up by cells in culture.
Specific uptake of [3H]NPPC was determined by incubation
with viable cells, whereas nonspecific uptake was determined by
incubation with paraformaldehyde inactivated cells. Results of uptake
assays indicate [3H]NPPC is taken up in a
dose-dependent manner by stably transfected PCG1-1 cells
(Fig. 2A). Uptake was not
unique to PCG1-1 Ile51 Arg transfectants, as the
parental cell line PCG1-1 H- and T15 H-expressing cells also
incorporated [3H]NPPC over 14 h (data not shown).
Uptake was inhibited by excess nonradiolabeled NPPC (Fig.
2B) and choline (data not shown), indicating that the uptake
was specific and not the result of passive diffusion. Although not
formally demonstrated, these data suggest that NPPC may be taken up by
a mechanism similar to that of choline. Choline is known to be taken up
by lymphocytes (26) and transported into neurons via a choline
transporter (27, 28). Measurement of NPPC uptake in cells cultured with
1 mM [3H]NPPC demonstrated a linear rate of
60 pmol h1/106 cells over 24 h (Fig.
2C, inset). In comparison,
[3H]choline is taken up by PCG1-1 Ile51 Arg cells at a rate approximately 1-2 orders of magnitude faster than
NPPC (data not shown), indicating that differences between choline and
NPPC uptake exist. Assuming a transport-mediated process, the apparent
Km of NPPC uptake calculated from the inhibition experiments was 14.5 ± 0.7 mM. Because uptake of
[3H]NPPC is via a slow, low affinity process, we examined
whether the time course of uptake paralleled the biological activity of Ig secretion rescue (Fig. 2D). A 4-fold increase in Ig was
detected in supernatant by 4 h, whereas a 27-fold increase was
detected by 10 h. These results demonstrate that, after a delay,
increased secretion parallels the uptake of labeled NPPC.
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Fig. 2.
Uptake of [3H]NPPC by the
PCG1-1 Ile51 Arg stable cell
line. A, dose-dependent uptake of
[3H]NPPC after 14-h incubation. Nonspecific uptake was
determined by incubating [3H]NPPC with
paraformaldehyde-fixed cells. B, uptake of
[3H]NPPC is inhibited by the addition of nonradiolabeled
NPPC. A nonlinear, one-site binding curve is plotted. C,
time course of uptake by cells incubated with 1 mM
[3H]NPPC. Uptake was normalized to the number of adherent
cells present at each time point. Inset, uptake was linear
over 24 h. D, kinetics of Ig secretion from PCG1-1
Ile51 Arg stable cell line incubated with or without 5 mM NPPC. Data are expressed as an average ± S.E. of
triplicate cultures from one experiment. Results are representative of
two to three experiments. Filled bar, + NPPC;
open bar, NPPC.
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NPPC Increases Intracellular H-L Assembly--
Because NPPC is
taken up by PCG1-1 cells, we investigated whether rescue of secretion
occurred via increased intracellular assembly and whether the secreted
Ig consisted of fully assembled H2L2
heterodimers. The kinetics and pathway of assembly were examined by
subjecting cells to [35S]Met/Cys-labeled pulse-chase
analysis either in the presence or absence of 5 mM NPPC.
Heavy chains were immunoprecipitated with protein A-Sepharose and
separated by nonreducing SDS-PAGE. Ig assembly was followed at 0-, 2-, and 10-h time points after a 20-min pulse. In the absence of NPPC, the
majority of the single Ile51 Arg H chain was arrested
at the H2 step in Ig assembly (Fig. 3A). As expected,
secretion-impaired mutant H chains co-immunoprecipitated with BiP (29,
30). A small amount of intracellular H2L was formed at 0 and 2 h, and then levels decreased by 10 h. In contrast, assembly of H2L2 in WT cells was essentially
complete by 2 h and co-immunoprecipitated BiP was not detectable
(Fig. 3C). In agreement with ELISA data (Table I), a small
amount of the mutant Ig was visible in the supernatant at the 2- and
10-h chase time points in the absence of NPPC (Fig. 3A). The
small amount of secreted mutant Ig was fully assembled
H2L2, as judged by two-dimensional electrophoresis (data not shown). The addition of NPPC markedly increased the amount of H2L2 in the mutant
supernatant. By 2 h there was a 2.2-fold increase in fully
assembled Ig, and by 10 h there was a 6.4-fold increase in
supernatant Ig (Fig. 3B). Importantly, NPPC stimulated an
increase in the amount of intracellular H2L in the
Ile51 Arg transfectant, indicating an intracellular
mechanism of rescue. The relative amount of H2L assembly
intermediate was increased by 1.7-fold at 2 h and 2.9-fold at
10 h in comparison to control cultures. In agreement with ELISA
data, the effect was specific to the mutated antibody, as NPPC did not
increase secretion of WT antibody nor was there an increase in WT
assembly intermediates (Fig. 3D). To independently confirm
that NPPC augments intracellular assembly, we inhibited secretion by
preincubation with brefeldin A (Fig.
