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J Biol Chem, Vol. 274, Issue 44, 31445-31455, October 29, 1999
From the Recombinant secretory immunoglobulin A containing
a bacterial epitope in domain I of the secretory component (SC) moiety
can serve as a mucosal delivery vehicle triggering both mucosal and systemic responses (Corthésy, B., Kaufmann, M., Phalipon, A., Peitsch, M., Neutra, M. R., and Kraehenbuhl, J.-P. (1996) J. Biol. Chem. 271, 33670-33677). To load recombinant secretory IgA
with multiple B and T epitopes and extend its biological functions, we
selected, based on molecular modeling, five surface-exposed sites in
domains II and III of murine SC. Loops predicted to be exposed at the
surface of SC domains were replaced with the DYKDDDDK octapeptide
(FLAG). Another two mutants were obtained with the FLAG inserted in
between domains II and III or at the carboxyl terminus of SC. As shown
by mass spectrometry, internal substitution of the FLAG into four of
the mutants induced the formation of disulfide-linked homodimers. Three
of the dimers and two of the monomers from SC mutants could be
affinity-purified using an antibody to the FLAG, mapping them as
candidates for insertion. FLAG-induced dimerization also occurred with
the polymeric immunoglobulin receptor (pIgR) and might reflect the
so-far nondemonstrated capacity of the receptor to oligomerize. By
co-expressing in COS-7 cells and epithelial Caco-2 cells two pIgR
constructs tagged at the carboxyl terminus with hexahistidine or FLAG,
we provide the strongest evidence reported to date that the pIgR
dimerizes noncovalently in the plasma membrane in the absence of
polymeric IgA ligand. The implication of this finding is discussed in
terms of IgA transport and specific antibody response at mucosal surfaces.
Clinically relevant IgAs can be produced by hybridomas and have
been shown in animal models to be protective against challenge with
mucosal pathogens (1-7). Association in vitro of
recombinant secretory component
(SC)1 and dimeric IgA
(IgAd) represents a convenient procedure to produce secretory IgA (8). Furthermore, the affinity of the reconstituted secretory IgA for its antigen is not affected at all, reflecting the
high specificity of association of the two partners (9). Hence, the
biological properties of a monoclonal IgA and of the cognate SC-IgA
complex can now be tested in parallel in different systems.
Recently, we designed a novel potential delivery shuttle for combined
active/passive immunization by assembling in vitro
IgAd with recombinant rabbit SC bearing a pathogen-derived
epitope in the E-F loop of domain I (10). Mice immunized orally with this complex developed an antibody response directed at both the heterologous SC and the epitope, which was surface-exposed. However, the antigenized SC was very poorly secreted and mostly retained in the
early secretion pathway. This study revealed a novel function for
domain I in SC/pIgR secretion, besides its role in the initial, high
affinity, binding to IgAd (11-17).
This unexpected drawback prompted us to identify other potential sites
of insertion for linear epitopes that will not affect secretion or
association with IgAd. We concentrated our mapping analysis
on domains II and III of SC because they do not play any documented
role in IgA binding and are even missing in certain rabbit alleles
coding for a truncated, yet active, pIgR (17, 18). Domain I of rabbit
SC exhibits two antiparallel A selection of loops was replaced by the FLAG octapeptide for two major
reasons. 1) The peptide is highly hydrophilic and thus more prone to
remain exposed on the molecule surface; 2) recognition of the peptide
by the commercial monoclonal antibody M2 confirms its surface exposure.
To facilitate the interpretation of the data, target sites were
modified individually. Surprisingly, epitope substitution in most
locations inside SC domains II and III induced the formation of SC
homodimers through disulfide bridges. In the SC dimer, structural
constraints or buried residues modulate the M2 reactivity against each
mutant differentially, yielding clues about its topology of
association. This implies that several insertion sites in the new
dimeric SC we have generated and analyzed either expose a peptide to
(ideal for B epitopes) or hide it from (valuable for T epitopes)
the external medium.
The unexpected tendency of mSC-FLAG mutants to dimerize prompted us to
extend the analysis to the full-length pIgR carrying the transmembrane
and the cytoplasmic regions missing in SC. We found that the pIgR
counterpart of one mutant substituted in the second domain formed
covalent dimers as the corresponding SC, reflecting the so-far
undetected dimeric nature of the receptor. We then examined the
behavior of the intact pIgR with either the 6xHis or the FLAG tag
sequence fused at the carboxyl terminus. Using coimmunoprecipitation,
we demonstrate that pIgR exists as noncovalent homodimers in the plasma
membrane of COS-7 cells and Caco-2 cells in the absence of polymeric
IgA ligand.
Computer-assisted Molecular Modeling of mSC Domains
Molecular models of domain I and of the domain II-domain III
module of wild type and mutant mSC were constructed as described by
Coyne et al. (17), and their quality was assessed by the three-dimensional/one-dimensional profile matching procedure of Lüthy et al. (19).
Construction of Expression Vectors for Mutant and Wild Type
mSC and mpIgR
The 3095-base pair-long cDNA-encoding murine pIgR (20), a
gift of Dr. Charlotte S. Kaetzel (Case Western Reserve University), was
cloned into the EcoRI site of Bluescript II KS+
(Stratagene). Because the authentic carboxyl terminus of mSC has not
yet been precisely mapped, we chose, based on comparison with human SC (21), to stop the translation of the protein after Arg596.
The 3' region coding for the transmembrane domain and the cytoplasmic tail was replaced by the double-stranded oligonucleotide coding for the
amino acids Pro589 to Arg596 containing half of
a 5' SmaI site and a 3' stop codon, an EcoRI site, and half of a 3' NotI site (Fig.
1A). The resulting plasmid was
named pBSmSC. The same approach served to generate the construct pBSmSC-FLAG:Cterm using a double-stranded oligonucleotide encoding the
FLAG octapeptide. Oligonucleotides were all obtained from Microsynth
GmbH (Balgach, Switzerland).
The 5'-untranslated region of the murine pIgR cDNA in pCB6 (22) was
deleted according to the strategy described in Fig. 1B,
using primers mSCBglMet(+) and mSCKpn1( We used recombinant PCR (23) to introduce into pCB6mSC(-5') one copy of
the FLAG coding sequence at sites defined according to the molecular
models (Table II and Fig.
