Membrane-anchored Human FcRn can Oligomerize

doi:10.1016/S0022-2836(02)00626-5 available online at http://www.idealibrary.com on
w
B
J. Mol. Biol. (2002) 321, 277–284
Membrane-anchored Human FcRn can Oligomerize in
the Absence of IgG
Asja Praetor1, Robert M. Jones2, Woei Ling Wong1 and Walter Hunziker1*
1
Institute of Molecular and Cell
Biology, Epithelial Cell Biology
Laboratory, 30 Medical Drive
Singapore, Singapore 117609
2
Institute of Biochemistry
University of Lausanne
CH-1066 Epalinges
Switzerland
FcRn is unique among immunoglobulin G (IgG) Fc receptors in that it is
structurally closely related to major histocompatibility complex class I
molecules and likewise consists of an a-chain and b2-microglobulin.
Crystallographic data show that rat FcRn a-chain/b2m heterodimers can
further dimerize via ionic interactions and a carbohydrate handshake.
Intriguingly, however, no dimers are found in crystals of human FcRn,
probably because the charged amino acids and the carbohydrate implicated in dimerization of rat FcRn are not conserved. Here, we show that
although a secreted soluble form of human FcRn does not dimerize, the
membrane-anchored receptor can form both non-covalent and covalent
dimers. Furthermore, dimerization of human FcRn occurs in the absence
of its ligand, IgG.
q 2002 Elsevier Science Ltd. All rights reserved
*Corresponding author
Keywords: IgG Fc receptor; dimerization; disulfide bond; oligomerization;
protein structure
Introduction
FcRn is an immunoglobulin G (IgG) Fc receptor
involved in the transfer of passive immunity from
the mother to the newborn or fetus.1,2 The receptor
is thought to transport IgG from the maternal
circulation across the placental syncytiotrophoblast
or yolk sac, and to transcytose IgG present in colostrum and milk across the small intestine of the
suckling neonate. In addition, FcRn is implicated
in the regulation of the IgG serum concentration
by binding internalized IgG and recycling it
back into the circulation, thus protecting IgG from
lysosomal degradation.
A typical feature of FcRn is the binding of IgG at
a mildly acidic but not at neutral pH, which, based
on the structural data and in vitro mutagenesis
studies, may reflect the difference in the protonation state of critical histidine residues in Fc
involved in the receptor– ligand interaction.3 – 7 The
transcytotic and protective functions of FcRn
appear to be linked intimately to its pH-dependent
IgG binding properties.8 Since FcRn binds IgG at a
mildly acidic pH, as found in the intestinal lumen
Abbreviations used: FcRn, Fc receptor neonatal; FcRn
dimer, dimer of FcRn a-chain and b-2 microglobulin
heterodimers; hFcRn, human FcRn; IG, immunoglobulin;
MHC, major histocompatibility complex; b2m, b-2
microglobulin.
E-mail address of the corresponding author:
[email protected]
or in endosomes, but not at neutral pH, the receptor is expected to bind IgG on the lumenal surface
and, following transcytosis, release the IgG into
the circulation upon exposure to the neutral serosal
pH. In the absence of a pH gradient, IgG internalized in the fluid phase may bind to FcRn in the
acidic milieu of endosomes, from where it could
either be transcytosed (syncytiotrophoblast) or
recycled (endothelia, mammary gland epithelium,
hepatocytes). Since in different epithelial cells
FcRn can transcytose in both directions,9 – 11 a pH
or IgG-concentration gradient between the lumenal
and serosal surface may determine the direction of
net IgG transport in vivo.
FcRn, like major histocompatibility complex
(MHC) class I, is assembled from an a-chain and
b2-microglobulin. The a-chain of FcRn has been
cloned from rat,12 human,13 mouse,14 cow15 and
possum,16 and shows a high degree of homology
to the MHC class I a-chain. On the basis of the
crystal structure of soluble forms of the rat17,18 and
human19 receptors, FcRn displays a folding similar
to that of MHC class I molecules. The peptidebinding groove found in MHC I, however, is
obstructed in FcRn by bulky amino acid sidechains projecting into the groove and a closer
juxtaposition of the two flanking a-helices, thus
restricting the accessibility for peptides.8
Dimers of FcRn a-chain-b2m heterodimers
(referred to as FcRn dimers) were observed in
crystals of rat FcRn, in the absence as well as in
the presence of bound Fc fragments.17,18,20 Such
0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved
278
Human FcRn Dimers
A
soluble Flag-hFcRn
soluble Myc-hFcRn
- + - +
- - + +
kDa
45
α-Flag
31
45
α-Myc
31
1
2
3
4
FcRn dimers in crystals and the 2:1 binding stoichiometry of hFcRn and IgG in solution have been
suggested to involve FcRn molecules binding to
both sides of the homodimeric Fc fragment rather
than FcRn dimers binding to a single Fc.22
Here, we show that although a secreted soluble
form of human FcRn does not dimerize, the fulllength, membrane-anchored receptor can form
non-covalent and covalent dimers in vivo. These
dimers were detected in the absence of IgG, indicating that ligand binding is not a requisite for
dimerization of human FcRn.