4A). In the presence of BFA,
NPPC caused a modest increase in intracellular H2L
(2.0-fold) and a substantial increase (4.1-fold) in
H2L2 by 10 h (Fig. 4C). The
formation of H2 was not altered by the presence of the
hapten and BFA, indicating that increased assembly is targeted to H and
L chain assembly. These data indicate that assembly occurs early within
the biosynthetic pathway. No H2L2 was detected
in the supernatant, indicating that secretion was completely blocked by
BFA treatment (Fig. 4A). This was independently confirmed by
a subsequent immunoprecipitation of free supernatant L chain (2-h chase
time point) from either BFA-treated or vehicle (methanol)-treated
PCG1-1 Ile51 Arg culture supernatants (Fig.
4B). The addition of BFA completely blocked L chain
secretion at both the 2-h (Fig. 4B) and 10-h chase time
points (data not shown). PCG1-1 L chains are secreted as both apparent
monomers and covalent dimers similar to other murine L chains (31, 32).
As observed for the WT antibody, NPPC had no effect on L chain
secretion or on the assembly of L chain into covalent dimers (Fig.
4B, data not shown). Taken together, these data indicate
that NPPC specifically rescues secretion of the mutant Ig by increasing
intracellular H2 H2L and H2L
H2L2 assembly early within the biosynthetic
pathway, presumably in the ER.
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Fig. 3.
Addition of 5 mM NPPC increases
Ile51 Arg mutant H chain but not
WT H chain assembly and secretion with endogenous light chain. Cells stably transfected with Ile51
Arg (A) or WT (C) H chains were
35S-pulse-labeled for 20 min and chased either in the
absence (NPPC) or presence of 5 mM NPPC
(+NPPC) for 0, 2, and 10 h. H chain and associated
proteins were immunoprecipitated from cell lysates or supernatants
using protein A-Sepharose CL-4B. Proteins were separated using 10%
SDS-PAGE under nonreducing conditions. B and D,
densitometry of H2, H2L, and
H2L2 present in the lysate or supernatant from
mutant (B) or WT (D) cells. -Fold increase caused
by NPPC treatment (black bars; + NPPC)
was determined by standardization to control (white
bars; NPPC) for each time point. Data
represent an average ± S.E. from three (WT) to five
(Ile51 Arg) experiments.
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Fig. 4.
Hapten-mediated intracellular assembly occurs
in the presence of brefeldin A. In the presence of brefeldin A, no
Ig or L chain is secreted into the supernatant, but the addition of
NPPC causes the assembly of H2L2, which is
detectable in the lysate. A, cells stably transfected with
Ile51 Arg H chains were cultured in the presence of
brefeldin A 1 h prior to a 20-min 35S pulse label,
followed by cold chase either in the absence (NPPC) or
presence of 5 mM NPPC (+NPPC) for 0, 2, and
10 h. H chains were immmunoprecipitated and separated using
electrophoretic conditions described in Fig. 3. B,
supernatants from control or BFA-treated Ile51 Arg
cells were re-immunoprecipitated for light chain. Secreted L chain
resolves under nonreducing conditions as a covalent dimer and monomer.
Data shown are representative of three experiments. C,
densitometry of H2, H2L, and
H2L2 present in the lysate at 10-h chase time
point. -Fold increase caused by NPPC treatment (black
bars; + NPPC) was determined by standardization
to control (white bars; NPPC). The
amount of H2L2 is increased significantly
(Student's t test; p = 0.016). Data
represent an average ± S.E. from three experiments.