2). "Inside" primers were designed to
contain the FLAG coding sequence as the overlapping region,
e.g. primers FLD1cdr3(
Covalent Homodimers of Murine Secretory Component Induced by
Epitope Substitution Unravel the Capacity of the Polymeric Ig Receptor
to Dimerize Noncovalently in the Absence of IgA Ligand*
§,
, and**
Institut Suisse de Recherches, Institut de Biochimie,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-pleated sheets shaped by nine rigid
successive -strands connected by protruding flexible loops (17).
Molecular models of mSC domains II and III were constructed and shown
to fold with a similar pattern.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Construction of expression vectors for murine
SC and pIgR. A, creation of novel carboxyl termini for
mSC by insertion of a double-stranded oligonucleotide. The stop codon
is underlined, half restriction sites are
italicized, and the EcoRI site is in
boldface. B, deletion of the 5'-untranslated region in the
eukaryotic expression vector pCB6 by PCR using primers mSCBglMet(+) and
mSCKpn1( 
) (Table I). B, BglII; E,
EcoRI; K, KpnI; N,
NotI; S, SmaI.
) (Table
I), yielding plasmid pCB6mpIgR(-5'). To
allow expression of wild type mSC and mSC-FLAG:Cterm, the 3'
KpnI-EcoRI fragment of the constructs in Fig.
1A was introduced into pCB6mpIgR(-5') cut with the same two enzymes.
List of primers used for PCR engineering of mSC/mplgR sequences
)
strand as indicated. Nucleotides within restriction sites are
underlined.
) and FLD1cdr3(+) (Table I).
"Outside" primers were designed to permit the amplification of
products exhibiting restriction sites adapted to the substitution of
wild type mSC sequences in pCB6. The pairs of outside primers and
restriction enzymes used were: mSCBglMet(+) and mSCNhe1(), with
BglII and NheI (domain I mutant); mSCKpn1(+) and
mSCNhe1(), with KpnI and NheI (domain II
mutants); mSCNhe1(+) and pCB6(), with NheI and EcoRI (domain III mutants). The FLAG coding sequence was
inserted between domains II and III (between residues 215 and 216) by
using two successive PCRs. The product amplified with primers
mSCKpn1(+) and D3D2FLin() was recovered from an agarose gel and
reamplified with primers mSCKpn1(+) and D3D2FLout() to elongate the
domain II carboxyl terminus by the FLAG sequence and a NheI
site. Insertion of the Kpn I-NheI-digested
product resulted in the generation of plasmid pCB6mSC-FLAG:D2-D3(-5').
The regions that have been amplified by PCR were sequenced (24). All
the mSC constructs, although keeping the original Kozak and signal
peptide sequences, lack both the 5'- and 3'-untranslated regions. The
plasmid encoding the mpIgR with the FLAG sequence replacing loop B-C of
domain II (pCB6mpIgR-FLAG:D2:BC(-5')) was obtained by cloning the
KpnI-NheI fragment obtained from the
mSC-FLAG:D2:BC(-5') construct into pCB6mpIgR(-5'). The polypeptides
expressed from these constructs are schematized in Fig.
3.
Sequence of loops in mouse secretory component replaced with the FLAG
epitope
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Fig. 2.
Ribbon representation of domains of wild type
and engineered mSC. Panel A represents a ribbon model
of domain II and III of wild type mSC. Panels B-F depict
ribbon models of mSC mutants carrying the FLAG epitope in
surface-exposed areas of domain II and domain III. B,
mSC-FLAG:D2:BC; C, mSC-FLAG:D2:DE; D,
mSC-FLAG:D2-D3; E, mSC-FLAG:D3:DE; F,
mSC-FLAG:D3:CC'. Amino acid sequences comprising the FLAG octapeptide
are colored in red. Orange rods connecting the
blue peptide backbone represent disulfide bridges.
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Fig. 3.
Schematic representation of the mouse SC and
pIgR constructs used in this study. Mouse SC, the ectodomain
portion of pIgR, consists of five immunoglobulin-like domains
(I-V). Open box, 18-amino acid signal peptide;
hatched box, transmembrane domain of pIgR; black
box, the FLAG sequence in mSC mutants; shaded box,
6xHis residues. The nomenclature on the left is that used in the text.
The carboxyl terminus of wild type mSC was set by introducing a stop
codon after the Arg596 codon, with numbering based on
Piskurich et al. (20).
For tagging the receptor with a FLAG epitope following
Asp748 (deleting the last five residues), mpIgR coding
sequences were first amplified by PCR using primers mSCSma1(+) and
mpIgFLin() (Table I). The gel-purified PCR fragment was reamplified
using primers mSCSma1(+) and mpIgFLout(), digested with
XmaI and XbaI, and cloned into Bluescript II
carrying the mpIgR KpnI/EcoRI fragment cut with
the same enzymes. This cloning intermediate was then treated with
KpnI and XbaI, and the resulting mSC fragment was substituted for the wild type fragment in pCB6mpIgR(-5') described above, yielding plasmid pCB6mpIgR:FLAG(-5').
The same strategy was followed to append five histidine residues after
His747 of pIgR (deleting the last six residues), using
primers mSCSma1(+), mpIgH6in(), and mpIgH6out() in the two
sequential PCR amplifications, yielding plasmid
pCB6mpIgR:His6(-5').
Antisera and Antibodies
The production of rabbit antiserum to denatured mSC has been described previously (25). The generation of rabbit antiserum against the native conformation of mSC and affinity purification of derived IgGs is decribed elsewhere (54). Rabbit IgGs were batch-purified using protein A-Sepharose CL-4B (Amersham Pharmacia Biotech). 2 mg of rabbit IgG to native mSC were biotinylated with biotin-LC-hydrazide (Pierce) according to the manufacturer's instructions. Purified mAb M2 and agarose-bound M2 (M2 affinity gel) were purchased from Eastman Kodak Co., and penta-HisTM mAb was from Qiagen. HRP-conjugated goat anti-mouse IgG (Fc-specific) was supplied by Sigma, and HRP-conjugated goat anti-rabbit IgG was bought from Cappel.