Results
B
α-Flag
α-Myc
b
c
d
soluble Flag& Myc-hFcRn
control
a
Figure 1. Characterization of cells co-expressing
soluble Flag- and Myc-hFcRn. (a) Western blot analysis.
Cell culture supernatants of control cells (lane 1) or
cells expressing soluble Flag-hFcRn alone (lane 2), MychFcRn alone (lane 3), or co-expressing Flag-hFcRn and
Myc-hFcRn (lane 4), were fractionated by SDS-PAGE,
blotted and probed with M2 anti-Flag (top) or 9E10 antiMyc antibodies (bottom). (b) Immunofluorescence
staining. (a and b) Control cells or (c and d) cells stably
co-expressing soluble Flag- and Myc-hFcRn were incubated at 20 8C to accumulate soluble proteins in the
secretory pathway in the trans-Golgi network. Cells
were fixed, permeabilized and stained by indirect
immunofluorescence with monoclonal anti-Flag (green)
and polyclonal anti-Myc (red) antibodies. Shown is one
of two representative cell clones used in subsequent
experiments.
FcRn dimers are consistent with data showing that
soluble forms of FcRn bind IgG with a 2:1 stoichiometry, possibly via two non-equivalent sites.21,22
On the basis of the crystal structure, a carbohydrate “handshake” and electrostatic interactions
between charged amino acid residues located in
the dimer interface probably mediate dimerization
of rat FcRn. In contrast to rat FcRn, however, no
dimers were observed in crystals of human
FcRn,19 possibly because the N-linked carbohydrate and the charged amino acid residues
involved in dimerization of rat FcRn are not conserved in the human receptor. The lack of human
Secreted soluble human FcRn does
not dimerize
To corroborate the absence of dimers in hFcRn
crystals,18 we analyzed directly the ability of
secreted soluble receptors to dimerize using
co-precipitation experiments. For this purpose,
MDCK cells stably expressing soluble forms of
Flag-hFcRn and Myc-hFcRn were generated. The
expression and secretion of the epitope-tagged
hFcRn constructs was analyzed by immunoblotting
of cell-culture supernatants. As shown in Figure
1(a), a band at , 40 kDa was detected by M2 antiFlag (top panel) and 9E10 anti-Myc antibodies
(bottom panel) in supernatants from cells transfected with Flag-hFcRn or Myc-hFcRn alone (lanes
2 and 3, respectively) and from cells transfected
with both Flag and Myc-tagged soluble hFcRn
(lane 4). As expected, the truncated hFcRn a-chain
migrated faster than the intact hFcRn (compare
lanes 2 and 3 in Figure 5(c)). No proteins were
detected in cell-culture supernatants of nontransfected control cells (lane 1).
Using immunofluorescence staining, we confirmed that individual cells within a population
expressed soluble Flag-hFcRn as well as MychFcRn. Since the bulk of soluble hFcRn was
secreted (data not shown), we cultured the cells
at 20 8C for 12 hours to accumulate newly
synthesized proteins in the cell. This allowed the
detection by immunofluoresence of receptors
present in the secretory pathway. As shown in
Figure 1(b), individual cells in a population homogeneously expressed the two differently tagged
soluble hFcRn constructs (c and d). Non-transfected control cells, in contrast, did not stain with
the antibodies (a and b).