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Kinetics of Rescue--
We investigated whether the presence of
NPPC was required early in biosynthesis (Fig.
5). A 27-fold increase in Ig secretion was achieved by addition of NPPC 12 h prior to and during the labeling (12 h, Fig. 5A). A 10-h pulse of NPPC ending
2 h prior to labeling increased secretion 14-fold, indicating that
hapten taken up by cells is necessary for rescue. Addition of NPPC at 2- and 4-h chase time points augmented secretion of labeled Ig only
3-and 2-fold, respectively. These data indicate that the presence of
NPPC within the cell is required during the early steps of H chain and
L chain biosynthesis for maximal restoration of secretion.
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Fig. 5.
Pretreatment of cells with NPPC prior to
metabolic labeling increases recovery of mutant Ig
(H2L2) from culture supernatant.
A, NPPC was added either during pulse labeling (0 h), 12 h prior and during pulse labeling (12 h)
or for 10-h period ending 2 h before labeling (12 to
2 h) with [35S]Met/Cys. B, NPPC
was added during (0 h) or after (2 or 4 h) labeling. Ig was immunoprecipitated and resolved using
nonreducing SDS-PAGE. -Fold increase in Ig was determined by comparison
to control wells lacking NPPC. Data shown are representative of three
experiments.
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Differential Rescue of Secretion of PCG1-1 Mutants--
To
examine whether the ability of NPPC to enhance secretion extended to
other nonsecreted Ig mutants specific for phenylphosphocholine binding,
we tested the original panel of four low secretion PCG1-1 mutants
(Fig. 6). Secretion-impaired mutants P35,
P34, P32, and P28, as well as secretion-competent mutant P20, were
incubated with or without NPPC, and the amount of supernatant Ig was
determined by ELISA (Fig. 6A). The addition of NPPC
significantly increased the amount of Ig present in culture
supernatants of three of the four multisite mutants; P35 (7.3 ± 0.6-fold), P34 (5.0 ± 0.4-fold), and P28 (3.9 ± 0.2-fold).
As with the PCG1-1 WT transfectant, NPPC did not increase the amount
of Ig present in culture supernatant from secretion-competent mutant
P20. Secretion of mutant P32 (1.6 ± 0.3-fold) was not improved by
inclusion of NPPC. Mutant P32 is of particular interest because antigen
binding is undetectable, presumably because of the substitution by Cys
at Trp52, a residue critical in PC protein binding (Fig.
5B) (7). Mutants P34 and P35 have reduced binding avidity
for PC protein as compared with PCG1-1 WT, whereas the binding
activity of P28 has not been determined. The secretion rescue of mutant
P34 demonstrates that NPPC can increase secretion of Ig from low
secreting mutants that have V-region mutations at positions other than
Ile51. These data also indicate that the mutations in P28
and P35 other than the mutation at Ile51 do not block the
ability of NPPC to rescue secretion. Taken together, these data
indicate that the capacity of secretion-impaired Ig to bind antigen
correlates with ligand-mediated rescue of secretion.
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Fig. 6.
Addition of NPPC increases Ig secretion from
three multisite, secretion-impaired mutants P28, P34, and P35.
A, addition of NPPC did not increase Ig secretion from
multisite mutant P32 that does not bind antigen, nor from
secretion-competent mutant P20 or WT PCG1-1 transfectants. The -fold
increase in secretion was calculated by dividing the amount of Ig
secreted in the presence of NPPC (+ NPPC) by the amount of
Ig secreted from control cultures ( NPPC). Ig
concentration was determined by ELISA after 24 h of incubation in
fresh medium. Data are expressed as an average ± S.E. of
triplicate cultures from one experiment. Results are representative of
three to six experiments. There was a statistically significant
increase in Ig production compared with paired cultures without NPPC,
as determined by Student's t test (***, p < 0.001). B, amino acid sequences of the second
complementarity determining region (CDR2) of the PCG1-1 WT and mutant
Igs. Secretion (S) data are from Ref. 7.