Cell Culture and Transfection
COS-7 cells (ATCC CRL 1651) were cultured under 5% CO2 at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 4.5 g/liter glucose, 20 mM HEPES, and 10 µg/ml gentamycin (all from Life Technologies, Inc.). All DNAs were prepared using the MaxiPrep kit of Qiagen and used without further purification. Two series of 107 cells in 0.4 ml of PBS were transfected by electroporation (400 V, 250 microfarads) for about 10 ms in 0.4-cm cuvettes (Bio-Rad) with 25 µg of the pCB6mSC constructs. Transfection of COS-7 cells with FLAG and 6xHis-tagged pCB6mpIgR constructs was performed with 20 µg of DNA when a single construct was used or with 10 µg of each DNA when they were used in combination. To allow expression of mSC and mpIgR derivatives, cells were then seeded into 15-cm dishes (~ 2 × 107 cells in 20 ml of medium), the medium was replaced after 20 h, and cultures were maintained in fresh medium for 4 more days.
Caco-2 cells (obtained from Dr. Eric Pringault, Pasteur Institute, Paris) were cultured under 5% CO2 at 37 °C in Dulbecco's modified Eagle's medium-Glutamax-I supplemented with 10% fetal calf serum (Seromed), 4.5 g/liter glucose, 10 mM HEPES, 1% nonessential amino acids, 0.1% transferrin (Life Technologies), and 10 µg/ml gentamycin. 2 × 107 cells were transfected with FLAG and 6xHis-tagged pCB6mpIgR constructs in several batches using the SuperFect reagent (Qiagen) according to the manufacter's instructions. To allow expression of mpIgR derivatives, cells were then seeded into a 175-cm2 T-flask (~2 × 107 cells in 20 ml of medium), and cultures were maintained in complete medium for 7 days.
Recovery of Recombinant mSC and mpIgR
Supernatants containing mSC proteins were harvested, clarified by centrifugation, buffered with 20 mM Tris-HCl (pH 8.0), and supplemented with 1% (v/v) Trasylol® (aprotinin; Bayer) and 0.02% (w/v) NaN3. Proteins were purified within 2 days after storage at 4 °C.
To analyze intracellular contents, cells were washed twice with PBS and
lysed by scraping in the presence of 2 ml of 10 mM Tris-HCl
(pH 8.0), 0.1 M NaCl, 1 mM EDTA, 0.5% (w/v)
Triton X-100, 0.1% (w/v) SDS, and CompleteTM (Roche Molecular
Biochemicals) reconstituted from 1 pellet in 50 ml of buffer. Lysates
were centrifuged at 4 °C for 15 min at 10,000 × g,
and supernatants were stored at 20 °C until use.
For expression conducted in the presence of 2-mercaptoethanol, fresh medium containing 2.5 mM of the reducing agent (Life Technologies, Inc.) was added the day after transfection, and cells were left for 36 h. Supernatants were harvested, clarified by centrifugation, buffered with 20 mM Tris-HCl (pH 8.0), and supplemented with 1% (v/v) Trasylol®, 0.02% (w/v) NaN3, and 30 mM iodoacetamide.
To recover pIgR proteins, cells were washed twice with PBS before
incubation for 5 min in 1 ml of hypotonic buffer (20 mM Tris-HCl (pH 7.5), 2 mM MgCl2). Swollen cells
were lyzed by scrapping in 1 ml of hypotonic buffer complemented with
0.05% Nonidet P-40 (Pierce), 1 mM leupeptin, and 50 µl
of CompleteTM reconstituted from 1 pellet in 50 ml of PBS.
The addition of leupeptin was required to preserve the integrity of the
pIgR (see "Results"). The cell lysate was transferred to a fresh
siliconized 1.5-ml tube, and cell debris and nuclei were pelleted at
3,000 × g by centrifugation. Aliquots of the clarified
lysate were stored at 20 °C before use.
Purification of mSC Proteins Engineered with the FLAG Epitope
All chromatographic steps were performed at 4 °C.
Glycosylated recombinant mSC-FLAG proteins were enriched from COS-7
cell-conditioned media on individual 2-ml ConA-agarose (Vector
Laboratories) columns equilibrated in binding buffer (10 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM
CaCl2, 1 mM MnCl2). After extensive
washing, elution was performed following an overnight incubation at
4 °C with binding buffer containing 0.5 M
-methyl-mannopyranoside (Sigma), and the eluates were concentrated
using Centricon-50 cartridges in PBS containing 0.02% (w/v)
NaN3.
A portion of each preparation was then applied to individual 1-ml M2 affinity gel columns equilibrated in TBS buffer (25 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl (pH 7.5)). After extensive washing with TBS, mSC-FLAG proteins were eluted with 7 column volumes of TBS containing 50 µg/ml FLAG peptide (DYKDDDDK; synthesized at the Institut de Biochimie, Université de Lausanne, Switzerland). The peptide was washed out by repeated passages over a Centricon-50 cartridge (Amicon), whereas the buffer was exchanged for PBS containing 0.02% (w/v) NaN3. Purified proteins were stored at 4 °C until further use. The columns could be used several times after sequential treatment with 0.1 M glycine (pH 3.0) and 1 M Tris-HCl (pH 8.0) without loss of binding capacity.
Gel Filtration Chromatography
Chromatography was performed at 4 °C. To study the monomeric or polymeric nature of mSC-FLAG polypeptides, 0.2-ml samples were injected into a 30 × 1-cm Superose 12 HR 10/30 column coupled to a fast protein liquid chromatography system (Amersham Pharmacia Biotech) and run in PBS at a flow rate of 0.5 ml/min. Fractions of 250 µl were recovered, and 15-µl aliquots were analyzed by immunodetection using the antisera described above.
Capture ELISA for mSC
The wells of Nunc MaxiSorp ELISA plates were coated overnight at 4 °C with 50 µl of affinity-purified IgG to native mSC (54) (2 µg/ml in 50 mM sodium carbonate/bicarbonate (pH 9.6)). Wells were blocked for 30 min at room temperature with 0.2 ml of TBS buffer containing 5% (w/v) nonfat dry milk and 0.05% (w/v) Tween-20 (Bio-Rad). Samples containing mSC proteins were serially diluted into 50 µl of PBS and incubated for 1 h at room temperature. 50 µg/well of purified mSC-FLAG:Cterm (0 to 200 ng/ml) was used as a standard. After washing with TBS containing 0.05% Tween-20, bound mSC was detected using biotinylated IgG (40 µg/ml) to native mSC. HRP-coupled ExtrAvidin (Sigma; 1:1,000) was developed with 50 µl of 1,2-phenylenediamine as a chromogen. The reaction was stopped with 50 µl of 2 M H2SO4, and plates were read at 492 nm using 620 nm as reference wavelength.