Next, we used co-precipitation experiments to
explore the ability of secreted soluble receptors to
dimerize. To exclude the possibility that residual
IgG present in fetal calf serum induced FcRn
dimerization, cells were cultured in serum-free
medium for at least 24 hours prior to all experiments. Flag-hFcRn was precipitated from the
cell-culture supernatants of cells co-expressing
soluble Flag-hFcRn and Myc-hFcRn, and analyzed
by Western blot for the presence of soluble
279
Human FcRn Dimers
Figure 2. Co-immunoprecipitation of soluble Myc and
Flag-tagged hFcRn. Culture supernatants from untransfected control cells (lane 1), cells expressing soluble
Flag-hFcRn (lane 2), soluble Myc-hFcRn (lane 3), or
co-expressing soluble Flag and Myc-hFcRn (lane 4) were
precipitated with immobilized M2 anti-Flag antibodies
and blotted with polyclonal anti-Flag (top panel) or
anti-Myc (bottom panel) antibodies. A representative
blot of at least three independent experiments carried
out with two different clones is shown.
Myc-hFcRn. As shown in Figure 2, anti-Flag
immunoprecipitates did not contain soluble MychFcRn (lane 4). Likewise, no soluble Flag-hFcRn
a-chain was associated with immunoprecipitated
soluble Myc-hFcRn (data not shown).
Thus, no dimers could be detected for secreted
soluble hFcRn, consistent with the reported
absence of dimers from crystals of hFcRn.19
Membrane-anchored human FcRn can form
non-covalent dimers
Since the transmembrane and/or cytosolic
domain could be required for dimer formation, we
next determined whether a membrane-anchored,
full-length hFcRn was able to dimerize. For this
purpose, we generated MDCK cell lines stably
co-expressing intact Flag and Myc-tagged hFcRn.
The expression of the two differently tagged receptors was confirmed by immunoblotting (Figure
3(a)). Two clones expressing comparable amounts
of Flag-hFcRn or Myc-hFcRn as compared to singly
transfected cells expressing Flag-hFcRn alone or
Myc-FcRn alone (compare lane 4 to lanes 2 and 3,
respectively) were chosen for further experiments.
As expected, precipitation of biotinylated cellsurface proteins with either anti-Flag-Sepharose
or IgG-agarose followed by streptavidin blots
revealed the FcRn a-chain and a 12 kDa protein,
which, in all likelihood, represents the endogenous
canine b2m that was precipitated bound to the
FcRn a-chain (Figure 3(b)).
Immunofluorescence experiments established
that the selected clones homogeneously expressed
the two differently tagged receptors. All cells in a
given population expressed Flag and Myc-tagged
hFcRn, and the two receptors co-localized extensively throughout the cell (Figure 3(c), (e) and (f)).
As expected, cells transfected with either FlaghFcRn (a and b) or Myc-hFcRn (c and d) alone
exclusively stained with M2 anti-Flag monoclonal
or anti-Myc polyclonal antibodies, respectively.
To explore the presence of hFcRn dimers, we
analyzed whether Myc-hFcRn could be co-precipitated with Flag-hFcRn and vice versa. To deplete
residual IgG present in the serum of the cellculture medium, cells were incubated in serumfree medium for 24 hours prior to all experiments.
As shown in Figure 4, Myc-tagged FcRn was
detected in anti-Flag immunoprecipitates from
cells expressing Myc-hFcRn and Flag-hFcRn, (lane
4), but not from cells expressing Flag-hFcRn (lane
3) or Myc-hFcRn (lane 2) alone. Importantly, the
differently tagged hFcRn molecules did not associate if cells expressing Myc-hFcRn only or FlaghFcRn only were mixed prior to the immunoprecipitation (lane 5), confirming that the observed
dimerization occurred in vivo and not during cell
lysis and sample preparation. Similarly, FlaghFcRn was detected when anti-Myc immunoprecipitates were blotted with anti-Flag antibodies
(lane 9). Using the same experimental approach,
hFcRn dimers were detected in FO-1 cells
expressing human b2m (see Discussion and
Supplementary Material).
Thus, co-precipitation experiments show that
the intact membrane-anchored hFcRn can form
homodimers.