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DISCUSSION |
A severe Ig secretion defect was specifically reversed in a panel
of single and multisite mutant antibodies by incubation of cells with
the cognate hapten ligand. NPPC increases intracellular assembly of
fully functional Ig and does not induce nonspecific release of
ER-retained Ig H or L chains. Secretion rescue was ligand-specific, as
evidenced by the inability of related haptens such as choline,
phosphocholine, or p-nitrophenol to rescue secretion. Failure to rescue secretion, at least for choline, cannot be attributed to lack of uptake. Rather, the rescue of Ig secretion correlated with
antigen binding capability. NPPC did not significantly rescue secretion
of a multisite mutant (P34) that does not exhibit detectable antigen
binding (7). Finally, hapten increased intracellular assembly of
H2L2 even when Ig secretion was blocked by
brefeldin A. Taken together, these data support the notion that NPPC
functions as a specific chemical chaperone to rescue assembly of Ig H
and L chains within the ER, thus leading to increased secretion of mutant Ig. To our knowledge, this is the first report of a hapten affecting the secretion of its cognate Ig. Furthermore, other chemical
chaperones (1% Me2SO or 1% glycerol) that appear to
function nonspecifically (9, 16) had no effect in this system (data not shown).
Although many steps in Ig assembly are known (reviewed in Refs. 33 and
34), the molecular events involved in H-L pairing are not clear.
Molecular chaperones BiP and GRP94 are associated with Ig H chain prior
to pairing (35, 36) and dissociation of BiP from H chain is temporally
linked to L chain arrival (37). Experiments using a truncated, two
domain heavy chain (VH-CH1) indicate that the disulfide
bond within the CH1 domain is not formed until BiP dissociates and L
chain associates with H chain (38). Furthermore, a mature L chain is
required to displace BiP from H chain (38). A possible mechanism of
NPPC action may be stabilization of correctly paired mutant
VH with VL within the ER, thereby facilitating
the dissociation of BiP and the folding of the CH1 and CL
domains. The requirement for antigen binding capacity supports this
mechanism. Both hapten and protein antigens have been shown to
facilitate the pairing and folding in vitro of several
VH and VL (22, 23, 39). Similarly, peptide has been shown to directly facilitate class I heavy chain association with
 2-microglobulin and folding in cell lysates in
vitro (40, 41). In addition, the thermal denaturation of
peptide-filled, purified Kb class I molecules results in
simultaneous loss of peptide and dissociation of the heavy chain and
2-microglobulin (42). The structural consequences of
ligand binding have been investigated by comparison of crystal
structures of ligand-bound and unbound antibodies (43). These
computational comparisons have identified that small antigens or
haptens compact the VH-VL interface in the
McPC603, 28B4, N1G9, and DB3 antibodies. The increase in compactness has been estimated to contribute 1.47 kcal mol1 to
stability, representing a 10-fold improvement in binding affinity between the VH and VL domains (43). An
improvement in affinity of this magnitude, occurring between the H and
L chains in the mutant PCG1-1 antibodies, may be sufficient to offset
the potentially destabilizing effect of the CDR2 mutation(s). WT Ig
assembly and secretion was not affected, presumably because of the
rapid and proper folding of the Ig H chain. We do not exclude the
possibility that NPPC may directly facilitate mutant H chain folding
prior to L chain pairing. In many antibody-antigen structures, H chain contributes a greater percentage of contacts than L chain (reviewed in
Ref. 44). We recently reported the crystal structure of a single chain
Fv, M3C65, complexed with NPPC (45). The M3C65 antibody
uses the same VH M141 gene as PCG1-1 but pairs with a V 1 L chain instead of a V1 L chain used by PCG1-1 (46). In the
M3C65-NPPC complex, residues in H chain CDR2 and CDR3 contact hapten,
although mutations in the CDR2 of the V1 L chains confer high
affinity binding (47). Thus, if the PCG1-1 combining site binds NPPC
in a similar orientation, it is likely that residues from both the
VH and VL contribute to binding. Further
analysis will be required to determine the molecular contribution of
hapten binding to mutant PCG1-1 Ig assembly.