Immunoblotting of mSC-FLAG Proteins
Crude lysates, mSC-FLAG preparations, affinity-purified proteins, or immunoprecipitated materials were separated in 6 or 8% denaturing polyacrylamide gels with or without 0.1 M dithiothreitol. SDS-PAGE and immunoblotting were performed as described in Rindisbacher et al. (8) using polyvinylidine difluoride membranes (BDH Laboratory supplies). Primary antibodies used were the rabbit IgG to denatured or native mSC (5 µg/ml, purified on protein A-Sepharose) or the mAb M2 to FLAG (0.75 µg/ml). Bound antibodies were detected using appropriate HRP-conjugated secondary reagents (all used at 1:3,000 dilution) and the ECL reagents from Amersham Pharmacia Biotech.
Matrix-assisted Laser Desorption/Ionization Mass Spectrometry (MALDI MS)
Sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid) was obtained from Aldrich, HPLC grade trifluoroacetic acid was purchased from Pierce; HPLC grade water came from Romil Ltd (Cambridge, U.K.), and acetonitrile was provided by Biosolve Ltd (Barneveld, Netherlands). All the other chemicals were of the highest purity and were used without further purification.
The buffer of protein samples was exchanged against 50 mM ammonium bicarbonate using a Centricon-50 cartridge, lyophilized, and dissolved in 0.1% aqueous trifluoroacetic acid at a concentration of 12.5 pmol/µl. 1 µl of the protein solution was mixed with 9 µl of sinapinic acid at a concentration of 10 mg/ml in 0.1% trifluoroacetic acid, H20:acetonitrile (2:1 v/v). 1 µl of the mixture was applied on the gold-plated target and air-dried before transfer into the source of the mass spectrometer. MALDI mass spectra were obtained on a Perseptive Biosystems Voyager RPTM mass spectrometer using a 337-nm nitrogen laser, a 25-kV accelerating potential, and a delayed extraction time of 300 ns. Samples were optically controlled using a video camera. External calibration of the MALDI spectra in the linear mode was carried out using bovine serum albumin.
Immunoprecipitations
mSC-- Identical amounts of each mSC protein were incubated for 4 h at 4 °C under gentle rocking with 0.5 ml of TBS buffer containing 60 µl of M2 affinity gel. After centrifugation, beads were washed with TBS and eluted overnight at 4 °C with 45 µl of TBS containing 100 µg/ml FLAG peptide. 15-µl aliquots of eluate were removed and boiled with 1 volume of gel-loading buffer (100 mM Tris-HCl (pH 6.8), 4% (w/v) sodium dodecyl sulfate, 0.2% (w/v) bromphenol blue, 20% (v/v) glycerol) for immunoblotting with rabbit IgG to denatured mSC.
mpIgR-- Cell lysates were diluted 6-fold with cold TBS (final volume 480 µl) and combined in siliconized 2.2-ml tubes with 60 µl of anti-FLAG slurry (M2 affinity gel) equilibrated in TBS, 0.02% sodium azide. After an overnight incubation at 4 °C, immunoprecipitates were washed 4 times with TENT buffer (50 mM Tris-HCl (pH 7.5), 5 mM EDTA (pH 8.0), 150 mM NaCl, 1% Triton X-100), and the beads were specifically eluted with 50 µg/ml FLAG peptide DYKDDDDK. The eluate was mixed with SDS-PAGE loading buffer with or without dithiothreitol, boiled for 3 min, and loaded onto an 8% SDS-polyacrylamide gel. Proteins were transferred to polyvinylidene difluoride membranes, and after blocking in TBS-0.05% Tween 20, the blot was sequentially incubated with 0.1 µg/ml Penta-HisTM mAb and 1:3,000 goat anti-mouse IgG (Fc-specific), HRP-conjugated. Enhanced chemiluminescence was used to visualize the precipitated receptors.
Alternatively, immunoprecipitation was performed with 1 µg/ml Penta-HisTM mAb. After an overnight incubation at 4 °C, the pIgR-antibody complexes were precipitated with 60 µl of precleared protein G-Sepharose bead slurry (Amersham Pharmacia Biotech). The beads were washed as above and specifically eluted with 50 µg/ml peptide DHHHHHHK. The associated pIgR-FLAG protein was detected using anti-FLAG mAb M2 at 2 µg/ml, with all other steps and reagents kept identical. Immunodetection with anti-mSC was performed as described above.
Surface Biotinylation
1 × 107 transfected COS-7 cells were washed in cold PBS, resuspended in 2 ml of cold PBS containing 0.5 mg/ml sulfo-NHS-LC-Biotin (Pierce), and incubated for 30 min at 4 °C. The cells were washed twice with PBS, resuspended in 1 ml of Dulbecco's modified Eagle's medium, and incubated on ice for 10 min to quench any residual biotinylation reagent. Cells were lysed using 1 ml of biotinylation lysis buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.2% bovine serum albumin, 1% Triton X-100, 1 mM leupeptin, 50 µl of CompleteTM reconstituted from 1 pellet in 50 ml PBS). Cleared lysates were incubated at 4 °C for 2 h with streptavidin-agarose (Sigma) to harvest biotinylated pIgR. The beads were washed three times with biotinylation buffer and boiled for 3 min in 2× SDS sample buffer, and the eluate was analyzed by immunoblotting using rabbit anti-mSC as described above.
Receptor Dimerization
Cross-linking by boiling in SDS was performed as described in Hirt et al. (26). 1 × 106 transfected COS-7 cells were washed in PBS, scraped, pelleted, lysed with 100 µl of hot 3% SDS, and then boiled for 5 min. The lysate was diluted with dilution buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100) then immunoprecipitated with anti-FLAG beads (M2 affinity gel) as above and analyzed by 8% SDS-PAGE. Immunodetection of blotted proteins was carried out with rabbit anti-mSC.
Other Analytical Procedures
Proteins were quantitated with the bicinchonninic acid assay
(27) using bovine serum albumin as a standard (Pierce). Silver staining
of SDS-PAGE gels was performed according to Morrissey (28).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Prediction of Surface-exposed Loops in mSC Using Molecular Modeling-- Based on the approach described in Coyne et al. (17) and Corthésy et al. (10), we constructed molecular models of domain II and III of wild type mSC (Fig. 2A). Both domains exhibit the characteristic structure of Ig superfamily members, consisting of nine strands (termed A, B, C, C', C'', D, E, F, G) connected by loops of variable size. Within each domain, a disulfide bond links strand B with strand F, and a second disulfide bridge constraints the C-C' loop. The folding pattern in the first four domains of pIgR has been classified in the V set (variable domain) of Ig-like domains (29).