Human FcRn can form covalent dimers
Interestingly, in addition to the , 47 kDa monomeric FcRn, a , 94 kDa band was observed if the
co-immunoprecipitates were analyzed on nonreducing gels (Figure 4), possibly reflecting
covalent FcRn dimers. To establish the nature
of the observed , 94 kDa band, cell lysates were
analyzed by immunoblotting following reducing
and non-reducing SDS-PAGE. The , 94 kDa band
was detected readily under non-reducing conditions (Figure 5(a)), using either anti-Flag (lane 3)
or anti-FcRn antibodies (lane 7), but was absent if
the samples were reduced with b-mercaptoethanol
(lane 2; b-ME). Since the , 94 kDa band was present in co-precipitation experiments (see Figure 4)
and only detected on non-reducing gels, it most
likely corresponds to a disulfide-linked hFcRn
dimer. Covalent hFcRn dimers were detected if
cells were lysed in the presence of the sulfhydrylmodifying reagents iodoactetamide (IAM; lane 4)
or N-ethylmaleimide (NEM; lane 5), indicating
that the disulfide-linked dimers did not result
from the reaction of free thiol groups during
sample preparation. Although covalent dimers
were consistently observed, the ratio of dimers
to monomers varied significantly between
experiments.
A detectable fraction of the covalent hFcRn
a-chain dimers reached the cell-surface and was
able to bind IgG. If cells were biotinylated with a
cell-impermeable biotinylation reagent to biotinylate cell-surface proteins prior to lysis, covalent
hFcRn dimers were detected under non-reducing
conditions on blots probed with labeled streptavidin (Figure 5(b), lane 2). Furthermore, if cell lysates
280
Human FcRn Dimers
A
-
Flag-hFcRn
Myc-hFcRn
-
+
+
+
+
-
kDa
α-Flag
45
α-Myc
45
1
2
B
pH
Flag- &
Myc-hFcRn
-
7.4
+
3
pH
4
7.4 H 6.5
p
+
+
kDa
45
14
6
1
2
α-Flag IP
3
4
IgG IP
SA-HRP
C
a
c
Flag-& MychFcRn
e
b
d
f
Myc-hFcRn
α-Myc
α-Flag
Flag-hFcRn
were incubated with IgG-agarose beads at pH 6.5
or 7.4, and bound proteins were analyzed by nonreducing SDS-PAGE and immunoblotting with M2
anti-Flag antibodies, covalent hFcRn a-chain
dimers were present in IgG-agarose precipitates at
pH 6.5 (lane 4) but not at pH 7.4 (lane 3).
To determine if soluble receptors were able
to form disulfide-linked dimers, cell-culture
supernatant containing secreted Flag-hFcRn was
analyzed by non-reducing SDS-PAGE and immunoblotting. As shown in Figure 5(c), only the
monomeric form of the soluble receptor was
detected (lane 3). Although covalent dimers were
Figure 3. Characterization of cells
co-expressing Flag and Myc-hFcRn.
(a) Western blot analysis. Lysates of
control cells (lane 1) or cells expressing Myc-hFcRn alone (lane 2),
Flag-hFcRn alone (lane 3), or
co-expressing Flag- and MychFcRn (lane 4), were fractionated
by SDS-PAGE, blotted and probed
with M2 anti-Flag (top) or 9E10
anti-Myc
antibodies
(bottom).
(b) Co-precipitation of the putative
canine b2m with the FcRn a-chain.
Control cells (lane 1 or cells coexpressing Flag and Myc-hFcRn
(lanes 2– 4) were surface biotinylated, lysed and lysates were incubated with immobilized anti-Flag
antibodies (lanes 1 and 2) or IgG
agarose at pH 7.4 (lane 3) or pH 6.5
(lane 4). Biotinylated proteins
bound to the anti-Flag or IgG
beads were fractionated by SDSPAGE, blotted and probed with
streptavidin-HRP. A , 12 kDa protein, most likely canine b2m, was
associated with the FcRn a-chain.
The top and bottom parts of the gel
were exposed separately for optimal detection of the a-chain
and b2m. (c) Immunofluorescence
characterization. Cells stably expressing (a) and (b) Flag-hFcRn alone,
(c) and (d) Myc-hFcRn alone, or
(e) and (f) co-expressing Flag- and
Myc-hFcRn were fixed, permeabilized and stained by indirect
immunofluorescence with monoclonal anti-Flag (green) and polyclonal anti-Myc (red) antibodies.
One of two representative cell
clones used in the subsequent
experiments is shown.
readily detected for intact hFcRn (lane 2), no
dimers were observed for the soluble receptor
(lane 3), even if the blot was overexposed (data
not shown).
Thus, in addition to non-covalent dimers, membrane-anchored hFcRn can form disulfide-linked
dimers.