At present the mechanism of uptake of NPPC into hybridoma cells is not
clear. Interestingly, oligopeptides can be delivered directly to the ER
of viable cells via a vesicular pathway resembling pinocytosis that
conveys small extracellular substances to the ER without traversing the
Golgi complex or the cytosol (48). This pathway is insensitive to
brefeldin A, a compound that disconnects the proximal and distal
portions of the Golgi, thus indicating that retrograde delivery of
endocytosed molecules to the ER is not involved (48, 49). In PCG1-1
cells, assembly of mutant Ig occurs in the presence of brefeldin A,
indicating that assembly is also not dependent on the retrograde
delivery of endocytosed NPPC. Pinocytosis of NPPC by PCG1-1 cells
would be consistent with the slow, low affinity uptake of NPPC and
insensitivity to brefeldin A inhibition (48). However, a pinocytotic
mechanism is inconsistent with the inhibition of [3H]NPPC
uptake by both NPPC (Fig. 2B) and unlabeled choline (data not shown). Rather, the specific inhibition of [3H]NPPC
uptake implicates the involvement of a transport-mediated process.
Choline is known to be taken up by lymphocytes (26), and we also have
evidence for choline uptake by PCG1-1 cells (data not shown). Recently
a choline transporter has been identified, although its expression is
restricted to cholinergic neurons (27, 28). Further experiments are
required to differentiate between these potential mechanisms. The
access of oligopeptides to the ER by a vesicular pathway (48) and our
data indicating that hapten can increase intracellular assembly, even
in the presence of brefeldin A, indicate that exogenous compounds may
find entry by incompletely understood mechanism(s), and thus
potentially stabilize subunit assembly of antigen receptor molecules.
The ability of a CDR mutation to disrupt assembly, and, of hapten to
repair this process suggest that VH-VL
interactions play an important role in quality control of normal Ig
secretion. Quality control processes are likely to be critical within
the bone marrow during formation of new antigen receptors as well as in
secondary lymphoid structures such as germinal centers in which high
numbers of somatic mutations are introduced into the variable regions of the H and L chains. The requirement of VH to pair well
with VL may prevent poorly paired H and L chains from being
expressed, thus avoiding generation of potentially unstable molecules
that could lead to heavy chain or light chain deposition diseases
(50-53). These mutants and the ability to modulate secretion provide a useful system for further elucidation of the molecular mechanisms governing B cell homeostasis and ER-mediated, Ig quality control.
We envision that cognate ligands may find application for increasing
assembly and secretion of Ig from poorly secreting hybridomas. A
drawback of NPPC, and most chemical chaperones, is that millimolar concentrations and long preincubation periods are required to rescue
assembly and secretion. In addition, it is unclear whether assembly and
secretion of antibodies that recognize naturally occurring peptides,
carbohydrates, and nucleic acids can be enhanced in a similar manner as
the ER access of larger molecules may be limiting. Future studies with
more penetrable ligands of higher affinity as well as molecules that
mimic larger and more complex antigen structures should clarify the
feasibility of this approach. Such compounds could ultimately be useful
in regulating Ig subunit assembly in cell lines and in vivo
(54). With over 200 Igs in current clinical trials (55), understanding
and maximizing Ig folding and secretion is of increasing importance.
| |
ACKNOWLEDGEMENTS |
We thank Drs. S. Landfear, M. Sanchez, J. Van Slyke, and K. Neve for helpful advice on uptake assays.
We also thank M. Brown and Drs. Q. Chen, M. Stenzel-Poore, B. Wiens,
and E. Whitcomb for critical comments. We especially thank Dr. C. Enns for helpful suggestions and commentary.
| |
FOOTNOTES |
*
This work was supported by a research award grant from the
Oregon Chapter of the American Cancer Society (to G. D. W.)
and by National Institutes of Health Grants AI-14985 and AI-26827 (to
M. B. R.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Dept. of Molecular
Microbiology and Immunology, L220, Oregon Health and Sciences University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97201-3098. Tel.: 503-494-2096; Fax: 503-494-6862; E-mail:
wiensg@ohsu.edu.
Published, JBC Papers in Press, August 30, 2001, DOI 10.1074/jbc.M104979200
| |
ABBREVIATIONS |
The abbreviations used are:
V-region, variable
region;
CDR, complementarity determining region;
ER, endoplasmic
reticulum;
VH, variable region of Ig heavy chain;
VL, variable region of Ig light chain;
PC, phosphocholine;
NPPC, p-nitrophenylphosphocholine;
APPC, p-aminophenylphosphocholine;
WT, wild type;
H, heavy;
L, light;
ELISA, enzyme-linked immunosorbent assay;
BFA, brefeldin A;
PAGE, polyacrylamide gel electrophoresis.
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ABSTRACT
INTRODUCTION