Guided by this model, we selected four internal loops for replacement with the FLAG octapeptide DYKDDDDK (Ref. 30; see Table II and Fig. 3). The FLAG sequence was aligned to optimally match the charge and hydropathy of amino acids in the loops. The tyrosine residue in the FLAG sequence served as an "anchoring point," always replacing a hydrophobic residue of SC (Fig. 2). Loops for substitution were also selected on the basis of (i) their length (6-8 amino acids), (ii) the degree of variability of the SC sequence between species including human, rat, and rabbit, and (iii) the absence of the N-linked glycosylation signal Asn-Xaa-Thr/Ser in the case of domain II. As a negative control, we engineered the F-G loop of domain I (CDR3-like), whose replacement with another sequence abolished binding of IgA to rabbit pIgR without strongly altering the overall conformation (17). Models of domains II and III modified with the FLAG epitope (Fig. 2, B-F) were constructed to take into account the local changes due to amino acid substitution.
The absence of a "linker" between domains II and III probably reduces their mobility in a back-to-back conformation. This prompted us to insert (rather than substitute) the FLAG sequence as a linker between domain II and III. Accordingly, the model of the resulting mutant shows an extension of the G strand of domain II projecting into the A strand of domain III (Fig. 2D). Finally, our positive control consisted of inserting the FLAG sequence at the carboxyl terminus of mSC (20).
Efficient Secretion of mSC-FLAG Mutants Produced In COS-7
Cells--
COS-7 cells were transiently transfected with the pCB6
expression vector comprising the wild type or mutated mSC constructs depicted in Fig. 3. The amount of recombinant mSC polypeptides secreted
into the cell culture medium was measured by capture ELISA as described
under "Experimental Procedures." All the recombinant proteins were
efficiently secreted by COS-7 cells (9 to 16 µg/ml) to a level
similar to wild type SC (12 µg/ml). Substitutions in domains II and
III were thus a suitable option, overcoming the limited production
resulting from replacement of the E-F loop of domain I (10).
Recombinant mSC-FLAG proteins in cell culture supernatants were
enriched by ConA-agarose chromatography, before fractionation by
SDS-PAGE under reducing conditions. Immunodetection with IgG to mSC
revealed a single band for the wild type and mutant mSC (Fig.
4A); consistently, mAb M2
recognized the FLAG epitope in the mutants (Fig. 4B).
Significant differences in the apparent Mr of
the various mutants were observed (Table
III). In particular, the two mutants
bearing a substitution of the D-E loop migrated much faster than the
other proteins. Such notable differences could not simply be explained
from the local sequence and charge substitutions imposed by the mere
presence of FLAG. Full deglycosylation with
peptide:N-glycosidase F, which cleaves N-linked
glycans, still resulted in mSC species with highly variable
Mr values in SDS-PAGE.2 Additional
analysis of mSC mutants with wheat germ agglutinin (31, 32) and
endo--N-acetylglucosaminidase H indicates that the
presence of the FLAG motif affects both the terminal glycosylation and/or the surface exposure of sialic acid residues in the protein mutants (summarized in Table III).
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Formation of Covalent Homodimers upon Introduction of the FLAG Epitope Within Domain I, II, or III of mSC-- When analyzed by nonreducing SDS-PAGE, mSC-FLAG:D1:FG, mSC-FLAG:D2:BC, mSC-FLAG:D2-D3, and mSC-FLAG:D3:CC' migrated as high molecular weight complexes along with the monomeric form (Fig. 4C). These high Mr forms are reversibly cross-linked by disulfide bonds because they were absent when samples were boiled in 0.1 M dithiothreitol before SDS-PAGE (Fig. 4A). The mere presence of the foreign sequence in mSC-FLAG:Cterm was not responsible for its lack of cross-linking since wild type mSC did not form oligomers either. Overexpression in COS-7 does not explain the phenomenon either, because not all the mutants are secreted as oligomers. The appearance of intermolecular disulfide bridges seems to correlate with the FLAG sequence introduced in the vicinity of the C-C' loop. The disulfide bond encompassing loop C-C' is the most surface-exposed in SC domains (Fig. 2) and, therefore, could be more prone to local changes.
To determine whether monomeric forms of mutants could exist as
noncovalent dimers, we chromatographed mSC-FLAG:D2:BC on a sizing
column run under native conditions. Column fractions analyzed by
immunoblotting showed that only the covalent dimer elutes early (Fig.
5A). mSC-FLAG:D2:DE,
mSC-FLAG:D3:DE, mSC-FLAG:Cterm, and wild type mSC were eluted at the
position of the monomer exclusively.2 Therefore, covalency
appears necessary to stabilize mSC as a dimer.
|
To provide conclusive evidence that the dimeric forms were not artifacts of electrophoresis, we subjected the purified mSC-FLAG:D2:BC protein to MALDI MS. Fig. 5B shows the MALDI spectrum obtained in the linear mode using sinapinic acid as a matrix. The spectrum displays a molecular ion peak centered at 77 kDa for the M+ ion and 38 kDa for the M2+. Note that a weak amount of monomer was present in this preparation as shown in the inset of Fig. 5B. In addition to the M+ ion, the 2M+ was obtained with a molecular weight of 153 kDa, corresponding to dimeric mSC-FLAG:D2:BC. The multiple signals reflect the presence of different glycoforms. This heterogeneity is attributed to native structure diversity and not to spectroscopic fragmentation (33). With a calculated Mr of 66,194, post-translational modifications contribute about 14% of the mass in both monomeric and dimeric mSC-FLAG:D2:BC.
Surface Accessibility of the FLAG Epitope Introduced Inside SC
Domains--
To test the surface exposure of the FLAG epitope in the
various mutants, we first performed immunoblotting under nonreducing conditions using the anti-FLAG mAb M2. Fig.