Discussion
The finding that FcRn is structurally closely
related to MHC class I led to considerable interest
Human FcRn Dimers
281
Figure 4. Co-immunoprecipitation of Myc and Flag-tagged
hFcRn. Cell lysates of control cells
(lane 1) or cells expressing MychFcRn alone (lane 2), Flag-hFcRn
alone (lane 3), or co-expressing
soluble Flag- and Myc-hFcRn (lane
4) were precipitated with either
immobilized M2 anti-Flag antibodies (a-Flag IP) or 9E10 anti-Myc
antibodies prebound to protein G
sepharose (a-Myc IP), fractionated
by non-reducing SDS-PAGE and
blotted with polyclonal anti-Flag
(top panel) or anti-Myc (bottom
panel) antibodies. In lanes 5 and
10, cells expressing only FlaghFcRn or only Myc-hFcRn were
mixed prior to immunoprecipitation to exclude that the formation
of dimers occurred during sample preparation. Asterisks in lane 7 indicate non-specific bands present in the antiMyc immunoprecipitates, one of which co-migrates with the FcRn a-chain. A representative blot of at least three
independent experiments carried out with two different clones is shown.
in the structure of the receptor and how it relates to
IgG binding. The crystal structure of the rat and
human receptors has been deduced,17 – 20 providing
detailed information on the overall folding of the
FcRn a-chain and its interaction with b2m. For the
rat receptor, structural information was obtained
also in the presence of ligand (i.e. IgG Fc fragments), identifying the contact sites between FcRn
and IgG, and showing that ligand binding does
not lead to dramatic conformational changes in
FcRn.
Despite the wealth of structural information for
FcRn and its interaction with IgG, the oligomeric
nature of the receptor remains controversial.
Evidence for FcRn dimers has been obtained only
for rat FcRn and is either based on SDS-PAGE and
Figure 5. Detection of covalent
disulfide linked hFcRn dimers.
(a) Full-length hFcRn. Control cells
(lanes 1 and 6) or cells expressing
Flag-hFcRn (lanes 2 – 5 and 7) were
lysed in the presence of IAM (lane
4) or NEM (lane 5). Lysates were
fractionated by SDS-PAGE under
reducing (lane 2; b-ME, b-mercaptoethanol) or non-reducing (lanes 1
and 3 – 7) conditions and blotted
with M2 anti-Flag antibodies (lanes
1 – 5) or with an a-hFcRn peptide
antibody
(lanes
6
and
7).
(b) Covalent hFcRn a-chain dimers
can reach the cell surface and bind
IgG. The plasma membrane of control cells (lane 1) or cells expressing
Flag-hFcRn (lane 2) was biotinylated and cell lysates were analyzed
by precipitatation with immobilized
M2 anti-Flag antibodies followed
by non-reducing SDS-PAGE and
blotting with streptavidin-HRP
(lanes 1 and 2). In lanes 3 and 4,
cells expressing Flag-hFcRn were
lysed and incubated with IgGagarose at pH 7.4 (lane 3) or pH 6.5 (lane 4). Bound proteins were analyzed by non-reducing SDS-PAGE and blotting
with M2 anti-Flag antibodies. (c) Soluble hFcRn does not form covalent dimers. Lysates of control cells (lane 1), cells
expressing full-length Flag-hFcRn (lane 2), or soluble Flag-hFcRn (lane 3) were fractionated by SDS-PAGE under nonreducing conditions and blotted with M2 anti-Flag antibodies. The data in (a)– (c) are representative for at least three
independent experiments carried out with two different cell clones.
282
electron irradiation studies of FcRn in rat neonatal
brush border23 or, more recently, on crystallographic data. Crystals of rat soluble FcRn show
dimers in which two a-chains contact each other
via their a-3-domains. Charged residues engaged
in electrostatic interactions and an ordered carbohydrate handshake in the dimer interface probably
mediate dimerization. Although the observation
that mimicking receptor oligomerization by immobilizing rat FcRn ectodomains on solid supports
increases the affinity for IgG has been used in
support of FcRn dimerization, no dimers of FcRn
ectodomains in solution could be detected.24
In contrast to rat FcRn, crystals of human FcRn
do not contain dimers, leading to the suggestion
that human FcRn may not be able to dimerize
because the charged amino acid residues and the
ordered N-linked carbohydrate implicated in rat
FcRn dimerization are not conserved. We now
show that intact, membrane-anchored human
FcRn can dimerize in vivo. The co-immunoprecipitation experiments show that human FcRn a-chain
can assemble into dimeric structures at the very
least. In addition to dimers, human FcRn may
assemble into higher-order non-covalent oligomers, which will not be detected in the experimental setup if the co-precipitated a-chains
dissociate in SDS. In any case, the charged residues
and the carbohydrate implicated in dimerization of
rat FcRn, but not conserved in human FcRn, are
apparently not required for dimerization of the
human receptor. Rather, since no dimers were
observed for a secreted soluble human FcRn, the
transmembrane and/or cytosolic domain appears
to be required for FcRn dimer formation, similar
to dimerization of the poly-Ig receptor.25
Interestingly, human FcRn was present in noncovalent as well as disulfide-linked covalent
dimers. The two types of dimers were observed in
MDCK cells, in which human FcRn associates
with canine b2m, and were present in FO-1 cells
expressing human b2m (Supplementary Material).