6A shows that mAb M2 detected
only a subset of the species observed in Fig. 4C using a
polyclonal antibody to mSC. Strong signals were observed for mSC-FLAG:Cterm, dimeric mSC-FLAG:D1:FG, and both forms of
mSC-FLAG:D2-D3. Dimeric mSC-FLAG:D2:BC and monomeric mSC-FLAG:D1:FG
yielded signals of intermediate intensity.
|
To directly assess the reactivity of the epitope in monomers and dimers in solution, we performed immunoprecipitation by using agarose-bound M2. Data in Fig. 6B indicate that all immunoreactive mutants in Fig. 6A were also precipitable, yet to various extents. In addition, detection of mSC-FLAG:D2:BC and mSC-FLAG:D3:CC' species was improved in this assay. This indicates that dimerization of mSC-FLAG:D1:FG, mSC-FLAG:D2:BC, and mSC-FLAG:D2-D3 does not mask FLAG-containing sites and maps those as candidates for B and T epitope insertion. The lack of binding to agarose-bound M2 of the two mutants with the D-E loop replaced with FLAG implies that the peptide epitope was probably in a constrained conformation no longer adapted for recognition by the mAb.
The same four mSC-FLAG mutants (D1:FG, D2:BC, D2-D3, D3:CC') could be reproducibly purified to homogeneity using the mAb M2 coupled to agarose beads and mild elution with the FLAG octapeptide. The purity and integrity of these preparations was verified by SDS-PAGE and subsequent silver staining (Fig. 6C, left panel). The protein preparations were homogenous, with the exception of mSC-FLAG:Cterm, which showed two bands, as observed for milk-derived mSC (34). Under nonreducing conditions (Fig. 6C, right panel), mSC-FLAG:D2:BC was shown to consist exclusively of dimers, whereas little monomeric mSC-FLAG:D1:FG and mSC-FLAG:D2-D3 accompanied the dimeric molecule. Thus, dimers were selectively enriched, although both monomeric and dimeric forms were immunoprecipitable (Fig. 6B). This discrepancy might be explained by the extended incubation time used for immunoprecipitation.
Formation of Covalent mSC-FLAG Dimers in the Early Secretory
Pathway--
Cell lysates were analyzed by immunoblotting under
nonreducing conditions to determine whether covalent dimers are formed intracellularly and how the presence of the FLAG affects secretion (Fig. 7A). Using anti-mSC
antiserum, we observed that all mutants forming dimers were retained
preferentially as compared with mSC-FLAG:D2:DE, mSC-FLAG:D3:DE, and
mSC-FLAG:D3:CC'. For mSC-FLAG:D2:BC and mSC-FLAG:D2-D3, the monomer and
the dimer accumulated to the same extent. In contrast, when the
immunoreactivity to the FLAG was examined, only dimers of
mSC-FLAG:D1:FG and mSC-FLAG:D2-D3 gave significant signals (Fig.
7B). Hence, monomers and most dimers that are present
intracellularly do not react with mAb M2, reflecting a distinct
conformation as compared with the secreted, mature forms analyzed in
Fig. 6A. This suggests that mSC mutants must undergo
important structural rearrangements before they are allowed to continue
their journey through the secretory pathway. Consistently, all the
mutants retained inside the cell were totally sensitive to
endo--N-acetylglucosaminidase H,2 indicating
that these glycoforms are not fully matured and reside in the
endoplasmic reticulum (ER) or the intermediate compartment (ERGIC)
(35). Note also that these intracellular forms are smaller than the
secreted proteins (Fig. 4C).
|
In an attempt to relieve inappropriate disulfide cross-linking of mutants leading to intracellular accumulation, cells were cultured for 36 h in the continued presence of 2.5 mM 2-mercaptoethanol. Such a treatment allowed the release of free immunoglobulin light chain (36) and of unpolymerized IgM (37) or IgA (38), which are normally sequestered in the ER. No dramatic effect on the degree of secretion and on the molecular aspect of recombinant mSC:FLAG could be seen (Fig. 7C) as compared with the material recovered from COS-7 cells kept under normal conditions (Fig. 4C). Exposure of cells to 2-mercaptoethanol produced a decreased reactivity of the FLAG in mSC-FLAG:D2-D3 molecular species, whereas dimeric mSC-FLAG:D3:CC' and, particularly, mSC-FLAG:D1:FG became more immunoreactive (Fig. 7D).
Covalent Dimerization of the Complete Receptor with a FLAG
Replacing Loop B-C of the Second Domain--
So far dimers were
obtained using recombinant mSC mutants, a nonnatural form of the pIgR
that normally exists as a transmembrane protein. To exclude the
possibilty that SC dimers represented an artifactual form appearing as
a consequence of the absence of the cytoplasmic and transmembrane
domain, we produced recombinant mpIgR with the FLAG replacing the B-C
loop of domain II (mpIgR-FLAG:D2:BC) in COS-7 cells. The structure of
pIgR was analyzed by immunoblotting of detergent-solubilized cellular
proteins under nonreducing conditions (Fig.
8A). Wild type and mutant
monomers both migrated with a Mr of 115. As
expected, the mpIgR mutant showed an additional band with a double
apparent Mr. Thus, dimerization induced by the
presence of the FLAG takes place whether the protein is being secreted
or integrated in the membrane, and the local change in charge and
sequence alone is responsible for the formation of novel disulfide
bond(s). Furthermore, only the dimeric form of mpIgR-FLAG:D2:BC was
immunoprecipitated by the anti-FLAG antibody.2
|
Coimmunoprecipitation Studies Identify pIgR Homodimers-- Our data provide evidence that pIgR forms homodimers within the plasma membrane; however, it remains to be demonstrated that this happens with wild type pIgR. So far, for this interaction to be detected, forced cross-linking or discrete modifications in the primary structure of the pIgR have had to be used (Refs. 26 and 39; this study). Furthermore, the possibility that pIgR associates with another protein of the same molecular weight could not be excluded. To demonstrate that pIgR indeed forms homooligomers, we performed co-immunoprecipitation studies on COS-7 cells cotransfected with pCB6 constructs encoding mpIgR:6xHis and mpIgR:FLAG carrying a different epitope tag at the carboxyl terminus (Fig. 3). We first determined that COS-7 cells expressed the tagged receptors similarly to the wild type untagged pIgR (Fig. 8B), yet a significant portion of pIgR (115 kDa) in the cell extract was rapidly converted into SC (85 kDa). Three cross-reactive bands were also detected in nontransfected cell extracts. Subsequent experiments were thus carried out in the presence of 1 mM leupeptin, a protease inhibitor known to block the conversion of plasma membrane pIgR into SC, and pIgR proteins were purified by immunoprecipitation.
COS-7 cell lysates expressing both mpIgR:6xHis and mpIgR:FLAG were
immunoprecipitated with the anti-FLAG M2 affinity gel. The
immunoprecipitates were separated by SDS-PAGE and subjected to
immunoblotting. The blots were analyzed with the anti-5xHis mAb
followed by anti-mouse IgG, HRP-conjugated. The results show that
mpIgR:6xHis coprecipitates with mpIgR:FLAG (Fig. 8C,
lanes 1 and 2). In either the presence or the
absence of dithiothreitol in the sample buffer, only the 115-kDa
species could be observed, indicating that the homodimer resulted from
the noncovalent interaction of the two partners. The same pattern was
obtained when the blot was analyzed with anti-SC antiserum (Fig.
8C, lanes 3 and 4), confirming that
the 115-kDa band was indeed pIgR. The same 115-kDa band was revealed
when the cell lysate was first precipitated with anti-6xHis mAb, then
analyzed with anti-FLAG mAb M2 (Fig. 8C, lanes
6-9). Immunodetection required the presence of the primary antibody (Fig. 8C, lanes 5 and 10),
showing the specificity of the interaction between the pIgR forms.
Plasma membrane localization of pIgR was assessed by the lack of
sensitivity to endo--N-acetylglucosaminidase H treatment
(Fig. 8D, lanes 1 and 2) and by cell
surface biotinylation (Fig. 8D, lane 3). Boiling
of the pIgR in 3% SDS resulted in partial dimerization that could be
detected under nonreducing conditions (Fig. 8D, lanes
4 and 5). This approach was chosen because chemical cross-linking with either glutaraldehyde or sulfo-SMBP
(sulfosuccinimidyl-4-[p-maleimidophenyl]- butyrate) was
unsuccessful (data not shown).
Although the experiments above all go to the direction of pIgR dimer
formation, one can argue that COS-7 cells are not representative of
epithelial cells normally expressing pIgR. Coimmunoprecipitation experiments performed with Caco-2 cells transfected with constructs pCB6mpIgR:6xHis and pCB6mpIgR:FLAG led to the same pattern as that
observed (Fig. 8C). We conclude that pIgR forms dimer-sized complexes in the plasma membrane of COS-7 cells and Caco-2 cells in the
absence of IgA ligand.
| |
DISCUSSION |
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|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
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Our attempt to seek favorable sites to integrate B and T epitopes in recombinant secretory IgA to be used as a mucosal vaccine (10) led us to the finding that the pIgR exists as a dimer in the absence of IgAd ligand. Initially, we chose to identify insertion sites based on modeling of domains II and III known to be dispensable to the pIgR function in rabbit. Five mSC mutants were obtained with the FLAG octapeptide replacing stretches of amino acid predicted to be surface-exposed. Two mutants were also produced containing the FLAG in between the second and third domain and at the carboxyl terminus, respectively. Unexpectedly, all mutants exhibited many particular structural features. First, the interaction between the FLAG and the cognate mAb was modulated depending on the selected site. Second, apparent sizes and glycosylation patterns were heterogenous. Third, several mutants were partly secreted as covalent dimers. The latter observation prompted us to extend our analysis to the full pIgR, and we could establish that the empty receptor dimerizes in a noncovalent fashion in the plasma membrane of both COS-7 cells and epithelial Caco-2 cells.
The molecular models predict that, in solution, the FLAG is displayed on the surface of individual domains. Mutants D1:FG, D2:BC, D2-D3, and D3:CC' could indeed be immunoprecipitated with the immobilized anti-FLAG mAb M2, indicating that the inner FLAG epitope was freely accessible, to an extent similar to that of construct Cterm. In immunoblots, the reactivity of mutants D1:FG, D2:BC, and D3:CC' toward mAb M2 was decreased under nonreducing conditions as compared with samples boiled in the presence of dithiothreitol. This behavior of mAb M2 has been observed before in another protein context (40) and might result from its ability to recognize multiple conformers. Two mutually nonexclusive possibilities, conformationdependent binding of mAb M2 or masking of the FLAG by interaction with residues of vicinal domains in the same mSC molecule, could explain the lack of reactivity of mutants D2:DE and D3:DE under nonreducing conditions. For those monomeric mutants that are poorly recognized by mAb M2, it can also be postulated that the five domains fold back into a U-shaped conformation, burying the FLAG located in the central portion of the protein. Consistently, mAb M2 reacts strongly with both the dimers and the monomers of mSC-FLAG:D1:FG and mSC-FLAG:D2:BC, exhibiting the FLAG in a more exposed environment. Finally, dimeric mSC-FLAG:D3:CC' is weakly immunoreactive, compatible with the C-C' loop being very close to the intermolecular disulfide bridge. Also, the monomeric form is poorly reactive, probably because the FLAG replacing the C-C' loop is strongly constrained by the disulfide bond connecting Cys254 and Cys261. We conclude that mutants D1:FG, D2:BC, D2-D3, D3:CC', and C-term should preferentially carry B epitopes to be exposed on the surface, whereas mutants D2:DE and D3:DE can be used as carrier of T epitopes solely.
We have shown that covalent homodimers of mutant mSC-FLAG proteins are formed early in the secretory pathway. It is likely that mSC exits the ER as multimeric cargo and that extra negative charges provided by a vicinal FLAG induce rearrangement of disulfide bonds. In addition to stabilizing the dimer, covalency might also mask thiols usually acting as intracellular retention elements for unassembled molecules (36). We were unable to detect cross-linking of mSC-FLAG mutants with ER matrix resident proteins. This argues in favor of a preferential reactivity of interchain thiols in SC mutants over other reactive groups. Biosynthesis of the mutants, performed in the presence of 2-mercaptoethanol added in the cell culture medium, did not abolish covalent dimerization nor improve secretion. The existence of covalent dimers was further established by resolution on sizing columns and mass spectrometry analysis. Interestingly, mutants D2:DE and D3:DE, which do not form covalent homodimers, have in common three conserved (KDD) and one related (R to K) residue found in the mSC D-E loop. Thus, too few sequence changes do not induce major structural differences required for reshuffling of disulfide bonds. In conclusion, the covalent dimers of mSC mutants described in the paper suggest that mSC molecules pair, at least transiently, via the first three domains. Model-based selection of surface elements and their substitution has enabled us to prove this so-far speculative property. In addition, we found that the pIgR counterpart of mutant D2:BC formed covalent dimers as well, indicating that the transmembrane and cytoplasmic domains do not contain motif(s) controlling dimer association.