The molecular mass of the covalent dimers corresponds to that of two human FcRn a-chains, but
association of one FcRn a-chain and a second
protein of a similar mass cannot be ruled out
completely. While consistently observed, the portion of disulfide-linked dimers among the total
human FcRn showed significant experimental
variability. At least a detectable cohort of covalent
dimers was present on the cell surface and able to
bind IgG in a pH-dependent manner, indicating
that they do not simply represent misfolded aggregates. Indeed, covalent FcRn a-chain dimers are
present in human kidney;26 confirming the
presence of these dimers in a physiological setting.
On the basis of the crystal structure, Cys48 and
Cys251 in human FcRn do not engage in intrachain
disulfide bonds and may thus be available for an
interchain link. Cys251, which is not conserved in
FcRn from other species, is located at the border
of the putative dimer interface in a human FcRn
dimer modeled after the rat FcRn dimer18 and
Human FcRn Dimers
thus a possible candidate for the formation of an
intermolecular disulfide bond.
Since human FcRn dimers were detected in the
absence of IgG, ligand binding is not a requisite
for receptor dimerization. Crystals of rat FcRn in
the presence of Fc reveal two types of complexes:
a “lying” complex, in which one IgG molecule
binds asymmetrically to only one of two FcRn molecules, and a “standing” complex, in which one
IgG molecule binds symmetrically to two FcRn
molecules.17,18 The formation of postulated FcRn –
IgG ribbons8 requires the alternating assembly of
lying and standing complexes. Since the standing
complexes presumably can assemble only in the
presence of IgG, ligand binding, while not required
for receptor dimerization per se, may be relevant for
the formation of FcRn – IgG ribbons. The formation
of receptor– ligand ribbons could modulate
IgG binding properties, intracellular routing, or
possible signaling functions of FcRn.
Materials and Methods
Reagents
Restriction enzymes were purchased from New
England Biolabs, Pharmacia, Gibco or Roche and Pwo
polymerase was obtained from Roche. All enzymes
were used according to the manufacturer’s instructions.
Tissue culture media and supplements were from Sigma
Chem. Corp. or Gibco. Fetal calf serum was purchased
from Hyclone and the antibiotics G-418 and hygromycin
from Calbiochem-Novabiochem. Corp. The protease
inhibitor cocktail contained 10 mg/ml each of chymostatin, leupeptin, antipain, and pepstatin A (from Sigma
Chem. Corp.) in DMSO and was used at a 1:1000
dilution. The protease inhibitor PMSF (Sigma Chem.
Corp.) was used at a concentration of 0.57 mM. SuperSignal chemiluminescence system was purchased
from Pierce. The alkylating reagents iodoacetamide
(IAM) and N-ethylmalemide (NEM) as well as protein
A-negative Staphylococcus aureus strain (Wood 46 strain)
were obtained from Sigma Chem. Corp. PVDF membranes were either from Macherey-Nagel or Bio-Rad
Laboratories. The Bradford assay kit was from Bio-Rad
Laboratories. Mowiol 4-88 (Calbiochem-Novabiochem.
Corp.) was used at 0.1 g/ml supplemented with 0.2%
(w/v) DABCO (Sigma Chem. Corp.).
Antibodies
Monoclonal M2 anti-Flag antibodies and M2-Sepharose were purchased from Sigma Chem. Corp., cellculture supernatant containing 9E10 anti-Myc antibodies
was kindly provided by R. Iggo (Epalinges, Switzerland).
For co-immunoprecipitation experiments, polyclonal
anti-Flag and anti-Myc antibodies from Zymed and
Santa Cruz Biotechnology Inc., respectively, were used.