Forced covalent dimerization of SC/pIgR mutants might reflect a physiological event involving homooligomerization of pIgR in plasma membrane. Only scant evidence supports the notion of pIgR being a dimer. First, polyclonal or mAb directed against the ecto domain of rabbit pIgR only recognized a subset of immature receptor transiting in the ER and/or Golgi (41). We concluded that pIgR oligomerization was compatible with masking of some epitopes, yet conformational changes or association with other components of the secretory machinery represent sound alternatives. Second, rabbit pIgR covalent dimers form upon boiling in 3% SDS (26). By analogy to glycophorin A, this phenomenon was originally attributed to transmembrane domain residues of pIgR, but the sensitivity to reduction points toward artifactual disulfide bonding (Ref. 39; this study). Similarly, disulfide-bonded dimers of the low density lipoprotein receptor (42) and the 46-kDa mannose 6-phosphate receptor (43) formed post-lysis as revealed by direct, nonreducing SDS-PAGE. In contrast, chemical cross-linking failed to detect the presence of pIgR homodimers in Madin-Darby canine kidney cells (44), COS-7, and Caco-2 cells (this study). Although not detectable by gel filtration at 4.5 µM concentration, dimers of mSC could be shown by mass spectrometry, indicating a tendency for association with increasing concentration (54). In human milk secretion, the concentration of free SC reaches 25 µM (2 mg/ml (45)), therefore suggesting that dimerization could be physiologically relevant. Detection of such dimers is precluded by a dissociation constant falling in the high micromolar to millimolar range, as exemplified also by the ectodomain of CD4 (46).
Recently, Singer and Mostov (39) reported on IgA-mediated dimerization of pIgR resulting in stimulation of their transcytosis and elicitation of cellular signal. In the discussion, they raised the possibility that pIgR preexists as a dimer, but as far as we know, there are no data on the oligomerization of intact pIgR on the membrane. Our data, using immunoprecipitation of membrane pIgR carrying two different epitope tags at the carboxyl terminus, demonstrate that noncovalent dimerization occurs in the absence of IgA ligand in both COS-7 cells and in epithelial Caco-2 cells normally expressing pIgR. The existence of pIgR dimers before IgAd binding makes it unlikely that dimerization per se induces the cascade events (stimulation, sensitization) recently reported in the Madin-Darby canine kidney cells model (47). In the absence of IgAd ligand, the short domain (amino acids 726-736 in rabbit pIgR) in the cytoplasmic tail regulating IgAd-stimulated transcytosis could be masked or assembled in a nonproductive conformation. Under these circumstances, constitutive transcytosis is reduced, enabling the cell to maintain enough of the pIgR available at the basolateral surface. Dimerization occurs equally on the surface of Jurkat cells (39) and COS-7 cells (this study), arguing in favor of an intrinsic, cell-independent property of the pIgR. Epithelial specific trafficking events seen in Madin-Darby canine kidney cells might be attributed to a so-far unknown partner protein (47).
It is possible that pIgR exists in an equilibrium of monomer and dimer, possibly distributed at specific locations within the cell. Studies with the 46-kDa mannose 6-phosphate receptor showed that the receptor is present in several oligomeric states (43), the distribution of which, however, was independent of recycling and binding of ligands (48). We propose that the function of IgAd may thus be 2-fold: (i) stabilization of pIgR dimers (displacing the equilibrium); (ii) triggering of conformational change(s) leading to activation of the signal transduction cascade and linked transcytosis (49, 50). In terms of mucosal protection, maintenance of a pool of pIgR dimers ready to achieve IgA transport and function could prevent the rate-limiting step occurring after massive production of IgA necessary to fight local infection. In addition, the existence of monomeric and dimeric pIgR on the cell surface could explain the differences observed in terms of affinity and number of binding sites revealed when using dimeric and tetrameric IgA (51). Up-regulation of pIgR expression by inflammatory cytokines might push the equilibrium toward the formation of pIgR dimers and promote transport of the larger IgA polymers carrying more antigen binding sites.
In conclusion, we have successfully designed a strategy to identify
useful sites for introducing 8-mer B and T epitopes and possibly larger
ones in the sequence of mSC. Surface-exposed, as well as buried sites,
both constitute valuable candidates for substitition. Remarkably,
except for the domain I mutant, the mSC-FLAG molecules had the expected
IgA binding properties (52). In addition, dimerization of certain
mutants might represent an asset, both in terms of IgA stabilization
(53) and as multiple epitope carriers. Forced covalent dimerization
provided indirect clues that SC/pIgR might assemble into oligomeric
structures. The biochemical and genetic approach we used gave the
strongest evidence reported so far that pIgR exists as noncovalent
dimers in the plasma membrane.
| |
ACKNOWLEDGEMENTS |
|---|
We acknowledge the technical assistance from Corinne Tallichet Blanc and Fabienne Peneveyre. We thank Drs. Walter Hunziker and Lucy Hathaway for critical reading of the manuscript.
| |
FOOTNOTES |
|---|
* This work was supported by Biotechnology Priority Program Grant 5002-38009 and Swiss National Science Foundation Grant 3100-050912.97.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.
§ Current address: Biozentrum, University of Basel, CH-4056 Basel, Switzerland.
To whom correspondence should be addressed: Division
d'immunologie et d'allergie, CHUV, BH18-701, Rue du Bugnon, CH-1011
Lausanne, Switzerland. Tel.: 41 21 314 07 83; Fax: 41 21 314 08 01;
E-mail address: blaise.corthesy@chuv.hospvd.ch.
2 P. Crottet and B. Corthésy, unpublished data.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: SC, secretory component; mSC, murine SC; ConA, concanavalin A; ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic reticulum; FLAG, the octapeptide DYKDDDDK; HRP, horseradish peroxidase; Ig, immunoglobulin; IgAd, dimeric IgA; IgAm, monomeric IgA; IgAp, polymeric IgA; mAb, monoclonal antibody; MALDI MS, matrix-assisted laser desorption/ionization mass spectrometry; pIgR, polymeric immunoglobulin receptor; mpIgR, murine pIgR; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; HPLC, high performance liquid chromatography.
| |
REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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