For immunofluorescence, polyclonal anti-myc antibodies
were purchased from Upstate Biotechnology. A polyclonal rabbit anti-FcRn peptide serum has been
described.9 Horse radish peroxidase (HRP)-coupled
secondary antibodies were purchased from Jackson
ImmunoResearch Labs., Inc. or Bio-Rad Laboratories.
Affinity-purified, fluorescently labeled, secondary antibodies were from Molecular Probes Inc.
283
Human FcRn Dimers
Epitope-tagged hFcRn constructs
hFcRn constructs carrying the Igk leader sequence and
an N-terminal Flag (Flag-hFcRn) or Myc (Myc-hFcRn)epitope tag on expression vectors with different selection
markers (e.g. G418 or hygromycin, respectively) have
been described.9,27 Corresponding constructs encoding
soluble forms of Flag- and Myc-hFcRn were generated
by PCR using oligonucleotides to introduce a stop
codon after amino acid residue 274 in hFcRn.
Cell culture and transfection
MDCK strain II cells were cultured as described.9 To
deplete the cells from residual IgG in fetal calf serum
(FCS), cells were washed with PBS and grown in
medium without FCS or in a defined serum-free MDCK
cell medium (Sigma Chem. Corp.) for at least 24 hours
prior to all experiments. MDCK cells stably expressing
Flag-hFcRn9 were transfected with the Myc-hFcRn
plasmid and selected in 0.5 mg/ml of hygromycin. Cells
stably expressing a soluble Flag-hFcRn or Myc-hFcRn
were generated as described9 and selected in 0.5 mg/ml
of hygromycin or G418, respectively. Cells stably expressing soluble Flag-hFcRn were then transfected with the
soluble Myc-hFcRn construct. Clones were analyzed
for expression by immunofluorescence and Western
blotting, and two clones for each transfection were used
for further experiments. Stably transfected cells were
maintained in 0.25 mg/ml of G-418 and/or 0.5 mg/ml
of hygromycin.
Co-immunoprecipitation and Western blot analysis
For analysis of cell lysates, cells were lysed in lysis
buffer (0.5% (v/v) Triton X-100 in PBS and protease
inhibitors) and sample preparation for SDS-PAGE was
carried out as described,9 except that the lysis buffer
was supplemented with 100 mM IAM or 10 mM NEM
where indicated. Protein concentrations were determined by the Bradford method. For co-precipitation
experiments, pre-cleared cell lysates were incubated
overnight at 4 8C with either M2-Sepharose (5 ml/100 mg
of lysate) or 9E10 (1 ml/100 mg of lysate) pre-bound to
protein G-Sepharose (5 ml/100 mg of lysate). Beads were
then washed three times with RIPA buffer (10 mM Tris
(pH 7.4), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1%
sodium deoxycholate, 1% SDS, 1% Triton X-100) and
bound proteins eluted by heating in sample buffer for
30 minutes at 40 8C. Samples were analyzed by reducing
or non-reducing SDS-PAGE on Tris – tricine 10% polyacrylamide gels, blotted onto PVDF membranes and
probed with polyclonal anti-Flag (2.5 mg/ml) or antiMyc (1 mg/ml) antibodies, monoclonal M2 anti-Flag
(5 mg/ml) or 9E10 anti-Myc antibodies (1:1000), or with
polyclonal anti-FcRn serum (1:500). Primary antibodies
were detected with HRP-coupled anti-mouse or antirabbit secondary antibodies (1 mg/ml) and chemiluminescence. Cell-surface biotinylation and IgG-agarose
binding experiments were carried out described.9
Immunofluorescence
Cells grown on coverslips were processed for immunofluorescence and labeled with M2 antibodies (5 mg/
ml) and/or polyclonal anti-Myc antibodies (5 mg/ml)
followed by fluorescently labeled goat anti-mouse
(Alexa488; 2 mg/ml) and goat anti-rabbit (Alexa568;
2 mg/ml) secondary antibodies as described.9
Acknowledgments
We thank Prasanna Kolatkar (IMCB, Singapore), and
past and present members of our laboratory for helpful
discussions. W.H. is an adjunct staff member at
the Department of Physiology, National University of
Singapore.
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Edited by I. Wilson
(Received 23 November 2001; received in revised form
14 June 2002; accepted 18 June 2002)
http://www.academicpress.com/jmb
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